Aortic arch replacement in acute type A aortic dissection patients remains the most challenging cardiovascular operation. Herein, we described our modified Y-graft technique using the Femoral Artery Bypass (FAB) and the One Minute Systemic Circulatory Arrest (OSCA) technique, and assessed the short-term outcomes of the patients.Between February 2015 and November 2017, 51 patients with acute type A aortic dissection underwent aortic arch replacement. Among them, 23 patients underwent FAB while 28 patients underwent both FAB and OSCA. The intraoperative data and postoperative follow-up data were recorded. The follow-up data of patients with traditional Y-graft technique were collected from previously reported studies.In the FAB group, two patients died due to pulmonary infection , and two patients were paralyzed from the waist down. Hemodialysis was performed for five patients (21.7%) before hospital discharge. Fifteen patients (65.2%) received respiratory support for more than 2-days and eight patients (34.8%) for more than 5-days. These follow-up results were comparable or better than the patients with traditional Y-graft technique. Furthermore, compared to the FAB group, the morbidity due to neurological dysfunction and acute renal failure was significantly reduced in the FAB+OSCA group. Moreover, the respiratory support, length of postoperative stay and ICU stay were shortened.This study clarified the feasibility of FAB and OSCA technique in modifying Y-graft technique. The acute type A aortic dissection patients showed less surgical complications and favorable short-term outcomes after this surgery. The first successful aortic arch replacement was reported more than fifty years ago. With the development of the surgical technique and the improvement of patient care, the aortic arch can be repaired more safely now. However, total aortic arch replacement in acute type A aortic dissection patients remains the most challenging cardiovascular operation, which incurs high risk of cerebral damage and acute renal failure, and consequently, a considerable risk of operative mortality. The 30-day mortality was as high as 18% and stroke rates were as high as 10% in 2011 [In 2002, Spielvogel and colleagues developed the Y-graft technique to enable antegrade selective cerebral perfusion , 4. HoweOur team modified the traditional Y-graft technique using Femoral Artery Bypass (FAB) and One Minute Systemic Circulatory Arrest (OSCA) technique to simplify arch reconstruction, reduce embolization and avoid cerebral ischemia.In the past 2 years, we have treated 51 cases of acute type A aortic dissection with our modified Y-graft technique for aortic arch replacement. The perioperative data and short-term follow-up data, such as perfusion time, 30-days survival rate, neurological dysfunction, length of respiratory support and acute renal failure, were recorded for all patients. The follow-up data of patients with traditional Y-graft technique were collected from previously reported studies. Herein, we described our modified Y-graft technique and assessed the short-term outcomes of the patients.We conducted a retrospective analysis of the data from 51 patients who underwent aortic arch replacement between February 2015 and November 2017 at our hospital. The clinical features of the patients are shown in Table The surgical procedure was divided into three stages.The femoral artery was exposed through a small incision in the inferior inguinal ligament. The left axillary artery was exposed through a small infra-clavicular incision. A median sternotomy was performed with extension of the incision superiorly along the medial border of the left sternocleidomastoid muscle and the brachiocephalic vessels were exposed.Intravenous heparin was administered to achieve an activated clotting time (ACT) > 350 s. A 10 mm graft (4 cm length) was end-to-side anastomosed to the femoral artery and then connected to the arterial tubes of the cardiopulmonary bypass (CPB) machine. Another 10 mm (15 cm length) graft was end-to-side anastomosed to the left subclavian artery and the other end of this graft was tunneled via the second intercostal space into the mediastinum on demand. The left common carotid artery was cannulated with an arterial catheter, which was connected to the arterial tubes of the CPB machine. Since the CPB machine did not work, the femoral artery-to-left common carotid artery bypass was completed. Then the left common carotid artery was transected. Another 8 mm graft was end-to-end anastomosed to the left common carotid artery. The previous 10 mm graft (connected to the left subclavian artery) was measured to the appropriate length and side-to-side anastomosed to this 8 mm graft (connected to the left common carotid artery). The free-end of the previous 10 mm graft (connected to the left subclavian artery) was tightly connected to the arterial tubes of the CPB machine. The free-end of the 8 mm graft (connected to the left common carotid artery) was clamped. This is how both femoral artery-to-left common carotid artery bypass and the femoral artery-to-left subclavian artery bypass were completed. As for the innominate artery, the procedures of cannulation and anastomosis were similar to the left common carotid artery. The innominate artery was cannulated with an arterial catheter, which was connected to the arterial tubes of the CPB machine. A femoral artery-to-innominate artery bypass was completed. Then the innominate artery was transected. A 12 mm graft was end-to-end anastomosed to the innominate artery. The free-end of the 12 mm graft was clamped.Finally, without the assistance of CPB machine, femoral artery-to-left common carotid artery bypass, femoral artery-to-left subclavian artery bypass and femoral artery-to-innominate artery bypass were all completed at room temperature, with continuous selective bilateral cerebral perfusion. The reconstruction of the three branches was also successfully completed. Every graft was carefully de-aired, and perfusion was restored to the head and upper extremities Fig. .The pericardium was opened after cannulation of the superior and inferior vena cava, and ensuring that the CPB machine worked and cooling was started. The ascending aorta was cross-clamped at 32 °C and the cardioplegic solution was usually perfused through a coronary sinus cannulation to arrest the heart. After the cardioplegic cardiac arrest, the aortic valve repair or replacement  was performed if significant aortic valve insufficiency was identified through TEE. The ascending aorta was also replaced by the graft, which was anastomosed to the aortic sinotubular junction or the artificial valve ring.The completion of the reconstruction of the proximal aortic root usually coincided with the end of core cooling (target temperature: 32 °C). At the beginning of this stage, the patient was placed in slight Trendelenburg position, and the head was packed in ice. The cross-clamp was moved to the distal aortic arch (between the innominate artery and the left common carotid artery), and the aortic arch was trimmed. Hypothermic circulatory arrest was started after removing the clamp. The intraoperative stent  was placed into the distal aortic arch and then the aortic arch was immediately cross-clamped after de-airing  was end-to-side anastomosed to the 12 mm graft (innominate artery). The free-end of the 12 mm graft (innominate artery) was end-to-side anastomosed to the ascending graft in ideal site , due to pulmonary infection. Two patients were paralyzed from the waist down, one of them was transient, while the other did not recover during the follow-up period. Hemodialysis was performed for five patients (21.7%) during the follow-up period. Fifteen patients (65.2%) received respiratory support for more than 2-days and eight patients (34.8%) for more than 5-days. As for the traditional Y-graft technique, we reviewed previous articles and obtained the corresponding data , male gender (78.6%), weight (80.8 ± 12.6 Kg), Marfan syndrome (7.1%), aortic valve regurgitation (28.6%), smoking, past or current (75.0%), hypertension (92.9%), renal dysfunction (7.1%), and pulmonary disease, are shown in Table  years, mThe operative variables are also shown in Table The operative mortality was 7.1%, and neurological dysfunction was 3.6%. Among the 28 patients, 10 patients (35.7%) had >2 days intubation and three patients (10.7%) had >5 days intubation. Only one patient (3.6%) had postoperative renal dysfunction and required temporary hemodialysis before discharge.p<0.05), except for the aortic clamp time and the skin-to-skin time. As for the short-term outcomes, the 28 patients who underwent modified Y-graft technique using FAB+OSCA had an operative mortality of 7.1% and the morbidity of neurological dysfunction was 3.6% . By this technique, we can reconstruct the brachiocephalic branches at room temperature, without cardiopulmonary bypass (CPB). Even the pericardium does not need to be opened. The left subclavian artery is difficult to expose and anastomose. Nerve injury, especially recurrent laryngeal nerve, can easily occur. Notably, reconstruction of brachiocephalic branches is easier to perform by our modified technique. Besides, it is easy to repair a leak immediately after anastomoses of the supra-aortic arteries because of optimal mobility and slight wall tension of the anastomoses. Twenty-three patients underwent this modified technique. Subsequently, we modified the Y-graft technique using FAB and One Minute Systemic Circulatory Arrest (OSCA) technique. The latter technique majorly decreases the systemic circulatory arrest time, thus reducing the ischemic time of spinal cord and kidneys. Moreover, the lowest nasopharyngeal/rectal temperature can be maintained at 32 °C. To summarize, our modified technique almost eliminated the systemic circulatory arrest time, and reduced the CPB time and other operative variables.Comparison of the traditional Y-graft technique with the modified Y-graft technique using FAB Table  showed tComparison of the FAB technique and FAB+OSCA technique Table  showed tThis study clarified the feasibility of FAB+OSCA technique in modifying Y-graft technique. The acute type A aortic dissection patients showed less surgical complications and favorable short-term outcomes by this surgery. Comparison of the traditional Y-graft technique with the modified Y-graft technique using FAB Table  showed t


