The purpose of this study is to assess the integrity of chondral defect repairs filled with a cartilage allograft and sealed with either allogeneic fibrin sealant or autologous fibrin sealants created with platelet-rich plasma (PRP) or platelet-poor plasma (PPP) in a cadaver model.Twenty-millimeter medial femoral condyle (MFC) chondral defects were created in five human cadaveric knees. The defects were filled with particulated cartilage allograft hydrated with PRP from human donors until slightly recessed. Sealants were applied until flush with the articular surface using PRP and autologous thrombin serum, PPP and autologous thrombin serum, or commercial allogeneic sealant. The MFC defects were cycled using a multiaxial testing system to simulate continuous passive motion undergone during rehabilitation. After testing, the repairs were assessed for integrity by quantitatively comparing defect exposure and qualitatively assessing sealant delamination.P = .227), and each group had significantly less defect exposure when compared to the critical clinically relevant value assigned to be 30% . No complete sealant delamination was observed, although the allogeneic sealant delaminated with a higher magnitude than did the autologous sealants.The mean defect exposures were 4.20% ± 5.02% for the PRP group, 4.60% ± 5.18% for the PPP group, and 1.80% ± 2.95% for the allogeneic sealant group. No significant differences were observed between groups (The PRP and PPP sealants were comparable to the allogeneic sealant for graft fixation when used in conjunction with an underlying PRP-hydrated particulated cartilage allograft. The autologous sealants had better delamination resistance than the allogeneic sealant.The time-zero model is critical in elucidating the retention properties of fibrin and allogenic sealants after cartilage repair and before healing processes help stabilize the repair. This is contrary to allogeneic commercial fibrin sealants, such as TISSEEL , that are developed from pooled human plasma. TISSEEL contains approximately 30-fold higher fibrinogen concentration than physiological levels present in autologous blood products.A previous study compared allogeneic and autologous fibrin sealants in a small-scale ex vivo cartilage repair model and demonstrated mechanical equivalence of autologous PPP-derived sealants and an allogeneic sealant.The purpose of this study is to assess the integrity of chondral defect repairs filled with a cartilage allograft and sealed with either allogeneic fibrin sealant or autologous fibrin sealants created with PRP or PPP in a cadaver model. We hypothesized that the fibrin sealants would be comparable when analyzing graft fixation and have the same level of delamination from the underlying graft after testing.Institutional Review Board (IRB) approval was obtained before collecting human blood samples for the autologous preparations . Ninety cc of whole blood anticoagulated with anticoagulant citrate dextrose solution  to a final concentration of 13.3% (vol/vol) was collected from five human donors consisting of two males and three females with an average age of 26.0 ± 3.5 years. PRP and PPP fractions were prepared with the Arthrex Angel cPRP system  set to 7% hematocrit. After centrifugation and automatic plasma separation by the machine, PRP was expanded to approximately 7 cc by pulling back on the PRP syringe until the desired volume was achieved. Complete blood counts (CBC) of whole blood, PRP, and PPP fractions were obtained with a Sysmex XE-5000 .2  and 4 cc PPP were injected into the device and allowed to clot for at least 15 minutes. When the defect was repaired and ready for fixation, the clot in the device was broken by shaking per the manufacturer’s instructions and an additional 0.2 cc CaCl2 and 8 cc PPP were injected into the Thrombinator device. One minute after clot reformation, the clot was shaken briefly, and the serum was extracted from the device through an 18-μm filter. A small sample of thrombin serum was used to evaluate comparative clotting time at a 1:1 ratio with a pooled fibrinogen source on a STart4 Hemostasis Analyzer .Autologous thrombin was prepared using the Thrombinator System  and PPP. Initially, 0.1 cc CaClFive, fresh-frozen human male cadaveric knee samples  of age ≥55 years were used in this study. Before use, each sample was evaluated radiographically and arthroscopically via NanoScope operative arthroscopy imaging system  to confirm there was no osteoarthritis exceeding Jäger-Wirth grade 2 on the distal MFC.The isolated femur was positioned upright and gripped securely in a vise containing serrated jaws. Using an Allograft OATS Harvester with a depth stop device , a-20 mm diameter defect was reamed normal to the distal medial articular surface to a depth of 1.5 mm. A ring curette was used to remove the calcified chondral layer and to establish the vertical defect walls. Microdrilling was then performed using a 1.5-mm, 45° PowerPick Drill, with a drill depth of 6 mm . The defect was cleaned of debris before chondral defect repair . For allThree fibrin sealant formulations were evaluated in this study: 1) PRP as a fibrinogen source with autologous thrombin, 2) PPP as a fibrinogen source with autologous thrombin, and 3) all-allogeneic fibrinogen and thrombin . Autologous thrombin (3 cc) was transferred to a 1:1 applicator assembly along with 3 cc of a fibrinogen source (PPP or PRP). A blending connector with mixer  was used to mix and dispense the sealant over the defect until flush with the articular surface , A-C. ThIn this study, each femoral defect was repaired and tested three consecutive times using the different sealants in a randomized order. Before each repair, the defect was thoroughly irrigated with phosphate-buffered saline (PBS) 1×  to remove remnant particulated cartilage and sealant from the previous round, similar to Bogunovic et al.3 foam block  using the appropriate instrumentation and polymethylmethacrylate (PMMA) cement . A dowel pin was used to traverse the width of the foam block and hold it via a clevis, enabling a rotational degree of freedom and minor lateral degree of freedom for the tibial implant to linearly track the MFC surface while cycling and, thus, be quasi-physiological for the flexion-extension mechanism  to simulate the cyclic shear forces experienced by the repair during postoperative continuous passive motion (CPM). An articulating iBalance Unicondylar Knee Arthroplasty tibial bearing (Arthrex) made of ultra-high-molecular-weight-polyethylene (UHMWPE) was used in place of a cadaveric tibia for consistency between groups and to circumvent concerns about variable tibial osteoarthritis in cadavers. The tibial bearing was implanted into a 30 lb/ftechanism B.Fig 3 to replicate the in vivo environment. Using WaveMatrix software, a compressive load of 44 N, an estimated tibiofemoral joint reaction force during passive motion,2).Immediately before and after testing, images were taken with a digital camera . The images after testing were analyzed with ImageJ software  to calculate the percentage of defect exposure. Defect exposure in this study was defined as visible loss or contractility of the particulated cartilage matrix  when compared to the pretest image. The scale was set by correlating pixels to the reamed 20-mm diameter of the defect. Using the polygon selection tool, the regions of loss were manually outlined and summed to determine the approximate area of defect exposure . The perComparable graft fixation was defined as having no observed significant difference between groups regarding defect exposure while also having significantly less than 30% defect exposure. The 30% value was calculated specifically for this study using the critical size defect of 6 mm, which is the defect dimension that is incapable of repairing without intervention, determined from well-accepted preclinical goat models of cartilage repair.12After testing, the sealant delamination was also assessed by two unblinded orthopaedic surgeons (of whom one is fellowship-trained) on a scale of 0-5, similar to Bekkers et al.,t-tests were used to evaluate any differences in cell content in PRP and PPP autologous fractions from the blood donors; if data were not normally distributed, the nonparametric Mann-Whitney rank sum test was used. Parametric independent t-tests were also used to compare clotting times of the autologous thrombin serum used with PRP and PPP sealants. A parametric one-way repeated measures analysis of variance was used to compare the defect exposures between the autologous and allogeneic sealant groups. Parametric one-sample t-tests were used to compare the mean defect exposures to the critical value of 30%. Interobserver reliability for sealant delamination was calculated via Kendall’s coefficient of concordance. The post hoc power analysis used a minimal statistical power of .80.Statistical analysis was performed in SigmaPlot 14.0  and Minitab 19  with all statistics using α = .05 and β = .20. For parametric data, independent P = .003), platelet count , and red blood cell count .Complete blood counts (CBC) of the PRP and PPP fractions derived from each patient’s whole blood were analyzed for confirmation that the autologous fractions had significantly different compositions and were consistent with known expected values for PRP and PPP . PRP hadP = .47, Power = .104) as seen in The comparative clotting times between the thrombin serum used to clot either PPP or PRP fibrinogen sources were confirmed to not be significantly different . Each group had significantly less defect exposure when compared to the critical value of 