Treatment Outcomes of Adolescents Compared to Younger Pediatric Patients with Acute Myeloid Leukemia: Do They Need a Special Approach?

BACKGROUND: The reports of studies that compare the survival of adolescents and young adults with younger children with acute myeloid leukemia (AML) are contradictory. PATIENTS AND METHODS: We retrospectively analyzed 220 AML patients aged 0-18 years treated in pediatric oncologic centers in Poland from 2015 to 2022. The evaluated group included 31 infants (below 1 year), 91 younger children (1-9.9 years), 59 older children (10-14.9 years), and 39 adolescents (15-18 years). RESULTS: A 5-year overall survival for adolescents was not significantly inferior compared to younger and older children (74.3 ± 7.6% vs. 80.5 ± 4.4% vs. 77.9 ± 5.1, p = 0.243). However, relapse-free survival was lower in adolescents compared to younger children (76.5 ± 7.8% vs. 65.7 ± 9.0%, p = 0.049), and treatment-related mortality tended to be higher (10.3% vs. 4.4%, p = 0.569). In the univariate analysis, high-risk genetics [HR, 2.0 (95% CI 1.1-3.6; p = 0.014)] and a leukocyte count at diagnosis above 100,000/µL [HR, 2.4 (95% CI 1.3-4.6; p = 0.004)] were found to be unfavorable prognostic factors for survival. CONCLUSIONS: Although we have not found that age over 15 years is an unfavorable factor for overall survival, the optimal approach to therapy in adolescents, as in other age groups, is to adjust the intensity of therapy to individual genetic risk and introduce targeted therapies when indicated.

Characteristics and Outcome of FLT3-ITD-Positive Pediatric Acute Myeloid Leukemia-Experience of Polish Pediatric Leukemia and Lymphoma Study Group from 2005 to 2022.

BACKGROUND: The FMS-like tyrosine kinase 3 (FLT3) gene mutated in 10-15% of pediatric acute myeloid leukemia (AML) is associated with an inferior outcome. The aim of the study was to analyze the outcome and characteristics of FLT3-ITD-positive pediatric AML. METHODS: We retrospectively analyzed the nationwide pediatric AML database from between 2005 and 2022. FLT3-ITD was found in 54/497 (10.7%) patients with available analysis. Three consecutive treatment protocols were used (AML-BFM 2004 Interim, AML-BFM 2012 Registry, AML-BFM 2019 recommendations). RESULTS: Probabilities of 5-year overall (OS), event-free (EFS) and relapse-free survival were significantly lower in the FLT3-ITD-positive patients compared to FLT3-ITD-negative (0.54 vs. 0.71, p = 0.041; 0.36 vs. 0.59, p = 0.0004; 0.47 vs. 0.70, p = 0.0029, accordingly). An improvement in the outcome was found in the analyzed period of time, with a trend of better survival in patients treated under the AML-BFM 2012 and AML-BFM 2019 protocols compared to the AML-BFM 2004 protocol (5-year EFS 0.52 vs. 0.27, p = 0.069). There was a trend of improved outcomes in patients treated with FLT3 inhibitors (n = 9, 2-year EFS 0.67 vs. 0.33, p = 0.053) and those who received stem cell transplantation (SCT) (n = 26; 5-year EFS 0.70 vs. 0.27, p = 0.059). The co-occurrence of the WT1 mutation had a dismal impact on the prognosis (5-year EFS 0.23 vs. 0.69, p = 0.002), while the NPM1 mutation improved survival (5-year OS 1.0 vs. 0.44, p = 0.036). CONCLUSIONS: It seems that SCT and FLT3 inhibitors have a beneficial impact on the prognosis. Additional genetic alterations, like the WT1 and NPM1 mutations, significantly influence the outcome.

Pediatric Acute Myeloid Leukemia Post Cytotoxic Therapy-Retrospective Analysis of the Patients Treated in Poland from 2005 to 2022.

