How metal films de-wet substrates - identifying the kinetic pathways and energetic driving forces

We study how single-crystal chromium films of uniform thickness on W(110) substrates are converted to arrays of three-dimensional (3D) Cr islands during annealing. We use low-energy electron microscopy (LEEM) to directly observe a kinetic pathway that produces trenches that expose the wetting layer. Adjacent film steps move simultaneously uphill and downhill relative to the staircase of atomic steps on the substrate. This step motion thickens the film regions where steps advance. Where film steps retract, the film thins, eventually exposing the stable wetting layer. Since our analysis shows that thick Cr films have a lattice constant close to bulk Cr, we propose that surface and interface stress provide a possible driving force for the observed morphological instability. Atomistic simulations and analytic elastic models show that surface and interface stress can cause a dependence of film energy on thickness that leads to an instability to simultaneous thinning and thickening. We observe that de-wetting is also initiated at bunches of substrate steps in two other systems, Ag/W(110) and Ag/Ru(0001). We additionally describe how Cr films are converted into patterns of unidirectional stripes as the trenches that expose the wetting layer lengthen along the W[001] direction. Finally, we observe how 3D Cr islands form directly during film growth at elevated temperature. The Cr mesas (wedges) form as Cr film steps advance down the staircase of substrate steps, another example of the critical role that substrate steps play in 3D island formation

A novel deep targeted sequencing method for minimal residual disease monitoring in acute myeloid leukemia

A high proportion of patients with acute myeloid leukemia who achieve minimal residual disease negative status ultimately relapse because a fraction of pathological clones remains undetected by standard methods. We designed and validated a high-throughput sequencing method for minimal residual disease assessment of cell clonotypes with mutations of NPM1, IDH1/2 and/or FLT3-single nucleotide variants. For clinical validation, 106 follow-up samples from 63 patients in complete remission were studied by sequencing, evaluating the level of mutations detected at diagnosis. The predictive value of minimal residual disease status by sequencing, multiparameter flow cytometry, or quantitative polymerase chain reaction analysis was determined by survival analysis. The sequencing method achieved a sensitivity of 10-4 for single nucleotide variants and 10-5 for insertions/deletions and could be used in acute myeloid leukemia patients who carry any mutation (86% in our diagnostic data set). Sequencing-determined minimal residual disease positive status was associated with lower disease-free survival (hazard ratio 3.4, P=0.005) and lower overall survival (hazard ratio 4.2, P<0.001). Multivariate analysis showed that minimal residual disease positive status determined by sequencing was an independent factor associated with risk of death (hazard ratio 4.54, P=0.005) and the only independent factor conferring risk of relapse (hazard ratio 3.76, P=0.012). This sequencing-based method simplifies and standardizes minimal residual disease evaluation, with high applicability in acute myeloid leukemia. It is also an improvement upon flow cytometry- and quantitative polymerase chain reaction-based prediction of outcomes of patients with acute myeloid leukemia and could be incorporated in clinical settings and clinical trials.This study was supported by the Subdirección General de Investigación Sanitaria (Instituto de Salud Carlos III, Spain) grants PI13/02387 and PI16/01530, and the CRIS against Cancer foundation grant 2014/0120. ML holds a postdoctoral fellowship of the Spanish Ministry of Economy and Competitiveness (FPDI-2013- 16409). PRP holds a postdoctoral fellowship of the Spanish Instituto de Salud Carlos III: Contrato Predoctoral de Formación en Investigación en Salud i-PFIS (IFI 14/00008).S

Novel deep targeted sequencing method for minimal residual disease monitoring in acute myeloid leukemia