30% .The mean defect exposures calculated for different sealant fibrinogen sources were 4.20% ± 5.02% for PRP, 4.60% ± 5.18% for PPP, and 1.80% ± 2.95% for allogeneic sealant . There wThe sealants were analyzed qualitatively on a scale of 0-5 for sealant delamination as seen in The main finding of this study is that no significant differences were observed between groups regarding defect exposure, and each group had significantly less defect exposure when compared to the critical value of 30%. No complete sealant delamination was observed after testing. However, the allogeneic sealant delaminated with a higher magnitude than did the autologous sealants. Thus, the hypothesis that the fibrin sealants would be comparable for graft fixation is accepted, while the hypothesis that the fibrin sealants would have the same level of delamination from the underlying graft is not accepted.The ex vivo cyclic testing model controlled the applied compressive load based on physiological approximations of passive tibiofemoral joint reaction forces.A previous in vitro cartilage explant study evaluated the mechanical properties of PRP, PPP, and allogeneic fibrin sealants used in conjunction with an underlying particulated cartilage allograft. The authors found no difference in bulk failure properties or microscale strains at the graft-cartilage interface between the PPP and allogeneic sealants. PRP sealants, however, were determined to be inferior because of their higher observed strains through the graft-cartilage interface depth.,A theory for the sealant displacement observations is derivative of that proposed by Irwin et al.The allogeneic sealant TISSEEL contains supra-physiological levels of fibrinogen with reported levels between 67 and 106 mg/ml. In contrast, both PRP and PPP have much lower fibrinogen concentrations that range from 2 to 4 mg/ml.,4 vs 10-15 U/ml14). In a wound-healing model, wounds treated with a fibrin matrix containing lower thrombin concentrations (4 IU/ml) appeared less severe and displayed more rapid wound healing than wounds treated with fibrin matrix containing higher thrombin concentrations (800 IU/ml).While unable to be evaluated as part of this study, there is evidence that autologous formulations may augment repairs from a biological perspective. For example, allogeneic sealants with supraphysiological fibrinogen concentration have been shown to create a dense barrier that hinders necessary functions in wound healing, such as cell migration and tissue formation.,Autologous sealants may also serve as a vector for enriched growth factor delivery within the particulated cartilage graft, which also contains bioactive factors supporting cell growth and chondrogenic differentiation.The allogeneic sealant investigated in this study (TISSEEL) contains thrombin and fibrinogen developed from pooled human plasma, as opposed to an all-autologous fibrin sealant. Although vapor heat and solvent detergent treatments are performed for viral reduction, no procedure has been proven to eliminate viral contamination, posing the risk of infectious transmission and immunogenic response.This study is not without limitations. One limitation of this model is that fibrin sealants were subjected to cyclic loading within 5 minutes following clot formation without immobilization to allow for repair stabilization before loading as is standard in clinical practice.Furthermore, the sample size was small; we are unable to rule out beta-error. A post hoc power analysis revealed that 72 specimens would be needed to achieve a power of .80. Because of considerations surrounding the use of cadavers, the study was not expanded further for increased power. Additionally, intraobserver reliability was not performed for the sealant delamination analysis. The surgeons were also not blinded, as color differences were required to observe regions of delamination.The PRP and PPP sealants were comparable to the allogeneic sealant for graft fixation when used in conjunction with an underlying PRP-hydrated particulated cartilage allograft. The autologous sealants had better delamination resistance than the allogeneic sealant.


The frequency-dependent contractile force generated by cardiac myocytes depends upon the characteristics of the Ca2+ transients. A stochastic computational local control model of a guinea pig ventricular cardiomyocyte was developed, to gain insight into mechanisms of force-frequency relationship (FFR). This required the creation of a new three-state RyR2 model that reproduced the adaptive behavior of RyR2, in which the RyR2 channels transition into a different state when exposed to prolonged elevated subspace [Ca2+]. The model simulations agree with previous experimental and modeling studies on interval-force relations. Unlike previous common pool models, this local control model displayed stable action potential trains at 7 Hz. The duration and the amplitude of the [Ca2+]myo transients increase in pacing rates consistent with the experiments. The [Ca2+]myo transient reaches its peak value at 4 Hz and decreases afterward, consistent with experimental force-frequency curves. The model predicts, in agreement with previous modeling studies of Jafri and co-workers, diastolic sarcoplasmic reticulum, [Ca2+]sr, and RyR2 adaptation increase with the increased stimulation frequency, producing rising, rather than falling, amplitude of the myoplasmic [Ca2+] transients. However, the local control model also suggests that the reduction of the L-type Ca2+ current, with an increase in pacing frequency due to Ca2+-dependent inactivation, also plays a role in the negative slope of the FFR. In the simulations, the peak Ca2+ transient in the FFR correlated with the highest numbers of SR Ca2+ sparks: the larger average amplitudes of those sparks, and the longer duration of the Ca2+ sparks.Calcium sparks are the elementary Ca A similar conclusion is also made from spark analysis; with the increase of SR Ca2+ ([Ca2+]SR), the mean as well as peak amplitudes of the Ca2+ sparks raise the force generated by the heart. With the increased adaptation of RyR2s, both peak and mean amplitudes start falling, giving the frequency-dependent decline in the force generation. This study explores the Ca-workers , in that2+dynamics from the rat ventricular myocytes model by Hoang-Trong and co-workers SR), modifies the channel opening rate. Therefore, the [Ca2+]SR available to be released plays a major role in the developing force-frequency relationship ds, Ca2+ concentration in dyadic subspace, and [Ca2+]jsr luminal Ca2+ concentration at the junction. In the resting phase, almost all RyR2s stay in the closed state  and 3 (O3) are open, and states 1 (C1) and 6 (C6) are closed. C5 is the voltage-dependent inactivated state . C4, the Ca2+-dependent inactivated state , is controlled by the subspace Ca2+ in each release site. Increased subspace Ca2+ increases the rate of inactivation of LCC and prevents Ca2+ overload in the myoplasm by limiting Ca2+ entry myo), NSR Ca2+ concentration ([Ca2+]nsr), and RyR2 opening probability (Popen) from each beat were measured. Finally, the peak values of each ionic current were collected and plotted against the pacing frequencies. Simulations using the guinea pig ventricular myocyte model studied Caransient . The fou2+ level was performed by calculating average numbers of Ca2+ sparks, Ca2+ spark amplitudes, and the Ca2+ spark duration. Ten beats were used to compute these data after the simulation arrived in a stable state. After that, an analysis algorithm chose the segments of the systolic phase only and excluded any data from the diastolic phase. From the systolic segment, the dyadic Ca2+ transient, which was at least 25 µM tall, was counted as a spark and used to sum the total sparks in that beat. Similarly, the sparks were counted from the rest of the beats, and the average numbers of the sparks were calculated. Likewise, the average amplitudes of the sparks from different beats of the same frequency were measured by adding the amplitude of each beat and dividing the total amplitude by the number of beats. After collecting average spark amplitudes of each beat, the mean amplitude was computed over 10 beats. The average spark duration was calculated using a similar protocol to that used for Ca2+ amplitudes. The total spark durations were combined in each beat, the sum was divided by the number of sparks, and the mean spark duration was concluded from the 10 beats.The subcellular and molecular analysis of the FFR at Ca2+ release units myo) rose with the increase in the pacing frequency, from 0.20 Hz (0.33 µM) to 4 Hz (0.64 µM), peaking at 4 Hz . The model allows an analysis of how Ca2+ spark frequency and Ca2+ spark amplitude regulate the Ca2+ transient and the resulting contractile force of a myocyte. Ca2+ spark properties show similar behavior to Ca2+ transients  also occurring at 4 Hz pacing and decreasing at higher pacing frequencies. The SR load also affects the Ca2+ spark amplitudes myo; and (c) increased SR Ca2+ load [Ca2+]sr available to be released in subsequent beats. The increase in SR Ca to Bers , in high2+ transients SR and RyR2 adaptation increases with increased stimulation frequency, giving rise to increasing, rather than falling, amplitude of the myoplasmic [Ca2+]myo transients. However, the model also suggests that the reduction of L-type Ca2+ current with an increased pacing frequency due to Ca2+-dependent inactivation also contributes to the negative part of the FFR. The model was able to give such insight because it also allowed the dissection of these frequency-dependent mechanisms down to the spark level, gaining a deeper perspective into the regulation of cardiac calcium dynamics. Simulations with the newly developed model for excitation-contraction coupling in guinea pig ventricular myocyte explored mechanisms behind the experimentally observed force-frequency relations. The continuous refilling of the SR by SERCA pump and enhanced SR Ca-workers , that di