The correlation of the in vitro dissolution of a drug with the pharmacokinetics of one of its metabolites was recently proposed by the authors of the article as an additional or alternative analysis to the usual in vitro correlations in vivo, mainly in the case of fast-absorbing drugs that have metabolites with a significant therapeutic effect. The model proposed by the authors considers that amiodarone has a slow dissolution, rapid absorption, and rapid metabolism, and before returning to the blood from other compartments, its pharmacokinetics is determined mainly by the kinetics of release in the intestine from the pharmaceutical formulation. Under these conditions, the rate of apparition of desethylamiodarone in the blood is a metric of the release of amiodarone in the intestinal fluid. Furthermore, it has been shown that such an estimated in vivo dissolution is similar, after time scaling, to the dissolution measured experimentally in vitro. Dissolution data of amiodarone and the pharmacokinetic data of its active metabolite desethylamiodarone were obtained in a bioequivalence study of 24 healthy volunteers. The elimination constant of the metabolite from plasma was estimated as the slope of the linear regression of logarithmically transformed data on the tail of plasma levels. Because the elimination of desethylamiodarone was shown to follow a monoexponential model, a Nelson–Wagner-type mass equilibrium model could be applied to calculate the time course of the “plasma metabolite fraction.” After Levi-type time scaling for imposing the in vitro–in vivo correlation, the problem became that of the correlation between in vitro dissolution time and in vivo dissolution time, which was proven to follow a square root model. To validate the model, evaluations were performed for the reference drug and test drug separately. In both cases, the scaled time for in vivo dissolution, t*, depended approximately linearly on the square root of the in vitro dissolution time t, with the two regression lines being practically parallel.Due to its very low water solubility and complex pharmacokinetics, a reliable point-to-point correlation of its  Amiodarone (AMD) has been shown to have variable oral bioavailability (20–80%). After absorption, AMD undergoes extensive metabolism, is distributed in the blood, lipids, and in deep compartments, and undergoes enterohepatic circulation . MetabolConcentrations in the myocardium have been shown to be 35 times higher than in the plasma . The phaIn vitro–in vivo correlations (IVIVCs) are correlations between in vitro dissolution data and in vivo release kinetics, estimated by the deconvolution of pharmacokinetic IVIVCs were constantly recommended by regulatory authorities in the last decades when developing extended-release formulations  at 100 rpm. The dissolution medium was sodium lauryl sulfate 10 g/L in ultrapure water . Samples of 5 ± 0.1 ml were collected at 5, 15, 30, 45, and 60 min and subsequently replaced with an equal volume of medium. AMD concentrations were determined at 242 nm on a V-530 UV-VIS spectrophotometer .In vivo data were obtained in a bioequivalence study by comparing a tested formulation (T) with reference (R) Cordarone 200 mg, Sanofi Synthelabo. The study was approved by the Romanian National Medicines Agency and Ethics Committee of the Army Center for Medical Research.Venous blood samples (5 ml) were collected into heparinized tubes through a catheter inserted in the antecubital vein before (time 0) and at 1, 1.5, 2, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 9, 10, 12, 24, 48, 72, 96, and 120 h. Blood samples were centrifuged at 5°C for 6 min at ∼3,000 rpm. Plasma was immediately frozen and stored at −30°C until analysis.Plasma samples  were transferred to 10 ml disposable polypropylene tubes, to which 50 µL internal standard (IS) solution (20 µg/ml fenofibrate in methanol), 500 µL pH 4.5 phosphate buffer, and 4 ml methyl tert-butyl ether were added. The tubes were vortex mixed for 10 min and then centrifuged for 10 min at 4,000 rpm. Of the organic layer, 3 ml were retaken and evaporated to dryness at 40°C under a gentle nitrogen steam. The sample was reconstituted into 200 μL of mobile phase. Of each sample, 100 µL were injected into the chromatographic column.2HPO4 and 11 mM KH2PO4, adjusted to pH 4.5 (Solvent A) and a 1:1 (v:v) acetonitrile methanol mixture (Solvent B), and delivered in a 20:80 (v:v) ratio. The mobile phase was prepared daily, filtered, and degassed before use. The flow rate was 1.0 ml/min, and all work was carried out at 40°C.The chromatographic analyses were performed on a Waters liquid chromatographic system  consisting of a 600E quaternary gradient system, an AF model in line degasser, 486 UV-VIS tunable absorbance detector, and a 717 plus auto sampler. Empower Pro software  was used to control the system and acquire and process data. The UV detector was set at 242 nm. A 15 cm × 4.6 mm i.d Microsorb-MV C18 column  and a guard column packed with C18 were used for separation. The mobile phase consisted of a phosphate buffer solution containing 7 mM NaThe method was validated in accordance with the bioanalytical method validation guidelines of the FDA, including linearity, limits of quantification, selectivity, accuracy, precision, recovery, dilution effects, and stability. The specificity was evaluated related to interferences from the endogenous matrix components of drug-free plasma samples of six different origins. The calibration curves of AMD and DAMD were constructed in the range in the range 20–1,000 ng/ml for both AMD and DAMD, by plotting the ratios between their peak areas and IS peak areas vs. concentration (ng/ml), using data obtained from triplicate analysis of the calibration standard solution. The lower limit of quantification (LLOQ) was set as the lowest concentration on the calibration curve. Within-run and between-run precision and accuracy were estimated by analyzing five replicates of the LLOQ and quality control (QC) samples in a single analytical run and on five consecutive days, respectively. The absolute recovery of AMD and DAMD was determined using five replicates of the three concentration level QC samples and was determined to be 74% for AMD and 97% for DAMD. Benchtop, extract, stock solution, freeze-and-thaw, long-term, and post-preparative stability studies were also performed to evaluate the stability of both analytes.max) were considered as random variables with the following structure  + C + eijk,Yk, j = index for period, k = index for sequence, F = the direct fix effect of the formulation in the kth sequence which is administered at the jth period, Cth period, where C = 0 and ΣC = 0, and eijk = the within-subject random error in observing Yijk.where μ = the overall mean, i = index for subject, i = 1, nAll parameters were evaluated by analyses of variance to determine statistically significant (α = 0.05) differences between the drug formulations using the program Kinetica, version 4.2. InnaPhase Corporation.0-τ and AUC0-∞ were shown to lie within the 80–125% interval.To demonstrate bioequivalence, the 90% confidence intervals for AMD (DAMD) test/reference ratios of AUCIn vitro dissolution data were modeled using a square root law and a power law model, used in linear forms, as previously described (escribed .r(t) is the ratio of cumulated released substance at the moment t. It should be noted that r(t) is sometimes written in the form r(t) = M(t)/M∞, where M∞ is the amount released at infinity; however, in all cases, this is not the total amount of diffusing component. In case of nanosystems, for example, the release most frequently involves only a part of the active substance, which we can consider as the “available fraction for release,” with another part of it remaining sequestered. Whatever the case, in practice, in most cases, the experimentally determined quantity tends to reach a saturation value. If this value remains constant for a sufficient period of time, it is reliable to consider it as M∞.The law of square root can result from a phenomenological model that involves the diffusion of the drug into the solvent that penetrates the matrix of the pharmaceutical formulation (Higuchi model) or from a model that considers release from the pharmaceutical formulation as an infinite reservoir across the interfaces with the solvent in a long diffusion path :r(t)=kt,Power law is an empirical law which combines two release kinetics as a result of the diffusion and the erosion of a matrix, is linearized in the formppas law .Analysis of time evolution of plasma levels of AMD and DAMD and estimation of the pharmacokinetic parameters was performed by both non-compartmental and compartmental methods, based on the data obtained in the 0–120 h time interval.There were estimated partial and cumulated areas under curves. It was tested if, after logarithmic transformation, a good regression line on the tail of the curve was obtained, in order to define an elimination constant. Mono- and bicompartmental modeling was tested for AMD and DAMD pharmacokinetics.Amiodarone, a lipophilic drug (logP = 7.24), undergoes substantial metabolism, being classified as BDDCS (biopharmaceutics drug disposition classification system) Class 2 compounds .t) could be considered an estimation of the absorption of the parent drug from the intestine FRA(ti). Based on this hypothesis, a correlation between in vitro dissolution and the in vivo pharmacokinetics of metabolites would be expected, which was indeed found in the case of diltiazem.The hypothesis of this article, presented previously by the authors , was thaBecause the pharmacokinetics was measured after a single dose, the return from the “deep compartment,” where accumulation occurs over time, was neglected. Furthermore, because metabolites occur at the same time as plasma AMD, metabolism is considered a rapid process.Consequently, the slowest, rate-determining step for the chain of kinetics leading to the apparition of metabolite in plasma remains the release kinetics of the parent drug in the gastrointestinal tract.Again, because AMD is lipophilic, the rate of transfer from the blood to the lipid compartment is higher than that of reverse transport; the return of AMD to the blood may be neglected, and the transfer from blood to lipids will become a component of the elimination of the parent drug.Consequently, in a simplified one-compartment model for DAMD, it was considered only two processes, corresponding to the appearance of the metabolite in the blood and its total elimination .cAMD and cdAMD are, respectively, the concentrations of the parent drug and metabolite in blood compartment.Where FRAAMD is the absorption fraction of AMD, FRD is the dissolution fraction, and correl denotes correlation.→ represents a slow process and →→ a rapid process, FRAp):FRApDAMD is the fraction of the apparition of the metabolized drug at time ti, cdAMD(ti) is the plasma concentration of the metabolite at time ti, and A modified, Wagner–Nelson-type equation  was applThe elimination rate constant was estimated as the slope of linear regression of the last points of the logarithmic transformed data. Integrals were approximated by areas under plasma levels of DAMD.The model could actually be much more general. In the case of compounds subject to extended metabolism (BDDCS classes 1 and 2 compounds), because the rate of absorption and metabolism are usually high, the rate of occurrence of metabolites in the plasma is determined by the rate and extent of the parent drug release from the pharmaceutical formulation.Since amiodarone is lipophilic (logP 7.24 ) [Amiodarone DrugBanK], its dissolution in water is very low, meaning that it is necessary to add surface-active agents in dissolution medium. The FDA recommends sodium lauryl sulfate (SLS) 1% or Tween 80 1% (accessdata.fda). In these conditions, the dissolution of AMD was rapid, being complete within 1 h in all cases. The mean amiodarone dissolution profiles are presented in Dissolution is forced by the addition of a high concentration of surfactant in the release medium, which is a good test for quality control, but dissolution in the presence of great concentrations of surface-active agents is not biorelevant .The modeling of release kinetics was performed using both the square root and power law model. It appeared that both models work well enough. Correlation coefficient was higher in the case of the power law, but the number of points approximated by the square root law was greater. Fitting with the square root law for tested and reference drug are presented in Individual pharmacokinetics curves for AMD and mean curves for AMD and DAMD, for the reference (R) and tested (T)formulations are presented in There is a great variability of concentrations between subjects from 12 h, but it is, at the same time, to note that the tails of curves are approximately parallel, suggesting a common pattern for elimination in all subjects. AMD has unpredictable absorption and therefore bioavailability .In the first phase, a rapid decrease in plasma levels appeared, with lipids and deep compartments becoming depots for both AMD and DAMD. Later, both of them return to the central compartment, and a long and variable terminal elimination half-life appears .A naked eye analysis suggests that the formulations are bioequivalent. Mean pharmacokinetic parameters and 90% confidence intervals for mean ratios As the formulations proved to be bioequivalent in spite of their high variability, starting from AUC and Cmax, a first analysis was performed on the entire set of data in the study .in vivo, the elimination constants for AMD and DAMD were estimated.To apply the mass balance of the Wagner–Nelson type in the calculation of the fraction of drug absorbed and, in our case, the fraction of AMD dissolved Half-time was not well defined in the case of AMD, with the result depending on the interval selected on