A high proportion of patients with acute myeloid leukemia who achieve minimal residual disease (MRD) negative status ultimately relapse because a fraction of pathological clones remains undetected by standard methods. We designed and validated a high-throughput sequencing method for MRD assessment of cell clonotypes with mutations of NPM1, IDH1/2 and/or FLT3-SNVs. For clinical validation, 106 follow-up samples from 63 patients in complete remission were studied by NGS, evaluating the level of mutations detected at diagnosis. The predictive value of MRD status by NGS, multiparameter flow cytometry, or quantitative PCR was determined by survival analysis. The method achieved a sensitivity of 10-4 for SNV mutations and 10-5 for insertions/deletions and could be used in acute myeloid leukemia patients who carry any mutation (86% in our diagnosis data set). NGS-determined MRD positive status was associated with lower disease-free survival (hazard ratio [HR] 3.4, p=0.005) and lower overall survival (HR 4.2, p<0.001). Multivariate analysis showed that MRD positive status by NGS was an independent factor associated with risk of death (HR 4.54, p =0.005) and the only independent factor conferring risk of relapse (HR 3.76, p =0.012). This NGS based method simplifies and standardizes MRD evaluation, with high applicability in acute myeloid leukemia. It also improves upon flow cytometry and quantitative PCR to predict acute myeloid leukemia outcome and could be incorporated in clinical settings and clinical trials.This study was supported by the Subdirección General de Investigación Sanitaria (Instituto de Salud Carlos III, Spain) grants PI13/02387 and PI16/01530, and the CRIS against Cancer foundation grant 2014/0120. M.L. holds a postdoctoral fellowship of the Spanish Ministry of Economy and Competitiveness (FPDI-2013-16409). P.R.P. holds a postdoctoral fellowship of the Spanish of Instituto de Salud Carlos III: Contrato Predoctoral de Formación en Investigación en Salud i-PFIS (IFI 14/00008).S

Management of adverse events in patients with acute myeloid leukemia in remission receiving oral azacitidine: experience from the phase 3 randomized QUAZAR AML-001 trial

Background Most older patients with acute myeloid leukemia (AML) who attain morphologic remission with intensive chemotherapy (IC) will eventually relapse and post-relapse prognosis is dismal. In the pivotal QUAZAR AML-001 trial, oral azacitidine maintenance therapy significantly prolonged overall survival by 9.9 months (P < 0.001) and relapse-free survival by 5.3 months (P < 0.001) compared with placebo in patients with AML in first remission after IC who were not candidates for transplant. Currently, the QUAZAR AML-001 trial provides the most comprehensive safety information associated with oral azacitidine maintenance therapy. Reviewed here are common adverse events (AEs) during oral azacitidine treatment in QUAZAR AML-001, and practical recommendations for AE management based on guidance from international cancer consortiums, regulatory authorities, and the authors’ clinical experience treating patients in the trial. Methods QUAZAR AML-001 is an international, placebo-controlled randomized phase 3 study. Patients aged ≥ 55 years with AML and intermediate- or poor-risk cytogenetics at diagnosis, who had attained first complete remission (CR) or CR with incomplete blood count recovery (CRi) within 4 months before study entry, were randomized 1:1 to receive oral azacitidine 300 mg or placebo once-daily for 14 days in repeated 28-day cycles. Safety was assessed in all patients who received ≥ 1 dose of study drug. Results A total of 469 patients received oral azacitidine (n = 236) or placebo (n = 233). Median age was 68 years. Patients received a median of 12 (range 1–80) oral azacitidine treatment cycles or 6 (1–73) placebo cycles. Gastrointestinal AEs were common and typically low-grade. The most frequent grade 3–4 AEs during oral azacitidine therapy were hematologic events. AEs infrequently required permanent discontinuation of oral azacitidine (13%), suggesting they were effectively managed with use of concomitant medications and oral azacitidine dosing modifications. Conclusion Oral azacitidine maintenance had a generally favorable safety profile. Prophylaxis with antiemetic agents, and blood count monitoring every other week, are recommended for at least the first 2 oral azacitidine treatment cycles, and as needed thereafter. Awareness of the type, onset, and duration of common AEs, and implementation of effective AE management, may maximize treatment adherence and optimize the survival benefits of oral azacitidine AML remission maintenance therapy. Trial registration. This trial is registered on clinicaltrials.gov: NCT01757535 as of December 2012

Redefining ‘Careers’ and ‘Sustainable Careers’: A Qualitative Study with University Students

Contemporary society challenges traditional linear career progressions with the emergence of the ‘sustainable career’ concept. This dynamic career path spans different societal domains over time and involves individuals actively shaping their paths through actions and the interpretation of their experiences. The evolving socioeconomic contexts demand a reevaluation of career development processes, necessitating an examination of individual perspectives on what makes a career authentically sustainable. Despite this, in the Italian and Spanish contexts, a definition of the concept of career and sustainable careers rooted in individuals’ interpretations is still absent. This qualitative study is designed to fill the existing gap by exploring the definition of the concepts of ‘career’ and ‘sustainable career’ in an initial sample of Italian (N = 197) and Spanish (N = 193) students (323 females, 67 males) aged 18–30 years (M = 20.13; SD = 2.13). Participants answered open-ended questions about ‘career’ and ‘sustainable career’. Qualitative data analysis software identified key themes, and correspondence analysis explored differences between the Italian and Spanish samples. The findings reveal that the concept of a career encompasses an evolving sequence of work experiences, incorporating training pathways, a continuous commitment to personal growth goals, and professional satisfaction. A sustainable career extends this, emphasizing a healthy work–life balance and the often-overlooked dimension of social empowerment. This study offers a perspective for designing research and interventions aimed at promoting careers and work environments perceived as authentically sustainable