FIP1L1::RARA fusion, are described in more detail.The first section of the bone marrow workshop of the European Association of Haematopathology (EAHP) 2020 Virtual Meeting was dedicated to pediatric myeloid neoplasms. The section covered the whole spectrum of myeloid neoplasms, including myelodysplastic syndromes (MDS), myeloproliferative neoplasms (MPN), myelodysplastic/myeloproliferative neoplasms (MDS/MPN), and acute myeloid leukemia (AML). The workshop cases are hereby presented, preceded by an introduction on these overall rare diseases in this age group. Very rare entities such as primary myelofibrosis, pediatric MDS with fibrosis, and MDS/MPN with JMML-like features and t;  Although less common in the pediatric age group, most myeloid neoplasms seen in adults can also occur in childhood where they may show differences from their adult counterparts. Some entities preferentially occur in childhood, such as several types of acute myeloid leukemia (AML) with specific recurrent genetic abnormalities, others exclusively in childhood such as juvenile myelomonocytic leukemia (JMML) and the provisional WHO entity refractory cytopenia of childhood (RCC). Furthermore, when dealing with children and young adults, one must be particularly aware of a possible underlying inherited or de novo germline mutation that might predispose to myeloid neoplasms.In this paper, we present an overview of the neoplastic cases submitted to the EAHP 2020 bone marrow (BM) workshop on pediatric myeloid neoplasms. The cases were grouped into the following sections: myeloproliferative neoplasms (MPN), JMML and related diseases, myelodysplastic syndromes (MDS), and AML. Each section is preceded by an introduction, focusing on the specific characteristics in childhood, followed at the end by a summary highlighting the lessons learned from the workshop.BCR::ABL1-positive MPN, characterized by leukocytosis due to proliferation of mainly the granulocytic lineage. CML is rare in childhood, accounting for less than 10% of all CML cases and less than 3% of all pediatric leukemias. The median age at diagnosis in children is 11–12 years, with 60–77% occurring in males /46,XX[2]} revealed a complex variant of the t translocation, involving both chromosomes 9 and 22 as well as three other chromosomes (in addition to 9q34 and 22q11). FISH:BCR/ABL dual-color, dual-fusion probe was positive for t in 67.5% of cells with deletion on derivative chromosome 9. Standard and variant t translocations in the current tyrosine kinase inhibitors era are thought to share a similar good prognosis.Four CML cases in chronic phase were submitted to the workshop Table , of whicBCR::ABL1 fusion gene may induce resistance to tyrosine kinase inhibitors, as was illustrated by case 744, containing multiple such mutations. On follow-up, this patient acquired an additional EVI1 rearrangement, indicating clonal evolution.In CML, mutations in the kinase domain of the BCR::ABL1 not detected by conventional karyotyping, but demonstrated by FISH and RT PCR, illustrating the need for additional investigation whenever the clinical and histological picture is suspicious for CML but conventional karyotyping is normal. Cryptic or masked BCR::ABL1 fusions occur in 2–10% of CML patients.Case 505 had a cytogenetically cryptic BCR::ABL1-negative MPNs, which include essential thrombocythemia (ET), polycythemia vera (PV), and primary myelofibrosis (PMF), are rare in children, with global incidences for children and young adults estimated to be around 0.6/100,000 patients/year (range 0.004 to 0.9) for ET, 0.18/100,000 patients/year for PV, and 0.53/100,000 patients/year (range 0.003 to 1.5) for PMF /46,XX[2]. A FIP1L1::RARA fusion was demonstrated as well as a bi-allelic deletion of CDKN2A/2B.The second case (case 472) describes a 16-month-old girl with a 1-month history of intermittent fevers, generalized malaise, and night sweats. Her PB showed marked leukocytosis (156.4 × 10FIP1L1::RARA fusion have previously been described in two adults and a child [FIP1L1::RARA fusion were diagnosed as acute promyelocytic leukemia (APL) [FIP1L1::RARA fusion showed a clinical picture most consistent with JMML [Myeloid neoplasms with a  a child –60. Theyia (APL) , 60. Oneith JMML .FIP1L1::RARA, other JMML mimics include cases with rare rearrangements, like ALK and ROS1 or CCDC88C::FLT3 fusions [FLT3).Besides cases associated with  fusions . PatientTET2, ASXL1, and DNMT3A) and RNA splicing  are rare in pediatric primary MDS (2%), whereas mutations in genes from the RAS/MAPK pathway are common (45%) [Pediatric MDS is rare, with an incidence of 1.8–4 cases/million . It can on (45%) .A relatively high percentage of pediatric MDS cases represent secondary MDS. This includes therapy-related myeloid neoplasms, MDS secondary to acquired aplastic anemia, and MDS secondary to inherited predisposition syndromes, including the inherited bone marrow failure syndromes Fanconi anemia, severe congenital neutropenia, dyskeratosis congenita, and Shwachman-Diamond syndrome.GATA2 mutation, myeloid neoplasms associated with bone marrow failure syndromes, myeloid neoplasms associated with telomere biology disorders, JMML associated with neurofibromatosis, Noonan syndrome or Noonan syndrome-like disorders, and myeloid neoplasms associated with Down syndrome [SAMD9) and its paralogue, SAMD9-like (SAMD9L) [Underlying inherited or de novo germline predisposing genetic aberrations are being recognized in an increasing amount of pediatric MDS cases. According to the revised 2016 WHO classification, myeloid neoplasms with germline predisposition can be classified in three groups: (1) Myeloid neoplasms with germline predisposition without a pre-existing disorder or organ dysfunction, (2) myeloid neoplasms with germline predisposition and pre-existing platelet disorders, and (3) myeloid neoplasm with germline predisposition and other organ dysfunction, which includes myeloid neoplasm with germline syndrome . Additio(SAMD9L) , 67. In GATA2 and SAMD9/SAMD9L syndromes currently form the most common predisposing conditions in pediatric MDS. In cases that originally were classified as primary pediatric MDS, 7% showed a GATA2 mutation and 8–17% a SAMD9/SAMD9L mutation [Together, mutation , 68, 69.mutation , 68.GATA2-related MDS typically shows BM hypocellularity with multilineage dysplasia –72. DyspSAMD9/9L syndromes are autosomal dominant syndromes with variable penetrance. They cause a spectrum of multisystem disorders that carry a markedly increased risk of developing myeloid malignancies with monosomy 7 . The medRCC is a provisional entity dealt with in the WHO MDS chapter . It is dIn total, 16 of the submitted cases were classified by the panel as MDS Table .Table 4MMLF1::NPM1, which is an aberration typically seen in younger pediatric patients with MDS or AML and is associated with an excellent response to SCT.Seven MDS cases had excess of blast  and two of them were accompanied by fibrosis. One of the MDS-EB2 cases (case 677) was a 7-year-old boy with a t; There were two cases of therapy-related MDS; one case of a 16-year-old boy with a history of Ewing sarcoma at the age of 15, and one case of a 14-year-old girl with a history of spinal neuroblastoma at the age of 2.One of the submitted cases was classified as MDS with single lineage dysplasia and fibrosis (case 322) and six cases were classified as MDS, unclassifiable (MDS-U), based on the presence of single lineage dysplasia combined with pancytopenia (4 cases) or either single or multilineage dysplasia combined with 1% blasts in the PB (2 cases). Two of the MDS-U cases (case 723 and case 754) also showed fibrosis (MF2) and were therefore classified as MDS-U-F. Case 754 is illustrated in Fig. In two of the MDS cases above (case 228 and case 723), the patients had a history of cytopenia, suggestive of an underlying germline condition: case 228 was a 10-year-old boy with MDS-U, known with congenital neutropenia, and case 723 was an 18-year-old young man with MDS-U-F with monosomy 7, who had a long history of neutropenia and thrombocytopenia. The latter patient received an allogeneic matched unrelated donor cord blood transplantation, 3 months after which he developed a donor cell MDS with again monosomy 7.RRAS and a concurrent del7/7q detected in his myeloid neoplasm. The presence of multiple phenotypic abnormalities, including scoliosis, craniosynostosis, and cardiac anomalies, as well as delayed milestones, was suggestive of an underlying RASopathy , but the fact that his RAS mutation was not detected by whole exome sequencing  that was performed on normal tissue to detect germline mutations was in favor of a somatic mutation.In one of the MDS-U cases (case 179), the 7-year-old boy had a de novo heterozygous mutation of None of the cases was felt by the panel to fit the diagnosis of RCC; these cases were at least moderately cellular, not displaying the typical low cellularity of RCC reminiscent of AA. They also did not display markedly enlarged erythroid islands with impaired maturation, which is a key feature for the diagnosis of RCC, including the normo- and hypercellular RCC variants . Rather,PML suggestive of trisomy 15 (case 329 classified as MDS-EB2-F), 1 case had monosomy 7 (case 723 classified as MDS-U-F), and the