the tail of the plasma level curves. Three, very different values were obtained: 7 h in the 7–12 h interval, 23 h in the 12–48 h interval, and 77 h in the 48–120 h interval. In the label of the AMD reference drug, a half-time of 53 days is reported. This evolution is a result of the distribution in lipids and enterohepatic circulation, as well as returning AMD back to the central compartment from the accumulations in lipid and deep compartments.in vitro and in vivo evaluations of three tablet formulations of amiodarone in healthy subjects were previously reported by in vitro dissolution, 120 min, and the in vivo time points of up to 18 h. He applied a time scale, following the FDA recommendation: “Time scaling may be used as long as the time scaling factor is the same for all formulations.” His conclusion was that “a point-to-point acceptable and reliable correlation was not achieved” and “dissolution data could be used only for routine and in-process quality control of amiodarone tablet formulations.”Comparative In the case of DAMD, as can be seen in By introducing this value in the proposed deconvolution formula and making the calculation, as can be seen in in vivo, an FRAp dependence on time similar to the model of dissolution kinetics in vitro could be expected. A naked eye examination suggests a linear model. A good fit of FRA as a function of the square root of time was also was obtained.As the pharmacokinetic model supposes that the apparition of DAMD in plasma equals the release of AMD The linear correlation is just slightly better, but the small lag time appeared in the square root of time scale; this was a good result since absorption and metabolism are not instantaneous.in vivo release is much slower.Following the low solubility of AMD and the small volume of GI liquids, dissolution had reason to be slow and limited. Release is also influenced by the secretion of bile salts and lecithine . Releasein vivo dissolution of the parent drug. In order to correlate the in vitro dissolution fraction with the in vivo appearance of metabolite, time scaling was performed. Time in the interval 0–60 min, corresponding to in vitro dissolution, was transformed in time t* in the interval 0–7 h.n circumstances of the model, the apparition of metabolite in plasma is correlated with An exponential dependence of the FRA on FRD  is diffiin vivo dissolution/T50% in vivo dissolution  was obtained, as can be seen in t (IA function een in t  seemed teen in t . This reeen in t .In the case of lipophilic drugs, due to slow dissolution, rapid absorption, and rapid metabolism, the pharmacokinetics of both the parent drug and metabolites before the return of the drug from other compartments in the blood is mainly determined by the kinetics of release in the intestine from the pharmaceutical formulation.For long-life lipophilic drugs, as shown for DMA, it is possible to estimate the absorption fraction of the parent drug from the simpler pharmacokinetics of the metabolite, in which case it is possible to calculate an elimination constant.in vitro dissolution and the in vivo estimated dissolution models as well as the similar dependence of scaled time on in vitro time in the case of bioequivalent formulations can be considered a validation of the metabolite approach of the in vitro–in vivo correlation model.The similarity between


Although numerous studies have presented potential mechanisms underlying its pathogenesis, the understanding of α-synuclein-mediated neurodegeneration remains far from complete. Here, we show that overexpression of α-synuclein leads to impaired DNA repair and cellular senescence. Transcriptome analysis showed that α-synuclein overexpression led to cellular senescence with activation of the p53 pathway and DNA damage responses (DDRs). Chromatin immunoprecipitation analyses using p53 and γH2AX, chromosomal markers of DNA damage, revealed that these proteins bind to promoters and regulate the expression of DDR and cellular senescence genes. Cellular marker analyses confirmed cellular senescence and the accumulation of DNA double-strand breaks. The non-homologous end joining (NHEJ) DNA repair pathway was activated in α-synuclein-overexpressing cells. However, the expression of MRE11, a key component of the DSB repair system, was reduced, suggesting that the repair pathway induction was incomplete. Neuropathological examination of α-synuclein transgenic mice showed increased levels of phospho-α-synuclein and DNA double-strand breaks, as well as markers of cellular senescence, at an early, presymptomatic stage. These results suggest that the accumulation of DNA double-strand breaks (DSBs) and cellular senescence are intermediaries of α-synuclein-induced pathogenesis in PD. Excess levels of a protein involved in Parkinson’s disease can impair the brain’s capacity to repair DNA damage, leading to a state of cellular aging that accelerates neuronal death. When aggregated, the α-synuclein protein plays a major role in Parkinson’s disease and other neurodegenerative disorders. A team from South Korea, led by He-Jin Lee of Konkuk University, Seoul, and Seung-Jae Lee of Seoul National University College of Medicine, showed that human neuronal cells and mouse models with elevated expression of α-synuclein develop double-stranded breaks in their genomes as a consequence of deficient quality control mechanisms. The accumulated DNA damage spurs the cells to enter a state in which they show canonical signs of cellular aging but remain metabolically active in ways that fuel neurodegeneration. Therapies that target these processes could help prevent or treat α-synuclein–linked diseases. This protein is abnormally folded and aggregated in several neurodegenerative diseases, referred to as α-synucleinopathies, such as dementia with Lewy bodies, multiple system atrophy, and Parkinson’s disease (PD)2. These diseases affect millions of people worldwide, with the distinct characteristics of intracytoplasmic protein aggregates and a gradual increase in neuronal death in particular areas of the brain.α-Synuclein is an abundant neuronal protein with an intrinsically disordered structure3. The central nervous system undergoes considerable changes with age. Brain autopsy studies of aged people without a PD diagnosis have reported brain and spinal cord atrophy; decreases in the volume of gray matter; accumulation of pathological protein aggregates, such as amyloid plaques, neurofibrillary tangles, and Lewy bodies; and inclusions of TAR DNA-binding protein 43 and senescent cells5.Aging is a serious risk factor for PD and related neurodegenerative diseases3. In addition, senescent cells undergo morphological alterations to become larger and irregular due to cytoskeletal rearrangements and changes in cell membrane composition7. Senescence is often triggered by irreparable DNA damage, which accumulates with aging. This accumulation may be responsible for the pathogenesis of age-related diseases, such as neurodegenerative diseases.Cellular senescence is defined by irreversible cell cycle arrest and resistance to apoptotic death, often accompanied by changes in cell metabolism, such as changes in protein synthesis, glycolysis, fatty acid oxidation, reactive oxygen species (ROS) generation, and the acquisition of senescence-specific phenotypes8. However, they are highly toxic and mutagenic, as chromosomal breakage may result in loss of genetic integrity9. Two major pathways are involved in repairing DSBs: homologous recombination (HR) and non-homologous end joining (NHEJ). HR is the predominant DSB repair pathway during embryonic development, meiotic recombination, replication fork stabilization, one-ended DSB repair, and two-ended DSB repair of the late S/G2 phase of the cell cycle12. The NHEJ pathway is the main pathway in mammalian cells acting on DSBs through all cell cycle phases, including the G1 interphase12. The primary DSB repair pathway for postmitotic neurons is NHEJ, although HR is important for other proliferating cells in the brain16. The initiation of DSB repair involves the formation of the MRE11-RAD50-NBS1 (MRN) complex, a central DSB sensor18. MRE11 plays an important role in the regulation of the choice of DSB repair pathway19. DNA end resection of DSBs leads to the loading of the RPA complex and RAD51 for HR repair, whereas blocking resection leads to NHEJ repair19. The binding of the Ku70/Ku80 heterodimer to DSB sites is recognized as blocking resection, which leads to NHEJ repair.DNA double-strand breaks (DSBs) arise infrequently, on the order of 10–50 per cell per day20. In addition to the direct restoration of DNA integrity, DDRs also activate several cellular processes, such as cell cycle checkpoints, gene expression, and protein turnover. p53/p21 and p16/p16INK4a-pRB are the two primary regulators of these responses. Poor execution of DDRs may trigger cellular senescence; persistent DDRs may lead to age-related neurodegenerative diseases.DNA damage activates a series of cellular pathways called DNA damage responses (DDRs). DSBs are the strongest triggers for such reactionsThis study aimed to assess the effects of α-synuclein on DDRs and their connections to cellular senescence. Our results showed that α-synuclein in human neuronal cells increased DSBs with impaired DNA repair. α-Synuclein-induced impairment of DSB repair leads to increased levels of senescence markers. These results suggest that α-synuclein induces cellular senescence with DSB accumulation via impaired DDRs.The following primary antibodies were used: α-synuclein monoclonal antibody (BD Biosciences), γH2AX, phospho-ATM, ATM, p53, H3, poly(ADP-ribose) polymerase (PARP), ERCC1, XRCC1, MRE11, Rad51, Ku80, DDB2, p16, p73, and H3K9me3 antibodies ; H2AX, phospho-p53, p21, and Ku70 antibodies ; α-tubulin and β-actin antibodies ; and 53BP1 antibody .Retinoic acid, poly-L-lysine, and glutathione were obtained from Sigma–Aldrich. NE-PER™ Nuclear and Cytoplasmic Extraction Reagents were obtained from Thermo Fisher Scientific . X-gal was obtained from Duchefa Biochemie .Total RNA was amplified and purified using the Target Amp-Nano Labeling Kit for Illumina Expression BeadChip. Detection of the array signal was carried out using Amersham Fluorolink Cy3 Streptavidin according to the bead array manual. The arrays were scanned using a bead array reader confocal scanner. The quality of the hybridization and overall chip performance were monitored manually by visual inspection of internal quality control checks and raw scanned data. Raw data were extracted using the software provided by Illumina Genome Studio v2011.1 and Gene Expression Module v1.9.0. Array probes were logarithm transformed and normalized using the quantile method. Filtered reads were aligned to the human reference genome (hg38 assembly) using the STAR mapper. The mapped reads were counted and converted to TPM values using RSEM. For differentially expressed gene analysis, fold-change and statistical significance were calculated using DESeq. Gene set enrichment analysis (GSEA) was performed in preranked mode. This dataset was obtained from the National Center for Biotechnology Information (NCBI) database (accession no. GSE149559).DAVID, Metascape, and Enrichr were used to infer the biological functions of the genes associated with the peaks. Default parameters were used.To evaluate global gene expression profiles, the RRHO test was performed on two sets of gene expression comparisons (10.3390/ijms20236098). In this algorithm, genes were ranked according to their differential expression between two sample groups, and then these ranked gene expression profiles were iteratively assessed for overlap.ChIP assays were performed according to the instructions provided by Upstate Biotechnology. For each assay, 50 μg of DNA was sheared by sonication (DNA fragment size 200–500 bp) and precleared with protein A magnetic beads (Upstate Biotechnology #16-661). Then, 50 μg of DNA was precipitated using γH2AX and P53 antibodies. After immunoprecipitation (IP), the recovered chromatin fragments were subjected to sequencing.TM DNA Library Prep Kit. Briefly, the chipped DNA was ligated to adaptors. After purification, PCR was