Acute P./myeloid leukemia post cytotoxic therapy (AML-pCT) is rare complication of cancer treatment in childhood. The objective of the study was to identify clinical characteristics and provide an analysis of the outcomes in pediatric AML-pCT. We retrospectively analyzed the data of 40 children with AML-pCT, treated from 2005 to 2020 within the Polish Pediatric Leukemia and Lymphoma Study Group. The most common primary malignancies were acute lymphoblastic leukemia (32.5%) and brain tumors (20%). The median latency period was 2.9 years (range: 0.7-12.9). Probabilities of overall (OS), event-free (EFS), and relapse-free survival (RFS) in the whole cohort were 0.49 ± 0.08, 0.43 ± 0.08, and 0.64 ± 0.10, respectively. Significant improvements in outcomes were observed in patients treated from 2015-2022 (two induction cycles followed by stem cell transplantation-SCT in 69% of patients) compared to 2005-2014 (four induction cycles followed by SCT in 49% of patients). The probability of EFS increased from 0.30 ± 0.10 to 0.67 ± 0.12 (p = 0.07) and RFS increased from 0.46 ± 0.11 to 1.0 (p = 0.01). The poorest outcome (OS and EFS 0.25 ± 0.20) was in AML post brain tumor, mainly due to deaths from toxicities. To conclude, treatment results achieved in patients with AML-pCT treated from 2015-2022, with two induction cycles followed by immediate SCT, were better than those reported by other authors, and comparable to the results in de novo AML.

Slow-cycling murine melanoma cells display plasticity and enhanced tumorigenicity in syngeneic transplantation assay.

Slow-cycling cancer cells (SCC) contribute to the aggressiveness of many cancers, and their invasiveness and chemoresistance pose a great therapeutic challenge. However, in melanoma, their tumor-initiating abilities are not fully understood. In this study, we used the syngeneic transplantation assay to investigate the tumor-initiating properties of melanoma SCC in the physiologically relevant in vivo settings. For this we used B16-F10 murine melanoma cell line where we identified a small fraction of SCC. We found that, unlike human melanoma, the murine melanoma SCC were not marked by the high expression of the epigenetic enzyme Jarid1b. At the same time, their slow-cycling phenotype was a temporary state, similar to what was described in human melanoma. Progeny of SCC had slightly increased doxorubicin resistance and altered expression of melanogenesis genes, independent of the expression of cancer stem cell markers. Single-cell expansion of SCC revealed delayed growth and reduced clone formation when compared to non-SCC, which was further confirmed by an in vitro limiting dilution assay. Finally, syngeneic transplantation of 10 cells in vivo established that SCC were able to initiate growth in primary recipients and continue growth in the serial transplantation assay, suggesting their self-renewal nature. Together, our study highlights the high plasticity and tumorigenicity of murine melanoma SCC and suggests their role in melanoma aggressiveness.

Gemtuzumab ozogamicin for relapsed or primary refractory acute myeloid leukemia in children-the Polish Pediatric Leukemia and Lymphoma Study Group experience.

Background: Gemtuzumab ozogamicin (GO), one of the first targeted drugs used in oncology, consists of an anti-cluster of differentiation 33 (CD33) monoclonal antibody bound to a derivative of cytotoxic calicheamicin. After the drug withdrawn in 2010 due to a significantly higher rate of early deaths, GO regained approval in 2017 for the treatment of newly diagnosed, refractory, or relapsed acute myeloid leukemia (AML) in adults and children over 15 years of age. The objective of the study was a retrospective analysis of clinical characteristics, treatment outcomes, and GO toxicity profile in children with primary refractory or relapsed (R/R) AML treated in Poland from 2008 to 2022. Methods: Data were collected through the Polish Registry of Acute Myeloid Leukemia. From January 2008 to December 2022, 35 children with R/R AML were treated with GO in seven centers of the Polish Pediatric Leukemia and Lymphoma Study Group. Results: Most of the children (30 of 35) received only one GO cycle in combination with various chemotherapy cycles (IDA-FLA, DOXO-FLA, FLA, FLAG, and others). Eighteen children (51%) achieved complete remission (CR), 14 did not respond to treatment, and three progressed. GO therapy was followed by allogeneic hematopoietic stem cell transplantation (allo-HSCT) in 18 children in CR. The 5-year overall survival (OS) after GO therapy was 37.1% ± 8.7% for the total cohort. There was a trend toward a superior outcome in patients with strong expression of CD33 expression (over 50% positive cells) compared with that in patients with lower expression of CD33 (OS, 41.2% ± 11.9% versus 27.8% ± 13.2%; p = 0.5; 5-year event-free survival, 35.4% ± 11.6% versus 25.7% ± 12.3%; p = 0.5, respectively). Children under 15 years have better outcome (OS, 34.9% ± 10.4% versus 30% ± 14.5%, p = 0.3). The most common adverse events were bone marrow aplasia, fever of unknown origin, infections, and elevated liver enzyme elevation. Sinusoidal obstruction syndrome occurred in two children. Conclusions: The use of GO in severely pretreated children, including those under 15 years of age, with previous failure of AML treatment is a feasible and effective bridging therapy to allo-HSCT with an acceptable toxicity profile.