fifth case (case 682 classified as MDS-EB1-F) showed on interphase FISH a translocation of chromosome 11q with unknown fusion partner. In literature, fibrosis in pediatric MDS has been associated with germline GATA2 mutations [Interestingly, as mentioned before, 5 of the 16 MDS cases showed marrow fibrosis Fig. . The meautations –72, but DNMT3A, TP53, and IDH1 or IDH2, are rare in pediatric AML [NRAS, KRAS, KIT, WT1, MYC, CBL, GATA2, SETD2, PTPN11, and FLT3 are more common in children, and sites of mutation often differ from those detected in adults [Although all types of AML can occur at the pediatric age, some forms occur more often or even exclusively in childhood, and the genetic landscape of pediatric AML differs from that of adult AML . Certaintric AML . Mutation adults . In addin adults .KMT2A::MLLT3 (20–22%), t; RUNX1::RUNX1T1 (9–15%), and inv(16)(p13.1q22) or t; CBFB::MYH11 (6–10%), of which t; RUNX1::RUNX1T1 and the two with involvement of chromosome 16 are associated with a favorable prognosis [KMT2A rearrangement, the prognosis depends on the fusion gene partner; some show a favorable prognosis, e.g., t, while others show an adverse prognosis, e.g., t, t, t, and t [KMT2A::MLLT3 , prognosis appears to be related to morphology, as those with acute monoblastic features have a significantly better outcome than cases showing other cytochemical types, at least in one study [The most common pediatric AML belongs to the group of AML with recurrent genetic abnormalities (AML-RGA). They include t; rognosis –81. In cq21;q23) . In t with t; RBM15::MKL1, AML with t; MNX1::ETV6, AML with t; KAT6A::CREBBP , and AML with inv(16)(p13.3q24.3); CBFA2R3::GLIS2 as reviewed by Quessada et al. [PICALM::MLLT10, AML with t; DEK::NUP214, AML with t; FUS::ERG, AML with t; RUNX1::CBFA2T3, AML with t; NUP98::NSD1, AML with t; KMT2A::MLLT4, and AML with t; PML::RARA [In addition to a et al. . Others ML::RARA . Other rML::RARA .GATA1 are an early event in the leukemogenesis and are characteristic for myeloid leukemia associated with DS [GATA1 mutation. This preleukemic clone, known as transient myeloproliferative disease (TMD) or transient abnormal myelopoiesis (TAM), may be morphological indistinguishable from Down-associated AMKL, but resolves spontaneously in 60% of cases at a mean of 58 days (range 2–194 days) [As already discussed above in the section on MDS, several inherited or de novo germline aberrations are known to predispose to the development myeloid neoplasms. One of the predisposition syndromes is Down syndrome (DS), which children have a markedly increased risk of developing AML in their first 5 years of life, especially acute megakaryoblastic leukemia (AMKL). MDS and AML in DS are biologically similar and are therefore grouped together under the diagnosis “myeloid leukemia associated with Down syndrome” . Somatic with DS , 86. App94 days) . About 294 days) .In total, 12 cases of pediatric AML were included in the workshop, covering a wide spectrum of AML subtypes Table . Four caRBM15::MKL1. This is a rare translocation occurring in infants and presenting as AMKL.The first AML-RGA case (case 458) was a 7-month-old girl with AML (megakaryoblastic) with t; CBFA2T3::GLIS2, which is equivalent to inv(16). This is a rare cryptic chromosomal translocation associated with treatment-refractory disease, occurring especially in infants, and presenting in 20% of cases as an AMKL. In this case, the blasts showed a so-called RAM phenotype , consisting of bright CD56 expression, dim or negative CD45 and CD38 expression, and lack of HLA-DR expression. The RAM phenotype is mainly seen in infants and very young patients, and an independent risk factor in pediatric AML patients associated with extremely poor outcome [The second case (case 511) was a 3-year-old girl with AML with  outcome .NPM1. The NPM1 mutation is one of the most common recurrent cytogenetic aberrations in AML, and, in contrast to the previous two cytogenetic aberrations, not specific for childhood. Its frequency is rare in AML in infants (2.5%), but it increases with age to up to 34% in adult AML [NPM1 mutation, the patient had several other mutations: RAD21 (VAF: 43%), KRAS (VAF: 27%), and two different PTPN11 mutations (VAF: 10% and 2%).The third case (case 583) was a 12-year-old boy with AML with mutated dult AML . In addiRUNX1T1::RUNX1, which interestingly showed myelomastocytic differentiation with increased serum tryptase (29 μg/L) and two KIT mutations (c.1251_1266delins16 and c.2466 T > A (p.Asn822Lys)), which have not been described in systemic mastocytosis. In both the AML blasts and the mast cells, the t could be detected, indicating they belong to the same clone.The fourth AML-RGA case (case 187) was a 10-year-old boy with AML with t; CDK6::MECOM aberration, which is a rare anomaly described in high-risk pediatric AML. The resulting overexpressed MECOM protein could be demonstrated by immunohistochemistry.Four of the submitted AML cases were classified by the panel as AML-MRC; one based on a previous history of MDS (case 773), two based on the presence of a complex karyotype (case 361 and 150), and the fourth (case 796) based on the presence of an MDS-related cytogenetic abnormality, being del(7)(q22). The latter case also showed a KMT2A (MLL) translocation (fusion partner not identified).Three of the submitted AML cases were classified as AML-NOS. The first case (case 282) showed megakaryoblastic differentiation and a normal karyotype, but on FISH a FLT3-ITD and a WT1 mutation, which concurrent presence forms a poor prognostic factor in de novo pediatric AML [The second AML-NOS case (case 766) had a tric AML .The third case (case 247) was considered AML-NOS. Although areas rich in proerythroblasts were noticed, the overall presence of > 20% myeloblasts supported a diagnosis of AML-NOS (over one of pure erythroid leukemia).RUNX1::RUNXT1.There were two cases of myeloid sarcoma Table , of whicRBM15::MKL1. Other commonly recurring rearrangements in non-Down syndrome pediatric AMKL are CBFA2T3::GLIS2, NUP98::KDM5A, HOX rearrangements, and KMT2A rearrangements, the latter being illustrated by case 282. These other recurring rearrangements are, however, not separate WHO entities but currently classified as AML-NOS (megakaryoblastic), although they do have prognostic value as outcomes vary considerably [Interestingly, among the submitted AML cases, there was a relatively high frequency of AMKLs Fig. , none ofiderably . Case 15EAHP 2020 virtual meeting was dedicated to pediatric myeloid neoplasms. Several interesting “take home messages” can be drawn from it:BCR::ABL1 variants and ABL1 TKI resistance mutations, as illustrated by the workshop cases 505 and 744.CML is rare in childhood and CML biology in adults and children is different as underlined by the different BCR breakpoint distribution seen in the latter. One must always be aware of cytogenetically cryptic BCR::ABL1-negative MPNs are rare and differ from the adult cases at several points:*Mild splenomegaly is common even in ET and should therefore not be used as a criterion for separating ET from PMF.*Thrombotic events are less frequent than in adults and are largely venous, with a high frequency of Budd-Chiari syndrome.*High cellularity and lack of iron storages are common, especially in young children, and are therefore less reliable criteria for the distinction of ET versus PV.*WHO diagnostic criteria for diagnosing adult PV need to be adjusted in children by using appropriate age-related thresholds for establishing presence of erythrocytosis.*Although it has been reported that pediatric MPNs have a lower frequency of driver mutations , this was not confirmed by the cases submitted to the workshop, in which a driver mutation was detected in 8 out of 11 (73%) cases. Further studies of adequately annotated cases diagnosed according to the WHO approach seem to be needed to clarify the exact frequency.*The features of pediatric PMF are overall similar to those seen in adult PMF, and the two submitted cases of prefibrotic (pre-)PMF underline the fact that that pre-PMF can also occur in children.Pediatric *JMML may present as AML-MRC.*RRAS2 is one of the less frequently mutated genes of the RAS-pathway in JMML.*SETBP1, JAK3, SH3BP1, SH2B3, or components of the polycomb repressive complex 2, including EZH2 and ASXL1.A second, often subclonal, mutation occurs in less than half of JMML cases; this mutation can occur in another RAS pathway gene or in *Some of the JMMLs occur in the setting of an underlying RASopathy, which is a group of developmental disorders characterized by typical phenotypic features, caused by a germline mutation in genes that encode components of the RAS/MAPK pathway. Of these, Noonan syndrome, neurofibromatosis type 1, and CBL syndrome predispose to JMML.*KRAS or NRAS mutation show an indolent clinical course and long-term survival, which entity is called RAS-associated autoimmune leukoproliferative disorder (RALD), (2) infants with Noonan syndrome develop a transient JMML-like myeloproliferation in 2.5–10% of cases, which is almost invariably associated with a PTPN11 mutation, (3) JMML with a germline CBL mutation may show spontaneous regression.JMML is generally an aggressive disease but milder forms and transient myeloproliferations occur: (1) Few cases with a *FIP1L1::RARA fusion are described. These cases manifest with monocytosis and an abnormal myelomonocytic proliferation similar to JMML but may be lacking some clinical features of JMML and do not contain identifiable mutations in the RAS pathway or other JMML defining genetic aberrations. FIP1L1::RARA fusion could also raise the possibility of acute promyelocytic leukemia, although typical features are lacking . Whether these pediatric cases should be classified as JMML in the future remains to be determined.Two rare cases of MDS/MPN with JMML-like features and t; Apart from JMML, the “adult types” of MDS/MPN are basically not seen at the pediatric age. The workshop cases illustrate the following:*Five of the 16 MDS cases submitted to the workshop had fibrosis, with a female predominance (M:F = 1:4) and a mean age of 12.2 years (range 2–18 years). All fibrotic cases showed chromosomal aberrations, including trisomy 8, trisomy 15, monosomy 7, and 11q translocation.