performed on the adaptor-ligated DNA with an index primer for multiplex sequencing. The library was purified using magnetic beads to remove all the reaction components. The size of the library was assessed using an Agilent 2100 Bioanalyzer. High-throughput 100 bp paired-end sequencing was performed using a HiSeq 2500 system. The dataset was submitted to the NCBI Gene Expression Omnibus database (accession no. GSE149558).The library was constructed using the NEBNext® UltraThe sequenced reads were trimmed using BBMap (BBDuk) and aligned to the human reference genome (hg38 assembly) using Bowtie 2. HOMER (findPeaks) was used to identify P53 binding sites (peaks) or rH2AX-enriched sites compared with the corresponding input samples in Con and SNCA-overexpressing cells with a false discovery rate-adjusted cutoff value of 0.001. The identified peaks were annotated using a known gene database (RefSeq). The annotated peaks were categorized into two groups (promoters and enhancers). Peaks located between −1 and +0.1 kb from the transcription start site were defined as promoter peaks, while the remaining peaks were defined as enhancer peaks. Superenhancer regions were also identified using HOMER (findPeaks with “super” option). The read coverage tracks for visualization were constructed using HOMER (UCSC file) with default options.Motif analysis of γH2AX-bound sequences depending on genomic locations (promoter and enhancer) was performed using HOMER (findMotifsGenome.pl) with the default option.21. B6.Cg-tg (Prnp-SNCA*A53T)23Mkle/J hemizygous mice overexpress mutant α-synuclein in the brain at levels approximately sixfold higher than the level of endogenous mouse α-synuclein. Three- and 8.5-month-old mice expressing A53T and control C57BL/6J mice were purchased from Jackson Laboratory . All mice were housed in pathogen-free facilities under 12-h light/12-h dark cycles with ad libitum access to food and water. All experimental animals were handled in accordance with the animal care guidelines of Konkuk University (IACUC KU16067-2).The human alpha-synuclein (A53T) transgenic line G2-3 was described previously22.The human neuroblastoma cell line SH-SY5Y (ATCC CRL-2266) was maintained and differentiated as described previously23.Differentiated SH-SY5Y cells were transduced with recombinant adenoviral vectors , as previously described24. Briefly, cells were rinsed with ice-cold phosphate-buffered saline (PBS), and ice-cold extraction buffer (PBS/1% Triton X-100/protease inhibitor cocktail/phosphatase inhibitor cocktail) was added. After incubating on ice for 10 min, the cell extracts were centrifuged at 16,000 × g for 10 min, and the supernatants and pellets were reserved separately for further analysis.Cell extracts were obtained as described previouslyg for 10 min. The supernatant fraction (cytoplasmic extract) was transferred to a prechilled tube. The insoluble pellet fraction was resuspended in nuclear extraction reagent and vortexed vigorously. The nuclear and cytoplasmic extracts were stored at −80 °C until further analysis.Nuclear extracts were prepared using the NE-PER Nuclear Cytoplasmic Extraction Reagent Kit according to the manufacturer’s instructions. Briefly, cells were rinsed with ice-cold PBS, and cytoplasmic extraction buffer was added. Cells were collected using a cell scraper. After incubating on ice for 10 min, the cell extract was centrifuged at 16,000 × 24. Chemiluminescence detection was performed using a FUJIFILM Luminescent Image Analyzer LAS-3000 and GE Healthcare Amersham Imager 680. Images were analyzed using the Multi Gauge (v3.0) software .Western blotting was performed as described previouslyThe cells were trypsinized and divided into two tubes. Accustain solution T containing detergent and propidium iodide (PI) was added to one tube to determine the total number of cells. Accustain solution N, containing PI but no detergent, was added to the other tube to label the damaged cells. The cells from both tubes were counted using an ADAM cell counter .Total RNA was extracted using the RNeasy Mini Kit . The RNA extract was reverse transcribed using the High Capacity cDNA Reverse Transcription Kit . Quantitative real-time PCR was performed on a LightCycler 480 II using LightCycler 480 SYBR Green I Master Mix , as recommended.The primers used were as follows: DDB2 forward: 5′-AAACCCAGAAGACCTCCGAG-3′, DDB2 reverse: 5′-ACATCTTCTGCTAGGACCGG-3′, BTG2 forward: 5′-AGGGAACCGACATGCTCC-3′, BTG2 reverse: 5′-GGGAAACCAGTGGTGTTTGT-3′, RPS27L forward: 5′-ACTACATCCGTCCTTGGAAGAG-3′, RPS27L reverse: 5′-GCTGAAAACCGTGGTGATCT-3′, p21 forward: 5′-CACCGAGACACCACTGGAGG-3′, p21 reverse: 5′-GAGAAGATCAGCCGGCGTTT-3′, GAPDH forward: 5′-GAGTCAACGGATTTGGTCGT-3′, GAPDH reverse: 5′-TGGAAGATGGTGATGGGATT-3′, p21_1 forward: 5′-CACCGAGACACCACTGGAGG-3′, p21_1 reverse: 5′-GAGAAGATCAGCCGGCGTTT-3′, GAPDH forward: 5′-GAGTCAACGGATTTGGTCGT-3′, and GAPDH reverse: 5′-TGGAAGATGGTGATGGGATT-3′.(tissue extract) p16 forward: 5′-TTCTTGGTGAAGTTCGTGCG-3′, p16 reverse: 5′-GCACCGTAGTTGAGCAGAAG-3′, p21_2 forward: 5′-ACAAGAGGCCCAGTACTTCC-3′, p21_2 reverse: 5′-GTTTTCGGCCCTGAGATGTT-3′, p53 forward: 5′-TGCTCACCCTGGCTAAAGTT-3′, and p53 reverse: 5′-AATGTCTCCTGGCTCAGAGG-3′.2) in a freshly prepared X-gal staining solution. The stained samples were rinsed with ice-cold methanol, air-dried, and then imaged using a digital camera.The cells were rinsed with ice-cold PBS and fixed in 4% paraformaldehyde (PFA) in PBS. The cells or tissues were incubated overnight at 37 °C . Primary antibodies were diluted in blocking solution and added to the cells. After washing in PBS, the cells were incubated with Alexa488, Cy2, rhodamine red X, or Alexa647 fluorescent dye-conjugated secondary antibodies  and then washed again in PBS. The nuclei were stained with TO-PRO-3 dye  or Hoechst 33342 (Sigma) and then mounted under coverslips using ProLongTM Gold Antifade reagent (Thermo Fisher Scientific). The stained cells were observed under an Olympus FV1000 confocal laser-scanning microscope or Zeiss LSM 900 with Airyscan 2.The cell staining procedure was described previously25. Briefly, 40-µm-thick floating brain sections were quenched with 0.3% H2O2 and then blocked with 4% BSA in PBST (0.1% Triton X-100). The samples were incubated overnight at 4 °C with primary antibodies against mouse anti-phospho-α-synuclein  and rabbit anti-γH2AX . After washing with PBST, the brain sections were incubated with biotinylated secondary antibodies  and treated with avidin–biotin peroxidase complex . Then, 3,3-diaminobenzidine (DAB)-developed sections were observed under a ZEISS AX10 microscope. All samples were evaluated by optical density analysis using the ImageJ program (NIH) with correction for background signal levels.Mouse brain tissue was kept in 4% PFA in cold 0.1 M phosphate buffer (pH 7.4) for 2 days, followed by incubation in 30% sucrose solution. For immunostaining, 40-μm coronal sections were cut on a sliding microvibratome . The details of the immunohistochemistry procedures have been described elsewhere26. Briefly, 40-µm-thick floating brain sections were quenched with 0.3% H2O2 and then blocked with 4% BSA in PBST (0.1% Triton X-100). Samples were incubated overnight at 4 °C with primary antibodies against mouse anti-phospho-α-synuclein , rabbit anti-γH2AX , and mouse anti-NeuN . After washing in PBST, the brain sections were incubated with Alexa488- or rhodamine red-X-conjugated secondary antibodies  and then washed again with PBST.Details of the immunohistochemistry procedures are provided elsewhereStained sections were mounted in a fluorescence mounting medium containing DAPI . The stained samples were observed under a Carl Zeiss LSM 700 confocal laser-scanning microscope.27.SH-SY5Y cells were transduced with adeno/α-syn or adeno/lacZ for 3 days. The sections were prepared as described previouslyP values were <0.05. The data were analyzed using repeated-measures one-way analysis of variance with posttests using the Prism 9 software . *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).All experiments were repeated at least three times. The values were expressed as the mean ± S.E.M. Null hypotheses of no difference were rejected if the P < 0.05). Downregulated genes were enriched in meiosis cytokinesis of unknown function . In contrast, γH2AX-enriched genes were slightly more often downregulated (five upregulated genes and eight downregulated genes) Fig. . A repre31.To verify whether p53 expression increased in α-synuclein-expressing cells, recombinant α-synuclein viral vector (adeno/α-syn) or control vector (adeno/lacZ) was transduced into differentiated SH-SY5Y cells. As shown in Fig. The p53/p21 pathway is involved in several cellular activities that lead to cell death or survival. These include apoptosis, cell cycle arrest, repair, and senescence. Our results in Figs. 32. SH-SY5Y cells were transduced with adeno/α-syn and adeno/lacZ for 3 days and stained for H3K9me3 me3 Fig. . The con33. Transmission electron microscopy results showed that the number of abnormal mitochondria was increased in α-synuclein-expressing cells (red arrows). The control and lacZ-expressing cells with normal mitochondria are indicated by blue arrows   Fig. .Fig. 5In37. The number of 53BP1 nuclear foci was also significantly increased in α-synuclein-expressing cells  and DSBs, was upregulated 24 h after α-synuclein expression but slowly decreased thereafter , DDB2, and BTG2. Cellular senescence and DNA DSBs were observed in cells overexpressing α-synuclein. Specifically, the NHEJ DNA repair pathway was partially activated, with a critical component of the pathway being reduced, resulting in a faulty DNA repair system. Histopathological analyses of α-synuclein transgenic mice showed increases in the levels of phospho-α-synuclein and DNA DSBs, as well as markers of cellular senescence at the presymptomatic stage, suggesting that cellular senescence and DNA damage are early events in synucleinopathy. The accumulation of DNA damage has been previously reported in α-synuclein-overexpressing cells and in several synucleinopathy mouse models41. These cells and tissues retained unrepaired DNA DSBs that were associated with DDR markers. Therefore, persistent DNA damage is probably a consequence of incomplete DDRs. Consistent with this idea, the levels of the DNA repair proteins Ku70 and Mre11 were decreased in aging human lymphocytes42. Neurons with prolonged DDRs showed typical characteristics of cellular senescence, such as mitochondrial dysfunction, production of ROS, and metabolic abnormalities. Neurons are particularly vulnerable to DNA damage because they are postmitotic and are highly metabolically active. A large proportion of neurons in several brain areas of aged mice showed severe DNA damage. Interestingly, the senescent-like phenotypes of a mouse model of premature senescence were rescued by deleting the p21 gene, a key signal transducer in cellular senescence43. These results suggest that senescence-like phenotypes can be induced by severe DNA damage in mature neurons.The mechanism by which DNA damage causes cellular senescence remains unclear. However, accumulating evidence suggests that these two phenomena are strongly associated. DNA damage accumulates with aging in both senescent cells and aged mammalian tissues45. Impairment of the base excision repair (BER) pathway, especially of the mitochondrial BER pathway, can cause various neurodegenerative disorders, such as Alzheimer’s disease46. Ataxia-oculomotor apraxia-1 (AOA1) and spinocerebellar ataxia with axonal neuropathy-1 (SCAN1) are associated with SSB repair defects48. AOA1 is associated with mutations in a novel human gene, aprataxin50, which encodes nucleotide hydrolases/transferases51. The encoded protein may play a role in single-stranded DNA repair through its nucleotide-binding activity and its diadenosine polyphosphate hydrolase activity. A mutation in the DNA repair protein tyrosyl-DNA phosphodiesterase 1 (TDP1), which can repair abortive SSBs created by topo1, is associated with SCAN152. Likewise, defects in DSB repair genes due to genetic mutations are also associated with neurological diseases, such as ataxia-telangiectasia (AT), AT-like