Go with the Flow-Early Assessment of Measurable Residual Disease in Children with Acute Lymphoblastic Leukemia Treated According to ALL IC-BFM2009.

Measurable residual disease (MRD) is a well-known tool for the evaluation of the early response to treatment in patients with acute lymphoblastic leukemia (ALL). In respect to predicting the relapse the most informative cut-off and time point of MRD measurement during therapy were evaluated in our study. Between 1 January 2013 and 31 December 2019, multiparametric flow cytometry (MFC) MRD was measured in the bone marrow of 140 children with ALL treated according to the ALL IC-BFM2009 protocol. The MRD cut-off of 0.1% and day 33, end of induction, were the most discriminatory for all patients. Patients with negative MRD on day 15 and 33 had a higher 5-year overall survival-OS (100%) and a higher relapse-free survival-RFS rate (97.6%) than those with positive levels of MRD (≥0.01%) at both time points (77.8% and 55.6%, p = 0.002 and 0.001, respectively). Most patients with residual disease below 0.1% on day 15 exhibit hyperdiploidy or ETV6-RUNX1 in ALL cells. Measurement of MRD at early time points can be used with simplified genetic analysis to better identify low and high-risk patients, allowing personalized therapies and further improvement in outcomes in pediatric ALL.

Heme Oxygenase-1 Has a Greater Effect on Melanoma Stem Cell Properties Than the Expression of Melanoma-Initiating Cell Markers.

Melanoma-initiating cells (MICs) contribute to the tumorigenicity and heterogeneity of melanoma. MICs are identified by surface and functional markers and have been shown to display cancer stem cell (CSC) properties. However, the existence of MICs that follow the hierarchical CSC model has been questioned by studies showing that single unselected melanoma cells are highly tumorigenic in xenotransplantation assays. Herein, we characterize cells expressing MIC markers (CD20, CD24, CD133, Sca-1, ABCB1, ABCB5, ALDHhigh) in the B16-F10 murine melanoma cell line. We use flow cytometric phenotyping, single-cell sorting followed by in vitro clonogenic assays, and syngeneic in vivo serial transplantation assays to demonstrate that the expression of MIC markers does not select CSC-like cells in this cell line. Previously, our group showed that heme-degrading enzyme heme oxygenase-1 (HO-1) can be upregulated in melanoma and increase its aggressiveness. Here, we show that HO-1 activity is important for non-adherent growth of melanoma and HO-1 overexpression enhances the vasculogenic mimicry potential, which can be considered protumorigenic activity. However, HO-1 overexpression decreases clone formation in vitro and serial tumor initiation in vivo. Thus, HO-1 plays a dual role in melanoma, improving the progression of growing tumors but reducing the risk of melanoma initiation.

The Assessment of Endovascular Therapies in Ischemic Stroke: Management, Problems and Future Approaches.

Ischemic stroke accounts for over 80% of all strokes and is one of the leading causes of mortality and permanent disability worldwide. Intravenous administration of recombinant tissue plasminogen activator (rt-PA) is an approved treatment strategy for acute ischemic stroke of large arteries within 4.5 h of onset, and mechanical thrombectomy can be used for large arteries occlusion up to 24 h after onset. Improving diagnostic work up for acute treatment, reducing onset-to-needle time and urgent radiological access angiographic CT images (angioCT) and Magnetic Resonance Imaging (MRI) are real problems for many healthcare systems, which limits the number of patients with good prognosis in real world compared to the results of randomized controlled trials. The applied endovascular procedures demonstrated high efficacy, but some cellular mechanisms, following reperfusion, are still unknown. Changes in the morphology and function of mitochondria associated with reperfusion and ischemia-reperfusion neuronal death are still understudied research fields. Moreover, future research is needed to elucidate the relationship between continuously refined imaging techniques and the variable structure or physical properties of the clot along with vascular permeability and the pleiotropism of ischemic reperfusion lesions in the penumbra, in order to define targeted preventive procedures promoting long-term health benefits.