Selective T cell subset depletion with anti-CD4 and anti-CD8 intact ricin immunotoxins

In the present study we have comparatively analysed the specific activity of a panel of immunotoxins (ITs) prepared by coupling Ricin to several monoclonal antibodies (MoAbs) directed against the CD3, CD4 and CD8 T cell membrane molecules. Peripheral blood and bone marrow mononuclear cells (PBMC and BMMC) were incubated with the different ITs for 2 h, in the presence of 0.1 M lactose, washed and subsequently stimulated with either phytohemagglutinin (PHA) or an anti-CD3 MoAb. Our results indicate that the proliferative response of PBMC to both stimuli was specifically inhibited (>95%) by either anti-CD3 IT or a combination of anti-CD4 and CD8 ITs, at concentrations comparable to those previously used for ex vivo T cell depletion (300 ng/ml). When used individually at the same dose, anti-CD8 and anti-CD4 ITs inhibited the PHA-induced PBMC proliferative response 40 and 70% respectively. When either anti-CD4 or anti-CD8 IT-treated cells were activated with PHA and cultured for 14 days in the presence of IL-2, less than 2% of the blasts expressed the corresponding antigens as assessed by flow cytometry analysis. Similar results were observed when BMMC were treated with the different ITs. In contrast, the growth of CFU-GM was minimally affected (0-25% inhibition). Our data indicate that ITs directed against T cell subsets are highly active and specific reagents that may be potentially useful for pre-transplant bone marrow purging.This work was supported by grants from INSALUD (FISS 87/1507). We are grateful to Fundación Valgrande for its cooperation.Peer Reviewe

High toxic efficency of ricin immunotoxins specific for the T cell antigen receptor of an human leukemia T cell line

Immunotoxins (ITs) were prepared by covalently coupling ricin to monoclonal antibodies (MAbs) directed against: (a) 2 different epitopes of the T-cell receptor (TcR) expressed by the Jurkat leukemia T-cell line (JTi2 and JTi4 MAb), (b) 2 epitopes of the CD3 complex (SpV-T3b and IID8 MAb), (c) the CD2 and the CD8 cell-surface molecules. Conjugates were assayed for their cytotoxic activity by pre-incubating the Jurkat cell line with different concentrations (10-250 ng/ml) of each IT for 2 hr at 37°C in the presence of 0.1 M lactose. After washing, cells were cultured for 24 hr and their protein synthesis and proliferative capacities were assessed. Doseresponse experiments indicated that JTi2, JTi4 and anti-CD3 (IID8) ITs inhibited by >90% the cell line proliferation at 50 ng/ml, a 5-fold lower concentration than that required to achieve a similar effect when anti-CD2 and anti-CD3 (SpVT3b) were used. After 4 hr of culture subsequent to treatment with JTi2 or JTi4 ITs (250 ng/ml), protein synthesis was inhibited (>80%). By limiting dilution analysis (LDA) we estimated that the frequency of proliferating Jurkat cells (1/1.5) was reduced to 1/20, 1/460 and 1/300 after treatment with anti-CD3 (SpVT3b), JTi4 and JTi2 ITs, respectively. Phenotypic analysis of 13 clones derived from JTi2 IT-treated Jurkat cells showed that 50% were CD7+ CD3− JTi− variants. When bone-marrow mononuclear cells, previously mixed with low concentrations of Jurkat cells, were treated with anti-JTi ITs, the toxic efficiency estimated by LDA was maintained whereas the growth of CFU-GM remained unaltered.INSALUD. Grant Number: FISS 87/1507.Peer reviewe

Defective interleukin 2 receptor expression is associated with the T cell disfunction subsequent to bone marrow transplantation