disorder (MRE11 gene mutations), and Nijmegen breakage syndrome (NBS1 gene mutations)9. These genes are essential for the recognition of DSBs at the initial stage of DSB repair. These findings suggest that the human nervous system is vulnerable to DNA damage and that impairment of DNA repair pathways can cause neurological diseases.Prolonged or irreparable DNA damage leads to various human diseases that are characterized by genomic instability, including neurodegenerative diseases53. The precise mechanism by which α-synuclein overexpression causes defects in DDRs remains unknown. The levels of MRE11 were decreased in cells overexpressing α-synuclein. Mre11 is the nuclease component of MRN/X, one of the primary complexes responsible for recognizing and repairing DSBs as well as transducing DSB signals in eukaryotes. Elucidation of the pathway leading to the reduction of Mre11 would reveal the mechanism by which DNA damage accumulates during synucleinopathy.Our study showed that the overexpression of α-synuclein leads cells to initiate DDRs. However, in this case, the DDRs are not fully functional, which leads to the accumulation of DNA damage, particularly DSBs. Lewy body-containing neurons in the brain tissues of human patients showed increased levels of DNA DSBs53. These authors also suggested that α-synuclein binds double-stranded DNA and facilitates NHEJ repair. Taken together, these findings indicate that both overexpression and deficiency of α-synuclein caused impaired DNA repair and accumulation of DNA damage. It remains unclear how these seemingly contradictory observations can be explained: either too much or too little α-synuclein leads to increased DNA damage. To maintain functional DNA repair systems, it may be essential to maintain α-synuclein levels within a specific range.Interestingly, Schaser et al. recently showed that the removal of α-synuclein in human cells and mice resulted in increased levels of DNA DSBs after bleomycin treatment and a reduced ability to repair DNA damage54. Alternatively, these results suggest that DNA damage may not solely be the result of the cell-autonomous actions of α-synuclein but may also be induced by a non-cell-autonomous mechanism. α-Synuclein and its aggregate form can be secreted by neurons55. These secreted forms of α-synuclein are the culprit of the non-cell-autonomous actions of this protein, affecting neighboring neurons and glia57. Neurons that were exposed to extracellular α-synuclein showed signs of apoptosis58, while glial cells treated with α-synuclein showed inflammatory responses57. The latter is particularly interesting because DNA damage can induce inflammatory responses in innate immune cells60. Our study paves the way for further studies on the mechanism of non-cell-autonomous neuronal degeneration and glial inflammation triggered by extracellular α-synuclein.In our study, DNA DSBs accumulated in both phospho-α-syn-positive cells and phospho-α-syn-negative cells in α-synuclein tg mice. Moreover, DNA DSBs have been found in nonneuronal cells and neurons. These observations may indicate the occurrence of senescence-induced senescence, in which senescent phenotypes spread to neighboring cellsIn conclusion, we propose that the accumulation of DNA damage and cellular senescence may be key components in the pathogenesis of PD and other α-synuclein-related neurological diseases. DNA damage appears to accumulate due to incomplete and hence impaired activation of DNA repair pathways. How α-synuclein impairs the DNA repair system is a critical question arising from the current study. Other important questions include how DNA damage leads to cellular senescence in neurons and what the consequences of senescence processes are in terms of neuronal function and viability. Pursuing these questions could ultimately lead to an understanding of the pathogenic mechanism of synucleinopathies.Supplementary Figures


Since the advent of TAVR , the transapical surgical approach has been affirmed as a safe and effective alternative access for patients with unsuitable peripheral arteries. With the improvement of devices for transfemoral approach and the development of other alternative accesses, the number of transapical procedures has decreased significantly worldwide. The left ventricular apex, however, has proved to be a safe and valid alternative access for various other structural heart procedures such as mitral valve repair, mitral valve-in-valve or valve-in-ring replacement, transcatheter mitral valve replacement (TMVR), transcatheter mitral paravalvular leak repair, and thoracic aorta endovascular repair (TEVAR). We review the literature and our experience of various hybrid transcatheter structural heart procedures using the transapical surgical approach and discuss pros and cons. Transcatheter Aortic Valve Replacement (TAVR) was introduced for the treatment of aortic valve stenosis in 2002 by Cribier , and theIn recent years, unfavourable outcomes have been reported, and reduction of sheath calibres for the transfemoral approach, together with the development of other alternative endovascular procedures, have led to a fall in the number of transapical TAVR procedures performed worldwide.Despite this, some researchers have developed consistent experience with the left ventricular apex manipulation and reported satisfactory results in large series of transapical TAVR. The left ventricular apex proved to allow safe access and had great advantages: short distance and good accessibility with good coaxiality with multiple heart structures, and thus good stability of the devices during implantation. For all these reasons, the trans-apical approach can thus also be a useful tool for multiple transcatheter structural heart procedures other than TAVR: transcatheter mitral valve-in-valve and valve-in-ring replacement, mitral valvuloplasty, transcatheter mitral valve replacement (TMVR), paravalvular leak occlusion, and thoracic endovascular aortic repair (TEVAR). Left ventricular outflow tract pseudo-aneurysm occlusion is also described.Transapical TAVR was introduced into clinical practice to treat patients deemed at high surgical risk for standard surgical aortic valve replacement and also at high risk of vascular complications for the transfemoral approach due to peripheral arteries occlusive disease, calcifications or excessive tortuosity. Transapical TAVR is a hybrid procedure in which arterial access for a transcatheter aortic valve implantation is obtained by surgical isolation of the left ventricular apex through a standard left anterolateral 5–7-cm-long thoracotomy in the fifth or sixth intercostal space. After opening the chest, the correct location of the apex is identified with the “finger test”: under transoesophageal echocardiography (TEE) guidance, the left ventricular apex is pushed with the finger to determine at the four chambers view the ideal place to insert the sheath in order to obtain the best possible coaxiality. We place two orthogonal full-thickness U-shaped 2–0 polypropylene stitches on the muscular portion of the myocardium to assure correct haemostasis after sheath removal . At thisSince the early days of TAVR, the transfemoral approach has been considered the treatment of choice, and patients undergoing transapical TAVR have concomitant severe peripheral artery occlusive disease and tend to be sicker and with a higher surgical risk profile due to significant comorbidities . SeveralThe “transfemoral first” approach has become more widespread over the years, and several randomised trials reported a consistent gap in terms of survival and adverse outcomes in favour of transfemoral over transapical TAVR ,7,8. TheConsidering that patients submitted to transapical TAVR are usually affected by significative peripheral artery occlusive disease, they tend to also be sicker due to other significant comorbidities . For thiOver time, however, many centres gained valuable experience in managing the left ventricular apex and thanks to improvements in materials, were able to report satisfactory results for the transapical approach in patients unsuitable for the transfemoral procedure . Some laIn the early days, larger sheaths were used, and access site complications were re-ported. In 2011 Bleiziffer et al. reported a rate of 7% of severe apical bleeding and 1% (2 patients) of apical pseudo-aneurysm, in one case requiring surgical revision, on a series of 143 patients undergoing transapical TEVAR . This leOur TAVR programme with a “transfemoral first approach” was started in 2009, and to date the multidisciplinary team has remained the same. Our preliminary results were collected in the Italian Registry of Transapical Aortic Valve Implantation and published by D’Onofrio and coll ,4. Up toAccording by the AHA/ACC focused update of the 2014 guidelines TAVR should be reserved for inoperable/high-risk patients (Class I recommendation) and intermediate-risk patients (Class IIa reccomendation) as defined by the STS score. The guidelines also state that a multi-disciplinary heart team approach is mandatory to define the risk profile of each patient and subsequently select the most appropriate procedure [Transcatheter mitral procedures can be performed with a venous transeptal endovascular antegrade approach, or a with a direct retrograde approach through the left ventricular apex isolated with a standard anterolateral minithoracotomy. As described for TAVR, in the transapical approach, the distance from the left ventricular apex to the mitral annulus is very short. Together with coaxiality of the catheter with mitral anulus, this guarantees easy access and high stability during implantation.After initial experience with transapical TAVR, surgeons started to report transcatheter transapical mitral valve replacement for degenerated mitral bioprosthesis and failed mitral valve repairs in which an anuloplasty prosthetic ring was used to reinforce the repair during the first intervention ,22,23. TSome authors report similar early and one-year clinical results among patients undergoing trans-septal, transapical mitral transcatheter ViV procedures and open surgical re-mitral valve replacement , while oThere have been attempts to treat mitral stenosis according to the same principles and with the same devices as TAVR , and manSystematic reviews of results reported in published studies and international conference presentations conclude that TMVR is a feasible alternative to open mitral replacement in high-risk patients ,31. ThesPromising results were reported by Muller et al. in the global feasibility trial of the Tendyne Mitral Valve System  . The valFurther studies with larger series of patients and other devices are necessary to better evaluate and validate the procedureSeveral devices for transcatheter mitral valve repair are nowadays commercially available. Most of them use the transvenous transeptal approach.Nechord DS100  is a device that allows artificial chord implantation on prolapsing mitral valve leaflets using the transapical approach on a beating heart without cardiopulmonary bypass. The surgical technique is described step by step by Colli et al. . The choAhmed et al. systematically reviewed the published literature on transapical beating-heart mitral valve repair with Neochord , and ideThranscatheter endovascular paravalvular leak (PVL) repair has become a reasonable alternative to open surgical reintervention for high risk patients. The procedure is performed with either arterial or venous femoral access, but in any case, it is technically demanding. The transapical approach offers a very easy alternative, although it involves surgical minithoracotomy and general anaesthesia. The transapical route guarantees direct access to the mitral annulus and an easy engagement of PVL, even those in the postero-medial position for which transvenous and transarterial approaches are particularly challenging .Some surgeons find that transapical access is associated with lower procedural and fluoroscopy time than other approaches .In 2014 Taramasso et al. report results of 139 patients undergoing open surgical (122 patients) and hybrid transapical transcatheter (17 patients) mitral