*GATA2 and SAMD9/SAMD9L are at the moment the most common.Especially in children and young adults, MDS and AML may develop secondary to aplastic anemia, an inherited bone marrow failure syndrome or in the context of another underlying germline mutation which predisposes to the development of a myeloid neoplasm, of which Pediatric MDS is rare. Most of the workshop cases were classified as MDS-U or MDS-EB1 or 2:*RBM15::MKL1 and AML with CBFA2T3::GLIS2 ) as illustrated by the workshop. Others are more common in adults, but can be seen in childhood as well, as illustrated by a case of AML with t; RUNX1T1::RUNX1.Some recurrent cytogenetic aberrations are typical of childhood, such as AML (megakaryoblastic) with t; *RBM15::MKL1 is the only recurrent cytogenetic abnormality in AMKL classified by the WHO as a separate entity, but the other recurrent cytogenetic aberrations in non-Down syndrome AMKL are also of prognostic importance and therefore deserve further investigation.The workshop contained a relatively high frequency of AMKL, not associated with DS. These can be classified as AML-RGA, AML-MRC, or AML-NOS. The t; All types of AML can occur at the pediatric age, and several cases of AML-RGA, AML-MRC, and AML-NOS, as well as 2 cases of myeloid sarcoma, were included in the workshop:The first section of the BM workshop of the


Multilayered carbon fiber-reinforced polymers (CFRP) are increasingly used in aircraft components because of their superior mechanical properties. However, composite materials are vulnerable to impact loads, resulting in delamination-type damage which, if unnoticed, could lead to catastrophic structural failure. The objective of this research was to investigate possibilities to improve damage detection and the localization using signal processing methods. Numerical modeling using the semi-analytical finite element (SAFE) method was performed to obtain guided wave dispersion curves and to perform modal analysis. From the modal analysis,  Composite materials such as carbon fiber-reinforced polymers (CFRP) and glass fiber-reinforced polymers (GFRP) are widely used in aerospace components, wind turbine blades, and other engineering structures ,2,3. TheHowever, multilayered CFRP structures are vulnerable to delamination and fiber breakages. Delamination-type defects due to impact damage can occur during assembly and exploitation. Key issues in aircraft wing structures are delamination and disbond-type damage. Such types of defects are difficult to detect during inspection, and this can lead to catastrophic structural failure.0 mode [In aerospace applications, structural health monitoring (SHM) could increase the safety, reliability, and quality control of the structure . However0 mode ,5, by Ca0 mode , and by 0 mode ,7. The m0 mode . The int0 mode ,10,11. S0 mode ,12.The primary advantages of guided waves compared to bulk waves for multilayered structure inspection are that these waves can propagate much further distances with less attenuation, and long-range inspection is possible . Hence, However, guided wave signals are complex to interpret due to multimode propagation, dispersive nature, and complex geometries, increasing the complexity even more ,16,17,18State-of-the-art reviews in the field of structural damage detection and localization using wavelet-based studies were conducted by several authors ,28,29,30On the basis of dispersion characteristics, the preferred mode can be selected to reduce the complexity. The development of the SHM technique based on guided waves for damage detection requires careful numerical modeling, because finite element analysis (FEA) of guided wave propagation and mode interaction with damage can provide useful information for further analysis.Currently, multi-sensor arrays are commonly used in SHM to improve the accuracy of damage localization. However, the weight issue of the SHM system should not be forgotten: the additional weight due to the SHM system should be as low as possible, which means that the number of sensors used should be as low as possible. Additionally, reducing the number of sensors is beneficial in terms of the amount of data to be processed. On the other hand, the accuracy of the localization of damage should not suffer when using fewer sensors.The objective of this research is to investigate a technique in which using only two receivers, it would be possible to determine if the delamination is located in between the transmitter and the receiver or in between the receiver and the edge of the structure. In addition, the accuracy of two different signal postprocessing techniques in improving damage localization is evaluated.2. The normal force was applied on an excitation zone of 8.4 mm to excite rX). Two cases of delamination positions were considered: the delamination position (dX) at 40 mm, i.e., between the excitation zone and the receivers, and at 120 mm, i.e., further than the receivers. Cases studied, positions, and distances are presented in The CFRP plate (USN150B) of 200 mm in length and 3.5 mm in thickness was selected for analysis. The CFRP panel was constructed from 18 plies, and the laminate orientation was The defect-free CFRP plate is presented in dX) was fixed at 100 mm, and the delamination location in the through-thickness direction (hX) was modified: 1.1, 1.7, and 2.3 mm. The cases investigated are presented in In another investigation, the delamination position in the through-thickness direction was changed. The delamination position  used in the numerical study are presented in he plate .µ are the Lamé constants, t is the time. The It is essential to obtain dispersion curves to select suitable modes and the central frequency for the inspection ,24,25,26In this analysis, the length and width of the plate were assumed to be infinitely long to consider the guided wave mode as symmetrically distributed in the through-thickness direction. Eighteen elements across the plate were assigned, and the thickness of one element replicates one ply of the multilayered plate. The thickness of each ply was 0.194 mm and the total thickness of the 18 plies of the CFRP plate was 3.5 mm.The SAFE method was derived to obtain the dispersion curves of guided wave phase velocity, The purpose of the SAFE method is not only to calculate dispersion curves, but in addition, to analyze displacement distribution in the through-thickness of the plate and to select a particular mode, which would be sensitive to delamination-type defects. In guided waves, out-of-plane displacements are dominant in asymmetrical modes, and in-plane displacements are dominant in symmetrical modes.The in-plane and out-of-plane displacements of The in-plane and out-of-plane displacements of the The Experimental inspection of every guided wave propagation scenario for different damages, loading, and environmental conditions requires many mock-up trials. Therefore, physics-based numerical modeling is an efficient way to understand the mechanism of guided wave propagation and interactions with delamination in composite structures.z-axis. The multilayered CFRP panels were anisotropic and the GW propagation velocity was angle-dependent. The 3D modeling could give propagation in all directions, which would be closer to a real investigation. However, 3D modeling of ultrasonic wave propagation, taking into account 20 elements per wavelength criterion, requires huge computational and time resources. In this research, numerical modeling was performed to investigate the interaction of guided waves with delamination-type defects at different depths. Therefore, modeling of guided wave propagation using the 2D plane strain approach was most suitable for the given task. The simulation was carried out with 2100 time-steps and the duration of each step was 0.1 µs. The total modeled duration of the The guided wave interaction with delamination was analyzed with the transmitter–receiver configuration, as shown in In FEA, mesh shape and size are important factors. The CPE4R-type elements were selected for the analysis in ABAQUS explicit. These types of elements are plain-strain quadrilateral and allow less integration time, and better hourglass control. In FEA, hourglass control attempts to minimize severe mesh distortion and provides better mesh control. On the other hand, a smaller mesh size improves the accuracy of the analysis but increases the computation time. In guided wave simulation, the mesh size is significantly dependent on the number of elements per wavelength The fundamental asymmetrical Guided wave analysis at 100 kHz is an appropriate way to achieve a good balance between dispersion, mode complexity, and attenuation. In order to properly identify u, and can be transformed into the wavenumber, k, and the frequency space using 2D FFT [x is the coordinate, t is the time, ω is the angular frequency, and k is the wavenumber.The dispersion characteristics of the guided waves propagating in the CFRP plate from B-Scan data were obtained using the