Blinatumomab as a Bridge Therapy for Hematopoietic Stem Cell Transplantation in Pediatric Refractory/Relapsed Acute Lymphoblastic Leukemia.

Despite the progress that has been made in recent decades in the treatment of pediatric acute leukemias, e.g., converting acute lymphoblastic leukemia (ALL) from a fatal to a highly curable disease, 15-20% of children still relapse. Blinatumomab, a bispecific CD3/CD19 antibody construct, has been successfully used in relapsed/refractory r/r B-cell precursor ALL (BCP-ALL) as a bridge to hematopoietic stem cell transplantation (HSCT). We retrospectively assessed the efficacy and toxicity of blinatumomab in 13 children with r/r BCP-ALL. Between 2017 and 2021, thirteen children, aged 1-18 years, with r/r BCP-ALL were treated with blinatumomab. Two patients were administered blinatumomab for refractory relapse without complete remission (CR), one due to primary refractory disease, and ten patients were in CR with minimal residual disease (MRD) ≥ 10-3. The response rate in our cohort of patients was 85%, with subsequent feasible HSCT in 11 out of 13 children. Ten children reached MRD negativity after the first blinatumomab administration. The three-year OS for the study patients was 85% (Mantel-Cox, p < 0.001) and median follow-up was 24.5 (range: 1-47). All responders proceeded to HSCT and are alive in CR, and MRD negative. Although our study had some limitations with regard to its retrospective design and limited patient population, it clearly showed blinatumomab as not only a feasible but also an effective therapeutic option in pretreated children with r/r BCP-ALL, with a tolerable toxicity profile, paving the way for an HSCT procedure.

Flipper Probes for the Community.

This article describes four fluorescent membrane tension probes that have been designed, synthesized, evaluated, commercialized and applied to current biology challenges in the context of the NCCR Chemical Biology. Their names are Flipper-TR®, ER Flipper-TR®, Lyso Flipper-TR®, and Mito Flipper-TR®. They are available from Spirochrome.

An Extensive Quality Control and Quality Assurance (QC/QA) Program Significantly Improves Inter-Laboratory Concordance Rates of Flow-Cytometric Minimal Residual Disease Assessment in Acute Lymphoblastic Leukemia: An I-BFM-FLOW-Network Report.

Monitoring of minimal residual disease (MRD) by flow cytometry (FCM) is a powerful prognostic tool for predicting outcomes in acute lymphoblastic leukemia (ALL). To apply FCM-MRD in large, collaborative trials, dedicated laboratory staff must be educated to concordantly high levels of expertise and their performance quality should be continuously monitored. We sought to install a unique and comprehensive training and quality control (QC) program involving a large number of reference laboratories within the international Berlin-Frankfurt-Münster (I-BFM) consortium, in order to complement the standardization of the methodology with an educational component and persistent quality control measures. Our QC and quality assurance (QA) program is based on four major cornerstones: (i) a twinning maturation program, (ii) obligatory participation in external QA programs (spiked sample send around, United Kingdom National External Quality Assessment Service (UK NEQAS)), (iii) regular participation in list-mode-data (LMD) file ring trials (FCM data file send arounds), and (iv) surveys of independent data derived from trial results. We demonstrate that the training of laboratories using experienced twinning partners, along with continuous educational feedback significantly improves the performance of laboratories in detecting and quantifying MRD in pediatric ALL patients. Overall, our extensive education and quality control program improved inter-laboratory concordance rates of FCM-MRD assessments and ultimately led to a very high conformity of risk estimates in independent patient cohorts.

Advances in the First Line Treatment of Pediatric Acute Myeloid Leukemia in the Polish Pediatric Leukemia and Lymphoma Study Group from 1983 to 2019.