paravalvular leak occlusion . The oveZorinas et al. review their single-centre experience of 19 patients undergoing transapical mitral PVL closure with the novel specifically designed device Occlutech PLD Occluder  . They reIn 2020 Onorato et al. report midterm results of 136 patients undergoing aortic or mitral PVL transcatheter closure in 21 sites in 9 countries with the same device (Occlutech PLD Occluder) . Access TEVAR is nowadays the treatment of choice for many acute aortic syndromes and elective aortic pathologies. Deployment of stent grafts is usually retrograde, through femoral and iliac arteries. Delivery sheath and catheter calibres range from 14 to 24 French and require large minimal inner arterial diameters. Thoracic pathologies or acute aortic syndromes often affect elderly patients with peripheral arterial occlusive disease, which precludes standard retrograde endovascular treatment. Alternative approaches, like common iliac arteries or infrarenal abdominal aorta, are often required. After reporting the feasibility of the technique in a pig model , MacDonaOur transapical TAVR program started in 2010, and we described our first case of a successful transapical TEVAR of a patient admitted for an acute aortic syndrome in 2012 . He was Antegrade delivery of the thoracic stent graft through a left anterolateral minithoracotomy was performed. A Dry-Seal 22 French sheath  was inserted into the left ventricular apex. The aortic valve was first inspected through trans-thoracic and trans-oesophageal echocardiography and presented no stenosis or regurgitation. Exclusion of the lesion was achieved, and the patient discharged on post-operative day 15.In 2018 we then reported a series of five patients affected by acute aortic syndromes undergoing transapical TEVAR: two patients presented with a post-traumatic aortic injury with signs of impending rupture, two patients with contained aneurysmatic aortic rupture and one patient with symptomatic PAU with large pseudo-aneurysm . All patWe also performed an elective transapical TEVAR as completion of a previous complete aortic arch replacement using the Elephant Trunk (ET) technique on a patient with extended aneurysmatic disease of the aortic arch and descending aorta, peripheral arteries occlusive disease and severe aortic tortuosity .Other researchers have described percutaneous transapical access to create a rail wire support for a very complex retrograde TEVAR . They inThe transapical antegrade approach proved to be a feasible option for experienced multidisciplinary teams for patients with complex thoracic aorta pathologies deemed unsuitable for standard retrograde TEVAR due to concomitant occlusive disease of the peripheral vessels. Considering the large size of the sheaths used for stent graft delivery, a preoperative aortic valve assessment with echocardiography is mandatory to exclude calcific aortic disease that could determine an acute impairment of hemodynamic conditions during the procedure.Transapical closure of left ventricular outflow tract (LVOT) pseudoaneurysm is also described in the literature ,49.Although in recent years the number of transapical procedures has decreased worldwide thanks to the size reduction of sheath calibres in the standard transfemoral approach, the transapical approach continues to be a valid alternative option for procedures normally performed using transfemoral access, such as TAVR and TEVAR, in elderly patients deemed to be at high surgical risk for standard surgical procedures with unsuitable peripheral arteries. The close vicinity and coaxiality of the left ventricular apex with several heart structures such as mitral valve and LVOT makes the transapical approach a safe and easy option that guarantees easy access and great stability and accuracy for several structural procedures.Possible left anterior descending coronary artery damage, apical bleeding and late left ventricular apical pseudoaneurysm formation are threatening possible complications of the transapical procedures. For this reason, considerable experience in ventricular apex manipulation as well as complications management and a multidisciplinary heart team evaluation are mandatory to properly evaluate patient risk profile, select the appropriate approach and safely perform a transapical procedure.Further improvements in devices and randomised control trials comparing TF vs. TA and TA vs. standard surgical procedures are necessary to standardise and validate the transapical approach in the subsetting of different structural heart procedures.We consider the transapical approach a valid and essential tool in the portfolio of a modern heart valve centre.


Objective: Since its outbreak, the rapid spread of COrona VIrus Disease 2019 (COVID-19) across the globe has pushed the health care system in many countries to the verge of collapse. Therefore, it is imperative to correctly identify COVID-19 positive patients and isolate them as soon as possible to contain the spread of the disease and reduce the ongoing burden on the healthcare system. The primary COVID-19 screening test, RT-PCR although accurate and reliable, has a long turn-around time. In the recent past, several researchers have demonstrated the use of Deep Learning (DL) methods on chest radiography (such as X-ray and CT) for COVID-19 detection. However, existing CNN based DL methods fail to capture the global context due to their inherent image-specific inductive bias. Methods: Motivated by this, in this work, we propose the use of vision transformers  for COVID-19 screening using the X-ray and CT images. We employ a multi-stage transfer learning technique to address the issue of data scarcity. Furthermore, we show that the features learned by our transformer networks are explainable. Results: We demonstrate that our method not only quantitatively outperforms the recent benchmarks but also focuses on meaningful regions in the images for detection (as confirmed by Radiologists), aiding not only in accurate diagnosis of COVID-19 but also in localization of the infected area. The code for our implementation can be found here - https://github.com/arnabkmondal/xViTCOS. Conclusion: The proposed method will help in timely identification of COVID-19 and efficient utilization of limited resources. I.A.The novel COronaVIrus Disease 2019 (COVID-19) is a viral respiratory disease caused by Severe Acute Respiratory Syndrome COronaVirus 2 (SARS-CoV2). The World Health Organization (WHO) has declared COVID-19 a pandemic on 11 March 2020 B.Motivated by the success of the Deep Learning in diagnosing respiratory disorders While there has been a large body of literature on use of Deep Learning for Covid detection, most of them are based on Convolutional Neural Networks (CNNs) 1)We propose a vision transformer based deep neural classifier, xViTCOS for screening of COVID-19 from chest radiography.2)We provide explanability-driven, clinically interpretable visualizations where the patches responsible for the model’s prediction are highlighted on the input image.3)We employ a multi-stage transfer learning approach to address the problem of need for large-scale data.4)We demonstrate the efficacy of the proposed framework in distinguishing COVID-19 positive cases from non-COVID-19 Pneumonia and Normal control using both chest CT scan and X-ray modality, through several experiments on benchmark datasets.II.A.Chest Computed Tomography (CT) imaging has been proposed as an alternative screening tool for COVID-19 infection The work in B.Although chest-CT has more sensitivity as compared to RT-PCR In C.Images can be naively represented using a sequence of pixels for analysis using transformers but that would lead to huge computational expenses with a quadratic increase in costs. This has led to a number of approximations. For example, III.Unlike the existing methods that incorporate CNNs, we propose a vision transformer (ViT) A.A Vision Transformer B.Unlike CNN based models that impose inherent bias such as translation invariance and a local receptive field, vision transformer (ViT) C.A domain and a task are the two main components of a typical learning problem. For the specific case of a supervised classification problem, the domain, Given a source domain, In the current problem, the target domain consists of chest radiography image data i.e., for xViTCOS-CXR, the target data is the COVID-19 CXR dataset and for the xViTCOS-CT model, the target data consists of the COVIDx-CT-2A dataset The first source domain The underlying distribution of clinical radiographic images is vastly different from an unconnected set of natural images like those in ImageNet, and distributional divergence is very high between the two domains. Hence in cases where the target dataset is of insufficient capacity, the pre-trained ViT model might find it highly difficult to bridge the domain shift between the learned source domain and the unseen target domain. However, with a sufficient number of training examples available from the target domain, the ViT model can overcome the gap between these two domains. Keeping this in mind, an intermediate stage of knowledge transfer is used in this paper to train our proposed model depending on the size of the target domain training data. The primary goal of this stage of transfer learning is to help the ViT model, pre-trained on a generic image domains With the COVIDx-CT-2A dataset D.A number of Vision Transformers architectures have been proposed in literature. In this paper we have tested our algorithm on architectures proposed in 1https://github.com/faustomorales/vit-kerasWhile training xViTCOS-CXR, for the intermediate finetuning step using CheXpert IV.A.Some of the existing works validate their methods using private datasets 1)To demonstrate the efficacy of xViTCOS-CT, we use COVIDx CT-2A dataset 2)To benchmark xViTCOS-CXR against other deep learning based methods for COVID-19 detection using CXR images, we construct a custom dataset consisting of three cases: Normal, Pneumonia, and COVID-19. Like in COVIDx-CXR-2 B.1)COVIDx CT-2A dataset 2)In the compiled dataset, the chest X-ray images are of various sizes. To fix this issue, all the images were resized to a fixed size of refer to . In addiC.To quantify and benchmark the performance of xViTCOS, we compute and report Accuracy, Precision , Recall (Sensitivity), F1 score, Specificity, and Negative Prediction Value (NPV) as defined and compared in the standard literature such as 1)The prowess of the proposed model can be further understood from examining the confusion matrix . The pro2)The observations regarding the performance of xViTCOS-CXR compared to its contemporaries are on the same lines as that of xViTCOS-CT, if not better. In terms of classification accuracy, xViTCOS-CXR achieves an accuracy of 96%, outperforming the baseline methods by a considerable margin as can be seen from Analysing D.1)To visually analyze how clustered the feature space is, we perform a t-SNE visualization of the penultimate layer’s features for both the models using the test splits. As can be seen from 2)For qualitative evaluation of xViTCOS we present samples of CXR images and CT scans along with their ground truth labels and corresponding saliency maps along with the prediction in Report corresponding to V.In this study, we introduce a novel vision transformer based method, xViTCOS for COVID-19 screening using chest radiography. We have empirically demonstrated the efficacy of the proposed method over CNN based SOTA methods as measured by various metrics such as precision, recall, F1 score. Additionally, we examine the predictive performance of xViTCOS utilizing explanability-driven heatmap plot to highlight the important factors for the predictive decision it makes. These interpretable visual cues are not only a step towards explainable AI, also might aid practicing radiologists in diagnosis. We also analyzed the failure cases of our method. Thus, to enhance the effectiveness of diagnosis we suggest that xViTCOS be used to complement RT-PCR testing. In the next phase of this project, we aim to extend this work to automate the analysis of the severity of infection using vision transformers.