two-dimensional Fourier transform. The propagated guided wave along the plate is expressed as a function of the coordinates and time, g 2D FFT :(2)H, and transmitted , the signals were acquired at three positions of the receivers, as shown in dX was 40 mm, the guided wave signals were received (rX) at 70, 80, and 90 mm with respect to the leading edge of the CFRP plate. The delamination location can be estimated by analyzing the reflected wave from the delamination.The guided wave interaction with delamination at 40 and 120 mm was analyzed. To analyze the delamination position (t1), and the reflection from the delamination must be considered as t2. The delamination position can be estimated considering the time of flight (ToF) between t1 and t2. The Hilbert envelope of the guided wave signals received at different receiver positions are presented in H(t), can be expressed using the following equation [In this case, the equation ,31,32,33The distance between the receiver and delamination can be estimated by calculating ToF between 1 and t2 :(4)Xe=ΔtaD and mD are the actual and measured values, respectively.The location of delamination was estimated by measuring the time difference between direct and reflected receiver b. TherefdX = 120 mm, the guided wave signals were received at 70, 80, and 90 mm with respect to the leading edge of the CFRP plate. The distance between delamination and the receiver (drX) was 50, 40, and 30 mm. In this approach, if the delamination is located after the receiver position, the excited t1) and the first reflection from the leading edge of the delamination is received as the second wave packet (t2) (In the case of ket (t2) c, as shodX) can be estimated by adding the distance between the edge of the sample and the receiver and the estimated distance between the receiver and delamination (eX). The localization of delamination and the relative error of this approach are presented in In this case, the estimated distance is the distance between delamination and the receiver. The delamination position  at 40 mm is post-processed and the estimated location using CWT is presented in The guided wave mode response obtained from the delamination position (dX) at 120 mm and the signals received at different receiver positions to estimate the delamination location using CWT are presented in The guided wave mode response obtained from the delamination position (The location of the delamination can be estimated using Equation (3). The maximum amplitude of the wave packet was used for the analysis. Therefore, the group velocity of The defect location measurement using CWT provided more accurate results compared with HT. Furthermore, the time–frequency distribution of the guided wave signal along with the amplitude were obtained simultaneously. The proposed method has the potential to measure the ToF of a signal by considering the excitation frequency as a reference even in the presence of other frequency content in the signal.In this section, the delamination position in the through-thickness direction was examined. The delamination position in the longitudinal direction was 100 mm, and the positions at different depths of 1.1, 1.7, and 2.3 mm were analyzed in the CFRP plate.For 1.1 and 2.3 mm, the delamination was close to the top and bottom surfaces of the plate. For 1.7 mm, the delamination was right in the center of the plate. The B-scan representation of the The amplitude of the transmitted and reflected guided wave mode varies depending on the position and size of the defect. Therefore, it is necessary to examine transmitted and reflected modes separately. In order to separate superimposed modes, the signal processing method was applied on the received guided wave signal. The flow chart of the signal processing method to separate reflected and transmitted modes is presented in t is the guided wave propagation time and x is the propagation distance.Initially, a two-dimensional fast Fourier transform (2D FFT) was applied to the B-scan data to obtain the wavenumber of the reflected and transmitted modes. The wavenumber of the signal using 2D fast Fourier transform can be expressed by the following equation :(7)U is the 2D bandpass filter of the selected mode. The results obtained using IFFT to separate the guided wave modes are presented in The bandpass filter with a cosine-tapered window function was applied to the frequency–wavenumber domain to separate the modes. The wavenumber of reflected ing IFFT :(8)uFUF′, respectively. As mentioned above, the delamination at 1.1 and 2.3 mm was at the same distance from the top/bottom of the plate. Hence, the transmitted y-direction and had a higher amplitude compared to The guided wave dispersion curves and modal analysis were performed using the one-dimensional SAFE method. In this analysis, the guided wave was bounded by two surfaces and propagated in a single direction. However, guided wave modes have displacements in three directions and are dominant in a single direction. Therefore, from the analysis, The plain-strain numerical simulation was performed on a multilayered CFRP panel to analyze the guided wave mode response with delamination. In this analysis, two different inspection setups were used to locate the delamination. In one case, the delamination was located between the excitation zone and the receiver. In the other case, the delamination was located after the receiver. In the conventional way, the defect location can be estimated by measuring the ToF between the converted and transmitted modes. In this analysis, the converted In guided wave inspection, the reflected mode could be useful for delamination localization when the converted mode does not provide any information due to the low amplitude. The location of the delamination can be estimated using ToF between direct t1) and the reflected (t2) t1 and t2. The CWT obtains the time–frequency and maximum amplitude of the signal simultaneously. The ToF between the maximum amplitudes of the two wave packets allows to estimate the defect location. In addition, the CWT has the potential to achieve accurate local transients in the signal.In this approach, the HT and CWT were used to calculate the ToF between the direct (ToF was similar in cases when the delamination was positioned between the excitation zone and the receiver, or after the receiver. Using three receivers at different positions enabled to determine the delamination location. In the case when the delamination was in between the excitation zone and the receivers, the estimated distances for all the receiver positions were similar and showed the delamination position from the leading edge. Meanwhile, in the case when the delamination was further than the receiver, the estimated distances for the three receiver positions were different and showed the delamination position from the respective receivers.When analyzing near-surface delamination at 1.1 and 2.3 mm in the through-thickness direction, strong reflections were obtained due to the delamination edge and the top/bottom surface of the CFRP plate. In the case of delamination at a depth of 1.7 mm, weak reflection was obtained.When considering the transmitted mode, a weak transmitted mode was obtained due to the delamination edge and the top/bottom surface of the CFRP plate. The presented method can be used to determine the characterization of near-surface delamination, but it has limitations when it comes to measuring the exact delamination depth.The In this investigation, the inspection setup was proposed to detect and localize the delamination. This method offers to locate delamination when the converted modes do not have a significant amplitude. The reflected mode provides useful information to precisely locate the delamination. The localization of the delamination was estimated using six different scenarios to assess the accuracy of the proposed inspection setup. All inspection scenarios have shown consistent results and obtained an accurate estimate of the location of the delamination, with a relative error of less than 7.5%. Using three receivers at different positions allowed to determine where delamination was located, either in between the excitation zone and the receivers, or further than the receivers.In this article, signal processing methods to improve damage detection and localization using guided waves have been proposed. The localization of delamination using HT and CWT was compared, where the relative error using HT and CWT was 2.5% and 1.2%, respectively. CWT obtained more accurate results to estimate the location of the defect compared to HT. In addition, CWT provided better linear time–frequency representation, higher resolution of time–frequency components, a finer spectrum, and it is superior for localizing the transients of the guided wave signal.While characterizing near-surface delamination at 1.1 and 2.3 mm in the through-thickness direction, a strong reflection was obtained from the delamination edge and the top/bottom surface of the CFRP plate. Such a mode response occurred as a result of the variation in propagation of mode speed along the upper and lower surfaces of the delamination. In the case of 1.7 mm, the distance between the upper and lower surfaces of the plate was identical. Therefore, the mode propagation speed was identical along the upper and lower surfaces of the delamination.Damage detection was improved using 2DFFT. When characterizing near-surface delamination at 1.1 and 2.3 mm, consecutive Future work will focus on the experimental verification of the proposed approach, which will be performed as the next step of this investigation.