In the present work we have used monoclonal antibodies (mAb) as probes to attempt a dissection of the mechanisms underlying the immunodeficiency subsequent to bone marrow transplantation (BMT). To this end we have studied 19 allogeneic BMT recipients, analyzing the proliferative response of peripheral blood mononuclear cells (PBMC) after activation with either phytohemagglutinin (PHA), anti-CD3 or anti-CD 2 mAb. All patients presented normal proportions of CD2+ and CD3+ lymphocytes, as assessed by flow cytometry. Our results indicated that in most cases both CD 2 and CD 3-mediated activation pathways were inefficient to trigger normal T cell proliferation. The addition of exogenous interleukin 2 (IL 2) did not restore in most cases the proliferative response, pointing out that additional defects contribute to the hyporesponsiveness. This was more evident in the group of patients studied during the first 6 months. To further dissect the T cell defect we analyzed the effect of a phorbol ester (phorbol myristate acetate, PMA), which activates protein kinase C, on the anti-CD 3-induced response. Our data showed that PMA synergized with anti-CD 3 similarly to exogenous IL 2, and restored the proliferative response only in certain cases. The expression of IL 2 receptors (CD 25) as assessed by cytofluorimetry, after either PHA or anti-CD 3 and PMA stimulation, was shown to be depressed, and the addition of IL 2 did not restore it. Finally, we observed that the early increase of intracytoplasmic Ca2+ after anti-CD 3 stimulation was comparable to that detected in normal PBMC. Altogether these results indicate that a diminished CD 25 expression is associated with the T cell defect, and cannot apparently be attributed to an inability of the CD 3 molecule to transduce early activation signals thus suggesting that either protein kinase C itself or an as yet undefined metabolic step preceding IL 2 receptor expression is abnormal in variable proportions of T cells after BMT, and constitutes another manifestation of this complex immunodeficiency.This work was supported by grants from INSALUD (FIS 86/825) and CAICYT (0456-84):Peer reviewe

Relation between the increase of circulating CD3+CD57+ lymphocytes and T cell dysfunction in recipients of bone marrow transplantation

Some patients undergoing bone marrow transplantation (BMT) persistently present increased proportions of circulating CD57+ T cells. We analysed the cell surface phenotype in peripheral blood mononuclear cells (PBMC) from 69 allogeneic and 11 autologous BMT recipients. In parallel samples from 49 patients, the proliferative response to T cell mitogens was assessed, either in the presence or absence of exogenous interleukin-2 (IL-2). PBMC samples from long-term allogeneic BMT patients with increased proportions of CD57+ cells displayed significantly (P < 0.001) lower proliferative responses, compared with samples from patients with normal proportions of CD57+ cells and from healthy subjects. Elimination of the CD57+ population by C'-dependent lysis did not normalize the proliferative response. After positive selection by cell sorting, CD57+ cells responded poorly, but in the presence of IL-2 the proliferation appeared to be similar to that displayed by the CD57- subset and still suboptimal compared with normal controls. These data suggest that the hyporesponsiveness to mitogenic stimuli in the presence of exogenous IL-2 of PBMC from allogeneic BMT recipients cannot be simply attributed either to the putative suppressor activity of CD57+ cells, or to a poor proliferative capacity of this subset. Supporting this notion we report that PBMC from long-term autologous BMT recipients containing high proportions of CD57+ T cells respond normally to T cell mitogens.This work was supported by grants from Insalud (FISS 88/1737).Peer Reviewe

A novel deep targeted sequencing method for minimal residual disease monitoring in acute myeloid leukemia

A high proportion of patients with acute myeloid leukemia who achieve minimal residual disease negative status ultimately relapse because a fraction of pathological clones remains undetected by standard methods. We designed and validated a high-throughput sequencing method for minimal residual disease assessment of cell clonotypes with mutations of NPM1, IDH1/2 and/or FLT3-single nucleotide variants. For clinical validation, 106 follow-up samples from 63 patients in complete remission were studied by sequencing, evaluating the level of mutations detected at diagnosis. The predictive value of minimal residual disease status by sequencing, multiparameter flow cytometry, or quantitative polymerase chain reaction analysis was determined by survival analysis. The sequencing method achieved a sensitivity of 10 for single nucleotide variants and 10 for insertions/deletions and could be used in acute myeloid leukemia patients who carry any mutation (86% in our diagnostic data set). Sequencing-determined minimal residual disease positive status was associated with lower disease-free survival (hazard ratio 3.4, P=0.005) and lower overall survival (hazard ratio 4.2, P<0.001). Multivariate analysis showed that minimal residual disease positive status determined by sequencing was an independent factor associated with risk of death (hazard ratio 4.54, P=0.005) and the only independent factor conferring risk of relapse (hazard ratio 3.76, P=0.012). This sequencing-based method simplifies and standardizes minimal residual disease evaluation, with high applicability in acute myeloid leukemia. It is also an improvement upon flow cytometry- and quantitative polymerase chain reaction-based prediction of outcomes of patients with acute myeloid leukemia and could be incorporated in clinical settings and clinical trials