Many living tissues achieve functions through architected constituents with strong adhesion. An Achilles tendon, for example, transmits force, elastically and repeatedly, from a muscle to a bone through staggered alignment of stiff collagen fibrils in a soft proteoglycan matrix. The collagen fibrils align orderly and adhere to the proteoglycan strongly. However, synthesizing architected materials with strong adhesion has been challenging. Here we fabricate architected polymer networks by sequential polymerization and photolithography, and attain adherent interface by topological entanglement. We fabricate tendon-inspired hydrogels by embedding hard blocks in topological entanglement with a soft matrix. The staggered architecture and strong adhesion enable high elastic limit strain and high toughness simultaneously. This combination of attributes is commonly desired in applications, but rarely achieved in synthetic materials. We further demonstrate architected polymer networks of various geometric patterns and material combinations to show the potential for expanding the space of material properties. Synthesizing architected materials with strong adhesion has been challenging. Here the authors fabricate architected polymer networks by sequential polymerization and photolithography, and attain adherent interface by topological entanglement. Many applications require soft materials to deform reversibly (high elasticity) and resist fracture (high toughness). High elasticity and high toughness, however, are often conflicting requirements in materials development. A highly elastic material loads and unloads without dissipating much energy, whereas a highly tough material resists the growth of a crack by dissipating a large amount of energy. Polymer networks have been synthesized to achieve either high elasticity or high toughness10, but rarely both. The difficulty in simultaneously achieving elasticity and toughness is evident on the plane of elastic limit strain  and toughness  , and highly tough materials have low elastic limit strain (εe < 100%)23. A large area in the top right of the plane is empty. This negative correlation originates from the commonly used toughening strategy: sacrificial bonds25. When a crack advances in such a material, the polymer network transmits high stress from the crack front to the bulk of the material, breaking sacrificial bonds in the bulk, which toughens the material. The sacrificial bonds, however, lowers the elastic limit strain.Soft polymer materials, such as elastomers and gels, are under intense development to enable emerging fields of  biointegration and bioinspiration, including tissue engineeringon) Fig. . The two26. An Achilles tendon has many parallel fascicles, and each fascicle consists of staggered collagen fibrils in a proteoglycan matrix27  = WD(εc) + WE(εc). For the soft gel, the hysteresis is small, WD(εc)/W(εc) = 1%, and the toughness comes from the rupture of the long polymer chains. For the hard gel, the hysteresis is pronounced, WD(εc)/W(εc) = 51%, and the toughness comes from both the dissipated work and elastic work. The former mainly comes from the rupture of the short-chain network, and the latter mainly comes from the rupture of the long-chain network. The synergy amplifies the toughness of the hard gel relative to the soft gel. For the TPN gel, the hysteresis is also pronounced, WD(εc)/W(εc) = 53%, and the toughness comes from both the dissipated work and elastic work. At the crack tip, the soft phase deconcentrates stress over the hard phase. This stress deconcentration further amplifies the toughness of the TPN gel relative to the hard gel.We record the stress–strain curves of the three gels with and without precut crack Fig. . For bot/m2 Fig. . The TPNels Fig. . We sepa33. The TPN gel demonstrates simultaneous improvement in elastic limit strain (seven times) and in toughness (three times) compared with the hard gel. The successful integration of high elasticity and high toughness needs to fulfill the following four requirements.(i)The soft phase needs to have high strength. The soft and hard segments in the stripe-patterned TPNs alternate in series, and are subjected to the equal stress. The soft segment is strong enough to make the hard segment fracture preferentially The soft and hard phases must adhere strongly. Interfacial adhesion is  the critical challenge for the design of composite materials. We attain strong interfacial adhesion by topological entanglement of polymer networks. The strong adhesion helps to smoothly transfer the stress between the phases. In all the mechanical measurements of TPNs, we have not observed any interfacial fracture.(iii)r of the hard phase. When r = 0, the TPN will regress to the soft gel, and lose high toughness. When r = ∞, the hard phase becomes continuous fibers, making the TPN lose high elastic limit strain. Therefore the TPNs must have an intermediate aspect ratio to reconcile the elastic limit strain and toughness. With the increase in r, the elastic limit strain decreases, whereas the toughness first increases to a peak value at r = 4, and then decreases slowly  and toughness (Γ = 4202 J/m2). The decrease of εe is attributed to the decreased volume fraction ϕs of the soft segment in the composite column when r increases. The toughness Γ is mainly determined by the energy dissipation in the process zone, which is localized in the hard block at the crack front CMBAA for the short-chain network. We prepare a series of TPNs with the different modulus ratio Eh/Es of the hard phase to the soft phase, and find they have the similar critical strain εc , the low εe is attributed to the large swelling of the hard segment and thus the decreased volume fraction ϕs of the soft segment , which greatly expand the space of material properties. The TPNs integrate diverse polymers and patterns by sequential polymerization, photolithography, and stacking. The TPNs resolve a longstanding challenge in fabricating architected materials with strong adhesion through topological entanglement. As a demonstration, we fabricate tendon-inspired TPNs that simultaneously achieve high elastic limit strain and high toughness. We further fabricate TPNs of various geometric patterns, material combinations, and multilayer stacks. We have fabricated TPNs using mask photolithography, but TPNs can also be fabricated using other methods, such as stereolithographyN,N-dimethylamino ethylacrylate methyl chloride quarternary . Crosslinker: N,N’-methylenebis(acrylamide) (MBAA). Initiators: 2-Oxoglutaric acid (OA), 2-hydroxy-2-methylpropiophenone (HMPP), and 2-hydroxy-4’-(2-hydroxyethoxy)-2-methylpropiophenone (Irgacure 2959). Dyes: Alcian blue and Amaranth. Patterned photomasks were designed by us and fabricated by GX Photomask (Shenzhen) Co. Ltd.Unless otherwise mentioned, all chemicals were purchased from Aladdin and used without further purification. Monomers: Acrylamide , 2-acrylamido-2-methylpropanesulfonic acid (AMPS), 2-hydroxyethyl acrylate (HEA), and 2) for 8 h. The as-prepared PAAm gels were leached in deionized water, and were used as scaffolds. We submerged the scaffolds in an aqueous precursor solution containing 1 M AMPS, 4 mol% MBAA, and 1 mol% HMPP to an equilibrium state. We sandwiched the precursor-containing scaffolds between glass and polyethylene terephthalate (PET) release film. The PET film was covered by patterned masks and subjected to UV irradiation  for several seconds in the air. The as-prepared PAAm/PAMPS gels were leached in deionized water to obtain patterned scaffolds. The patterned scaffolds were further immersed in the aqueous precursor solution containing 4 M AAm, 0.001 mol% MBAA, and 0.01 mol% OA to an equilibrium state, and then covered by two parallel glass plates. After the UV irradiation  for 8 h in a nitrogen atmosphere, the TPN gels were leached in deionized water, and were ready for various characterization tests.TPNs were prepared through three-step sequential polymerization and photolithography Fig. . The scaThe patterned TPN gels consisted of soft phase (PAAm/PAAm) and hard phase (PAAm/PAMPS/PAAm). The modulus ratio of the hard phase to the soft phase was tuned by changing the MBAA concentration in the second precursor. As the control cases to the TPN gels, the soft gels were prepared without the second precursor, and the hard gels were prepared without photomasks. PHEA-based and PDMAEA-Q-based TPN gels were synthesized by replacing the PAMPS precursor. Note that the UV initiator Irgacure 2959 was used in the third precursor for PDMAEA-Q-based TPN gels. Bilayer TPN gels were obtained by stacking two layers of patterned scaffolds and then polymerizing the third PAAm network.To visualize the hard phase in TPN gels, the charged hard phase was selectively dyed by adsorbing dye molecules with the opposite charges  in the elastic tensile region. The strain ratio εs/εh was estimated through the in situ observation of deformation . Fully swollen stripe-patterned TPN gels were cut into a dumbbell shape by a standardized cutter with width of 2 mm and height of 12 mm. Both ends of the dumbbell-shaped samples were clamped and stretched at a constant velocity of 100 mm/min, by which the stress–strain curves were recorded. The tensile process of the stripe-patterned gels obeys the series model (isostress model), so the modulus ratio of the hard phase to the soft phase equals to the inverse ratio of their strains  and a 20 mm one-edge crack were used. The sample was clamped and stretched along the H direction at a constant velocity of 30 mm/min, by which the stress–strain curve was recorded. The strain at which the precut crack started to propagate was defined as the critical strain εc, and the integrated area under the stress–strain curve from 0 to εc was defined as the critical work of extension W(εc). The toughness was given by Γ = W(εc)H. Note that the stress was corrected by the initial effective sample width of 30 mm. With this correction, the stress–strain curve of the precut sample coincided with that of the uncut sample with the same geometry in the range of 0 ≤ ε ≤ εc  including seven columns of hard phase (width 1.5 mm and height 6 mm) (Supplementary Fig. Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2Supplementary Movie 3Supplementary Movie 4Supplementary Movie 5


Non-Hodgkin lymphoma is one of the most frequently occurring hematologic diseases in the world. Current drugs and therapies have improved outcomes for patients with lymphoma, but there is still a need to identify novel medications for treatment-resistant cases. The aim of this review is to gather the latest findings on non-Hodgkin lymphoma in children, including genetic approaches, the application of therapy, the available treatment options, and resistance to medications.One of the most common cancer malignancies is non-Hodgkin lymphoma, whose incidence is nearly 3% of all 36 cancers combined. It is the fourth highest cancer occurrence in children and accounts for 7% of cancers in patients under 20 years of age. Today, the survivability of individuals diagnosed with non-Hodgkin lymphoma varies by about 70%. Chemotherapy, radiation, stem cell transplantation, and immunotherapy have been the main methods of treatment, which have improved outcomes for many oncological patients. However, there is still the need for creation of novel medications for those who are treatment resistant. Additionally, more effective drugs are necessary. This review gathers the latest findings on non-Hodgkin lymphoma treatment options for pediatric patients. Attention will be focused on the most prominent therapies such as monoclonal antibodies, antibody–drug conjugates, chimeric antigen receptor T cell therapy and others. Non-Hodgkin lymphoma (NHL) is one of the most frequently occurring hematologic disease in the world . In chilOverall survival (OS) rates in children, adolescents and young adults diagnosed with NHL increased to 80–90% during the last 30 years, giving the opportunity to investigate the long-term effects of prior chemo- and radiotherapy (RT). Children and adolescent NHL survivors are at significant risk of late mortality from secondary neoplasms, recurrent/progressive disease and chronic health conditions , and late morbidity of multiple organ systems and poor health-related quality of life. These risks are similar to other long-term risks of childhood and adolescent acute lymphoblastic leukaemia (ALL), Wilms tumor and Hodgkin lymphoma (HL) survivors . IncreasNovel approaches are required to reduce the burden of late morbidity and mortality in childhood and adolescent NHL survivors, and obtain methods to identify at-risk patients who are at significantly increased risk of these