The Ca2+ channels are linked to synaptic vesicles which are tethered by active zone proteins. Ca2+ entrance into the active zone triggers: (1) the fusion of the vesicle and exocytosis, (2) the replenishment of the active zone with vesicles for incoming exocytosis, and (3) various types of endocytosis for vesicle reuse, dependent on the pattern of firing. These time-dependent vesicle dynamics are controlled by presynaptic Ca2+ sensor proteins, regulating active zone scaffold proteins, fusion machinery proteins, motor proteins, endocytic proteins, several enzymes, and even Ca2+ channels, following the decay of Ca2+ concentration after the action potential. Here, I summarize the Ca2+-dependent protein controls of synchronous and asynchronous vesicle release, rapid replenishment of the active zone, endocytosis, and short-term plasticity within 100 msec after the action potential. Furthermore, I discuss the contribution of active zone proteins to presynaptic plasticity and to homeostatic readjustment during and after intense activity, in addition to activity-dependent endocytosis.Within 1 millisecond of action potential arrival at presynaptic terminals voltage–gated Ca F. F2+ sen2+ sensors: Synaptotagmin-1 and -2 are expressed on the SV membrane, while synaptotagmin-7 is more concentrated on the presynaptic plasma membrane or internal membrane . T. T114]. membrane ,179. 2+ channels in the RRP [2+-sensor synaptotagmin, the activator/regulator complexin, the assembly factors Munc18 and Munc13, and the disassembly factors NSF and SNAP are the core molecules of the fusion machinery [2+-bound synaptotagmin [2+ sensors for slow and asynchronous SV fusion.The AZ protein assembly promotes SV states of tethering, docking and priming, and it sets release-ready SVs nearby Ca the RRP . The relachinery . AP-evoktotagmin ,65. Synatotagmin ,65. Synatotagmin ,72. Synatotagmin  and -7 [totagmin ,79, and totagmin ,81 are h2+ dynamics that control SV states, synchronous and asynchronous fusion, and undocking, redocking, and priming [2+ dynamics activate multiple protein cascades via Ca2+-sensor molecules [2+ channel coupling, is regulated by RIM and RIM-BP, which are required for clustering the Ca2+ channel at the release site [AP triggers SV dynamics in the AZ, following millisecond Ca priming . The und priming . AP-induolecules  to controlecules . Neurotrase site ,30,31. Tase site ,53,106. ase site ,40,42.2+-dependent regulation of the release probability via SV fusion machinery has been proposed for the generation of presynaptic plasticity [Presynaptic plasticity is dependent on flexible neurotransmitter release. Residual Caasticity . Howeverasticity ,127,128.asticity ,133,134.asticity , RIM-BP asticity , and Basasticity  in presySVs are recycled within nerve terminals. After exocytosis, the SVs are recovered via either fusion pore closure “kiss-and-run”  at the AFor summarizing this review discussing the mechanisms of SV exo- and endocytosis, molecular players act in the presynaptic release site AZ, the exocytosis, the replenishment of the release site with SVs, and the endocytosis are listed in the


Strongyloides stercoralis, and to a lesser extent Strongyloides fuelleborni which mainly infects non-human primates. Zoonotic sources of infection have important implications for control and prevention of morbidity and mortality caused by strongyloidiasis. Recent molecular evidence suggests that for S. fuelleborni, primate host specificity is variable among genotypes across the Old World, and consequently that these types likely vary in their capacity for human spillover infections. Populations of free-roaming vervet monkeys (Chlorocebus aethiops sabaeus), introduced to the Caribbean Island of Staint Kitts from Africa, live in close contact with humans, and concern has arisen regarding their potential to serve as reservoirs of zoonotic infections. In this study, we sought to determine the genotypes of S. fuelleborni infecting St. Kitts vervets to explore whether they are potential reservoirs for human-infecting S. fuelleborni types. Fecal specimens were collected from St. Kitts vervets and S. fuelleborni infections were confirmed microscopically and by PCR. Strongyloides fuelleborni genotypes were determined from positive fecal specimens using an Illumina amplicon sequencing-based genotyping approach targeting the mitochondrial cox1 locus and 18S rDNA hypervariable regions I and IV of Strongyloides species. Phylogenetic analysis of resultant genotypes supported that S. fuelleborni from St. Kitts vervets is of an exclusively African variety, falling within the same monophyletic group as an isolate which has been detected previously in a naturally infected human from Guinea-Bissau. This observation highlights that St. Kitts vervets may serve as potential reservoirs for zoonotic S. fuelleborni infection, which warrants further exploration.Human strongyloidiasis is an important neglected tropical disease primarily caused by the nematode   •S. fuelleborni.Free-roaming vervets on the Caribbean Island of Saint Kitts are infected with •S. fuelleborni from St. Kitts vervets to assess the zoonotic potential of these worms.We genotyped •S. fuelleborni type that has been found previously in a human.St. Kitts vervets are infected with an African •S. fuelleborni infection.St. Kitts vervets may therefore represent a reservoir of human  The clinical characteristics of strongyloidiasis are of variable severity, but generally include abdominal pain, diarrhea, skin rash, eosinophilia, and respiratory symptoms; disseminated strongyloidiasis or hyperinfections caused by S. stercoralis infection can be life-threatening because of autoinfection , while S. fuelleborni has been recognized as a cause of strongyloidiasis in humans and non-human primates throughout the Old World  which has been reported in sub-Saharan Africa, South Asia, and southeast Asia (S. fuelleborni kellyi (S. f. kellyi), which is an enigmatic species that possesses an ambiguous relationship with S. f. fuelleborni and is only found in Papua New Guinea  separately from Central and West African isolates, with human infections caused by both East and West African varieties (xposures . Anthropan types . Among Aarieties . Asian varieties .Chlorocebus aethiops sabaeus) exist as free-roaming populations on the Caribbean island of Saint Kitts, descended from a small group originally introduced by Europeans during the 17th century -based multi-locus-sequence-typing (MLST) method targeting the mitochondrial cox1 locus, and hypervariable regions (HVRs) –I and -IV of the 18S rDNA  from the island of Saint Kitts. Fecal specimens were collected from the transport cages of vervets trapped for other purposes  and stored at 4 °C until an initial screening . Samples identified with Strongyloides in this initial screen (n = 34) were placed directly into 90% ethanol (1 g–3 mL) and stored at −20 °C until shipment to the Parasitic Diseases Branch (PDB) at the US Centers for Disease Control and Prevention (CDC) for all subsequent analyses, including S. fuelleborni genotyping, and a secondary microscopic screen (400× magnification) of wet mounts for the presence of Strongyloides eggs and/or larvae. The specimens were stored at 4 °C prior to subsequent molecular analyses. A single bronchoalveolar lavage (BAL) fluid specimen from a human patient who tested positive for S. stercoralis in the parasitology reference laboratory at CDC (by PCR and microscopy) was included in this study as a positive control.The study population included multiple troops of free-roaming African vervet monkeys  with some minor modifications. As the fecal specimens had been preserved in 70% ethanol, they were washed in PBS prior to DNA extraction. To do this, approximately 0.25 g of feces suspended in ethanol was placed in a 2 mL Eppendorf tube and centrifuged in a benchtop