complications. Nowadays, we can distinguish several therapeutic substances that work in various ways. These include immunomodulatory drugs, monoclonal antibodies (mAbs), immune checkpoint inhibitors (ICI), antibody–drug conjugates (ADCs) and genetically modified chimeric T cell receptor antigens (CAR) . In addiMore detailed treatment of the different types of NHL in pediatric patients is summarized in Monoclonal antibodies target specific markers on cancer cells by activating the patient’s immune system. In this way, the therapy avoids widespread non-specific cytotoxic effects . MoleculSome mAbs, such as obinutuzumab and ofatumumab, did not improve outcomes in NHL patients . NeverthThe following generation of mAbs are bispecific antibodies (BiAbs), which are derived from mAbs. They consist of two single-chain variable fragments (scFv) that target tumor-associated antigens. BiAbs engage the cells of immune system to attack indicated tumor cells . BlinatuADCs comprise of a mAb connected to a small cytotoxic molecule. When attached to the cell-surface antigen of cancer cells, the ADC is internalized; next, the cytotoxin is released, causing cell cycle termination and cell apoptosis. The drug can also kill adjacent cells by “bystander killing”  40]. Th. Th40]. Polatuzumab vedotin, whose clinical trial (NCT02257567) ended in FDA approval for treatment of R/R DLBCL in adults, is the combination of the CD79b antibody and MMAE . PolatuzCAR-T cell therapy uses T lymphocytes, which are engineered with synthetic chimeric antigen receptors (CAR). The CAR-T cell recognizes and then eliminates specific cancer cells, independently of major histocompatibility complex molecules . The creIn a recent study of Juan Du, an eight-year-old child suffering from R/R BL showed no clear response after being treated with CD19-specific CAR-T cells. After the attempt to treat the malignancy with CD22-specific CAR-T cells, the disease reoccurred. Subsequently, CD20 CAR-T cell treatment was applied, and that action resulted in the achievement of CR. What is more, CAR-T cell therapy targeting CD23 and the tyrosine kinase-like orphan receptor brought about promising results in the improvement of R/R NHL treatment in children . One of DNMT inhibitors belong to a family of enzymes that catalyze the methylation of DNA . There aThe conclusions of Han Weidong’s study on children under 16 years of age inform us that decitabine-primed CAR-T cells can recognize and kill the CD19 negative malignant cells, which leads to death of lymphoma tumor cells. Decitabine increases tumor antigens and human leukocyte antigen expression, enhances antigen processing, promotes T cell infiltration and boosts effector T cell function; therefore, it can be used in DLBCL, high grade B-cell lymphoma and other aggressive B-cell lymphomas in pediatric patients . DecitabFor many years, the modulation of epigenetic mechanisms was the subject of trials and studies . The HDAAt the time of this article’s publication, there are two ongoing pediatric clinical trials involving vorinostat in NHL. They cover combination with chemotherapy before donor stem cell transplant (NCT04220008) and a potential graft-versus-host disease (GVHD) incidence reducing drug NCT03842696) ,68. Prom6 ,68. PrIn the case of panobinostat, the results of only one clinical trial (NCT01321346) were published by Goldberg et al. Among 22 pediatric patients, only one was diagnosed with NHL. The researchers observed several adverse effects, predominantly regarding the gastrointestinal tract. Moreover, the clinical activity of panobinostat was unsatisfactory, and now more efficient therapies are available .The following examples of HDACIs were registered in trials in patients at least 16 years old. With a positive outcome of these studies, we may assume the age of enrollment to these trials will be lowered in the future.Chidamide is being tested in R/R peripheral T cell lymphoma with encouraging results and a satisfying safety profile . MoreoveHDACIs are undeniably under rapid development. Certainly, we will observe more clinical trials of either HDACIs as single agents or in combined therapies with novel drugs .ICIs have become a great milestone as a cancer cure. Pediatric malignancies, such as leukemias and solid tumors, also benefit from ICIs, with their expansion in our sight ,76. DespAs for nivolumab, which is a fully human IgG4 mAb targeting the PD-1 receptor , there aIn terms of other checkpoint inhibitors, the fully human IgG1 PD-1L antibody, atezolizumab, has become the object of clinical trials . HoweverThere are several active clinical trials, which will determine the usage of the mentioned ICIs, along with others, such as anti-CTLA-4. The National Cancer Institute is conducting studies (NCT02304458) on combined nivolumab with ipilimumab (which is the anti-CTLA-4 antibody) in patients with R/R NHL .EZH2 is a histone methyltransferase, which comes from the gene family containing epigenetic regulators that inhibit transcription . EZH2 alWhen EZH2 mutation occurs, the suppression of GC output genes and checkpoints persists, which in turn leads to hyperplasia . Until nSince 2012, several specific EZH2 inhibitors have been investigated. Their task was to inhibit H3K27 methylation, reactivate silenced PRC2 target genes, or inhibit the survival of B-cell lymphoma cells (GC-derived and containing the EZH2 activating mutation) . These sEZH2 inhibitors have the potential to be useful in the treatment of lymphomas, but there is still insufficient research to draw specific conclusions. It is possible that in the future it will be possible to use compounds from this group in combination therapy, e.g., with chemotherapy, which would result in improved patient outcomes. However, for this to happen, a lot of research has yet to be conducted.IDH is an enzyme that catalyzes the conversion of isocitrate to α-ketoglutarate (αKG) . RecentlEnasidenib and ivosidenib are orally selective inhibitors of mutant IDH, with enasidenib blocking IDH2 and ivosidenib blocking IDH1. Both of these drugs have already been approved by the FDA for the treatment of acute myeloid leukemia (AML) ,107,108.Further clinical trials are currently underway to test these compounds in other diseases as well. One of these studies, on ivosidenib, is currently being conducted in the age group 12 months to 21 years. It focuses on patients with R/R NHL, advanced solid tumors, or histiocytic disorders that have IDH1 genetic alterations .Expression of anti-apoptotic proteins from the BCL-2 family can be disrupted by several different mechanisms, including gene amplification, chromosomal translocation, increased gene transcription, or altered post-translational processing . In the Currently, several compounds that are inhibitors of BCL-2 can be distinguished. One of them, ABT-263 (navitoclax), initially showed good efficacy. However, its use has been limited due to its main toxicity: thrombocytopenia. Despite this, research can still be found in the context of NHL and navatoclax. In one study, although each patient had at least one treatment-related adverse event, the safety profile of navitoclax in R/R FL was assessed as acceptable . In turnIn the context of the future, VOB560 (65487) and MIK665 (S64315) may also be significant. Both of these compounds were designed to potently and selectively block BCL-2 and MCL-1, respectively. Their combination in preclinical studies has shown strong anti-cancer properties . Undoubtedly, the discovery of inhibitors of the BCL-2 family opened a new path in the targeted therapy of many cancers. There is the need for new clinical trials comparing the effectiveness of these substances in similar and the same diseases. Their toxicity profile is also extremely important. As already proven, the side effect of individual BCL-2 inhibitors is not only the abovementioned thrombocytopenia. In addition, with venetoclax, diarrhea, upper respiratory tract infections, neutropenia and tumor lysis syndrome may occur ,126,127.Approximately 90% of pediatric ALK+ ALCL is due to t  chromosomal translocation, which entails the emergence of oncogenic fusion protein nucleophosmin activating several proliferation and survival pathways . The reaCrizotinib is a first-generation ALK inhibitor . Based oCeritinib is a second-generation ALK inhibitor that binds to ALK with a higher affinity than crizotinib , but itsBTK is a kinase with a protein structure involved in the regulation of B-cell signaling . As one Several generations can be distinguished among these drugs. The first-generation BTK inhibitors include ibrutinib, which is administered orally. It binds irreversibly to a cysteine residue (C481) in the active site of BTK, thereby inhibiting B-cell receptor signaling  ,143. As Studies to date have shown that ibrutinib is well tolerated in many B-cell tumors, including DLBCL ,146. HowIt is worth mentioning that, as in the case of other drugs, the possibility of combining BTK inhibitors with, e.g., BCL2 inhibitors, is being considered. In one such study, Constantine et al. proved that the combination of ibrutinib + venetoclax was highly effective in patients with MCL. They received an OS rate of 79% at 12 months and 74% at 18 months . In turnBortezomib (PS-341) is a peptide aldehyde derivative. It is the first potent, selective, and reversible inhibitor of the 26S proteasome in its class  161]. . 161]. Its role is to inhibits the ubiquitin–proteasome pathway by binding directly to the active sites of the proteasome, which in turn disrupts targeted protein proteolysis ,162. TheAlthough most studies on bortezomib in NHL patients concern adults, for several years there have also been studies in which the target group are children. One of them was conducted by Horton et al. and related to the use of combination therapy of bortezomib with ifosfamide/vinorelbine (IVB) in pediatric NHL patients. Although few patients achieved the primary goal , the CR after two cycles was 83% ,173. RecTemsirolimus is one of the mammalian targets of the rapamycin (mTOR) inhibitor, which, as a derivative of the sirolimus ester, exhibits antibacterial, immunosuppressive, and antitumor properties ,175. TheCurrently, temsirolimus is approved in the European Union for the treatment of R/R MCL, but not in the USA ,178,179.For this reason, combinations of temsirolimus with other agents, both cytotoxic drugs and other targeted inhibitors, are currently at the forefront of NHL treatment . One sucAs can be seen, despite the approval of temsirolimus for treatment in MCL, there is still a space for improvement in its effectiveness in this and other types of NHL. It is possible to achieve this by developing better biomarkers, which would allow for better stratification of the patient prior to drug administration. Moreover, such biomarkers could help to elucidate the mechanisms of resistance and to develop new therapeutic combinations, which would certainly improve the effectiveness of treatment .Our knowledge of the refractory NHL’s treatment has grown significantly. Novel drugs trial results give hope to those who suffer from the mentioned hematologic dis-eases and make up a large percentage of people.There are many approved drugs for NHL therapy, e.g., temsirolimus for MCL or bilinatumomab for R/R B-NHL in children and adults. The application of second-generation CD19 CAR-T cells has shown significant positive outcomes in the treatment of FL, PMBCL, DLBCL, MCL, and splenic MZL. Moreover, CD20 CAR-T cell treatment resulted in CR in BL.EZH2 inhibitors in combination with chemotherapy will probably improve patients’ outcomes, if examined more closely. When the effective doses have been improved and the side effects suppressed, BCL-2 inhibitors will be a promising class of drugs to fight NHL. Additionally, combination of ibrutinib and venetoclax is considered highly effective in treating MCL. Lastly, chidamide indicated encouraging results in its effectiveness and safety profile in T cell lymphoma treatment; for that reason, we can expect many clinical trials validating this method.There is a necessity to develop better biomarkers, which could help with elucidation of resistance mechanisms and stratification of the patient before administrating the drug.It is likely that the majority of the compounds described in this review will be used in widespread NHL therapies; however, much more research needs to be conducted and many years have to pass to establish personalized treatment options. As always, future pediatric treatment regimens will arrive after clinical trials take place in adults. Thus, we expect rapid progress in this branch of medicine. All figures presented in this article have been created with Biorender.com.