centrifuge (Eppendorf Centrifuge 5424) at maximum speed . The supernatant was discarded. Next, the tube was filled to 2 mL with PBS and vortexed to disrupt the pellet. The suspension was centrifuged at maximum speed once more and the supernatant was removed again. The resulting pellet was transferred to a PowerBead Tube and subjected to Bead beating for 3 min at 1400 RPM on an Eppendorf Thermomixer. After adding solution C1 to the tube containing the PowerBeads and feces, we also added 20 mL of proteinase K taken from a DNEasy Blood & Tissue Kit (Qiagen). The solution was gently vortexed and left to digest at 65 °C for 30 min. The digested specimen was vortexed again following digestion. The DNA was eluted in a volume of 50 μL of Solution C6.2.4c oxidase subunit 1 (cox1), 18S rDNA hypervariable region I (HVR-I), and 18S rDNA hypervariable region IV (HVR-IV). The primer sequences used to amplify each locus are provided in S. stercoralis in the diagnostic parasitology reference laboratory at CDC.Three genotyping loci were amplified from each specimen by PCR; Cytochrome 2.5www.geneious.com). Briefly, this workflow performed read quality control and identification of the haplotype composition of each specimen at the three loci sequenced. This workflow is described in detail in the studies by The sequencing methods employed here were modified from those described by 2.6S. fuelleborni genotypes compiled as part of previous work (Strongyloides species identified in Bornean slow lorises (S. fuelleborni: one from a human and 23 from pig-tailed macaques (Macaca nemestrina) (S. fuelleborni cox1 sequences generated by Macaca mulatta), Japanese macaques (Macaca fuscata), and sequences from four primate species housed in zoological parks in Japan; siamang , red-shanked douc (Pygathrix nemaeus), Francois’ langur (Trachypithecus francoisi), and Bornean Orangutan (Pongo pygmaeus) . A detailed overview of all specimens within this dataset, including information on hosts, genotype, and GenBank accession numbers, is provided in File S1, Tab A.We used 128 ous work , plus 18 lorises . Our datestrina) . We alsoygmaeus) . To servenotypes , plus a 2.7Strongyloides sp. .To characterize the genotypes from our reference isolates (and from the St. Kitts vervets), all cox1, 18S HVR-I, and HVR-IV sequences were assigned a haplotype name following earlier haplotype naming conventions developed for ides sp. , with soides sp.  – see ht2.8Barratt's heuristic was used to compute pairwise genetic distances for phylogenetic tree construction . Barratt2.9https://github.com/Joel-Barratt/Eukaryotyping. Our final calibrated HDS was used as input for distance computation and subsequent tree construction. The resultant pairwise matrix was clustered via the ‘agnes’ R package using Ward's method  or prepared in-house for annotation of dendrograms. Maps were generated in R using ggplot. Images were rendered using the GNU Image manipulation program (https://www.gimp.org).Genetic distance computation was performed using Barratt's heuristic via the scripts and instructions available here: s method  to geners method  from the33.1Strongyloides sp. were observed microscopically in 29 of the 34 fecal specimens during the secondary microscopic screening performed at CDC  included hits to S. fuelleborni 18S sequences. Haplotypes XVI and XVII possessed a ‘T’ SNP at position 119, and a ‘C’ SNP at position 305 relative to the alignment in S. fuelleborni. The HVR-IV haplotypes L, T, M, O, R, and P detected in these samples are also characteristic of S. fuelleborni from African primates , and 5 or 6 HVR-IV haplotypes  were detected in all specimens  were generated from St. Kitts vervets and each was used to construct a single genotype, including the cox1 sequence and the conserved segments of HVR-I and HVR-IV.The pecimens . Therefopecimens . Additiopecimens . Subsequ3.3S. fuelleborni genotypes from St. Kitts vervets were of an African variety, forming a unique cluster within the larger monophyletic African clade. This African clade includes S. fuelleborni types causing infections in chimpanzees, gorillas, baboons, and humans (S. fuelleborni genotype sequenced previously from a human in Guinea-Bissau (S. fuelleborni types A through G. Cluster H (introduced here) includes S. fuelleborni from St. Kitts vervets and the type detected in a human in Guinea-Bissau.Two of the 287 genotypes failed to meet the minimum data requirements for distance computation (fewer than 14 cox1 segments) and were excluded, leaving 285 genotypes for phylogenetic analysis. A robust phylogenetic reconstruction was achieved by analyzing any HVR data available for these 285 types plus cox1 segments I1 through O1 , which constitutes an additional five cox1 segments beyond the 14 segments defined within the 217 bp amplicon . The pairwise distance matrix  generated from this HDS was clustered using Wards method to construct a hierarchical tree , and usid humans  and 2. Aa-Bissau  clusterea-Bissau . The Neia-Bissau  and 3 fo4S. fuelleborni from St. Kitts vervets and confirmed that they are genetically similar to a S. fuelleborni type found previously in a human from Guinea-Bissau   . TherefoS. fuelleborni populations display genetic population sub-structuring based largely on geography, with Asian and African varieties being monophyletic. Sub-structuring based on the distribution of specific hosts seems of lesser importance versus geographic origin, suggestive of allopatric divergence. Strongyloides fuelleborni includes a diverse array of geographically and genetically distinct subpopulations (A through H), lending further support to the hypothesis that S. fuelleborni is not a single species but rather exists as a species complex as previously proposed  that could be included in a clustering meta-analysis, despite that the combinations of loci sequenced varied between studies included in this meta-analysis  sequenced between studies, highlighting the utility and potential benefits of this method.The prior studies by S. fuelleborni from St. Kitts vervets sheds further light on the global population structure of the S. fuelleborni complex while also revealing preliminary evidence of zoonotic risk. Our phylogenetic reconstructions conformed with prescient knowledge of the global population structure of the S. fuelleborni complex. Though reconstruction of complete genotypes based on Illumina sequenced PCR products from fecal DNA was not possible, our data successfully confirmed the African origin of S. fuelleborni from St Kitts and was sufficient to identify S. fuelleborni type H as a previously unrecognized sub-population of the S. fuelleborni complex. Notably, a member of S. fuelleborni type H was observed previously in a human from Guinea-Bissau, highlighting the potential for zoonotic spillover of S. fuelleborni from St. Kitts vervets. Surveillance for human infections on St. Kitts and genotyping of isolates will help to further elucidate the role of vervets as potential reservoirs of human S. fuelleborni infection on the island. Overall, these data add to the growing understanding of S. fuelleborni as a species complex with more nuanced geographic and host relationships than what was assumed prior to the molecular era.Our genetic characterization of The findings and conclusions in this manuscript are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention/the Agency for Toxic Substances and Disease Registry.JB: Conception, study design, PCR, sequencing, data analysis, bioinformatics, data interpretation, manuscript editing. TR: Study design, PCR, sequencing, drafting of manuscript. JKK: Collection and screening of samples, review of manuscript SM: Conception and review of manuscript. SS: microscopy, manuscript editing. YQ: study coordination and funding, review of manuscript.The authors of this manuscript have no conflicts of interest to disclose.