BACKGROUND: From 1983, standardized therapeutic protocols for pediatric acute myeloid leukemia (AML) based on the BFM group experience were introduced in Poland. We retrospectively analyzed the results of pediatric AML treatment in Poland from 1983 to 2019 (excluding promyelocytic, therapy-related, biphenotypic, and Down syndrome AML). METHODS: The study included 899 children suffering from AML treated with the following: AML-PPPLBC 83 (1983-1993, n = 187), AML-PPGLBC 94 (1994-1997, n = 74), AML-PPGLBC 98 (1998-2004, n = 151), AML-BFM 2004 Interim (2004-2015, n = 356), and AML-BFM 2012 (2015-2019, n = 131). RESULTS: The probability of three-year overall survival was 0.34 ± 0.03, 0.37 ± 0.05, 0.54 ± 0.04, 0.67 ± 0.03, and 0.75 ± 0.05; event-free survival was 0.31 ± 0.03, 0.34 ± 0.05, 0.44 ± 0.04, 0.53 ± 0.03, and 0.67 ± 0.05; and relapse-free survival was 0.52 ± 0.03, 0.65 ± 0.05, 0.58 ± 0.04, 0.66 ± 0.03, and 0.78 ± 0.05, respectively, in the subsequent periods. A systematic reduction of early deaths and deaths in remission was achieved, while the percentage of relapses decreased only in the last therapeutic period. Surprisingly good results were obtained in the group of patients treated with AML-BFM 2012 with unfavorable genetic abnormalities like KMT2A-MLLT10/t(10;11)(p12;q23) and DEK-NUP214/t(6;9)(p23;q24), while unsatisfactory outcomes were found in the patients with FLT3-ITD. CONCLUSIONS: The use of standardized, systematically modified therapeutic protocols, with the successive consideration of genetic prognostic factors, and advances in supportive care led to a significant improvement in AML treatment outcomes over the last 40 years.

Genetically Encoded Supramolecular Targeting of Fluorescent Membrane Tension Probes within Live Cells: Precisely Localized Controlled Release by External Chemical Stimulation.

To image membrane tension in selected membranes of interest (MOI) inside living systems, the field of mechanobiology requires increasingly elaborated small-molecule chemical tools. We have recently introduced HaloFlipper, i.e., a mechanosensitive flipper probe that can localize in the MOI using HaloTag technology to report local membrane tension changes using fluorescence lifetime imaging microscopy. However, the linker tethering the probe to HaloTag hampers the lateral diffusion of the probe in all the lipid domains of the MOI. For a more global membrane tension measurement in any MOI, we present here a supramolecular chemistry strategy for selective localization and controlled release of flipper into the MOI, using a genetically encoded supramolecular tag. SupraFlippers, functionalized with a desthiobiotin ligand, can selectively accumulate in the organelle having expressed streptavidin. The addition of biotin as a biocompatible external stimulus with a higher affinity for Sav triggers the release of the probe, which spontaneously partitions into the MOI. Freed in the lumen of endoplasmic reticulum (ER), SupraFlippers report the membrane orders along the secretory pathway from the ER over the Golgi apparatus to the plasma membrane. Kinetics of the process are governed by both the probe release and the transport through lipid domains. The concentration of biotin can control the former, while the expression level of a transmembrane protein (Sec12) involved in the stimulation of the vesicular transport from ER to Golgi influences the latter. Finally, the generation of a cell-penetrating and fully functional Sav-flipper complex using cyclic oligochalcogenide (COC) transporters allows us to combine the SupraFlipper strategy and HaloTag technology.

Immune cells lacking Y chromosome show dysregulation of autosomal gene expression.

Epidemiological investigations show that mosaic loss of chromosome Y (LOY) in leukocytes is associated with earlier mortality and morbidity from many diseases in men. LOY is the most common acquired mutation and is associated with aberrant clonal expansion of cells, yet it remains unclear whether this mosaicism exerts a direct physiological effect. We studied DNA and RNA from leukocytes in sorted- and single-cells in vivo and in vitro. DNA analyses of sorted cells showed that men diagnosed with Alzheimer's disease was primarily affected with LOY in NK cells whereas prostate cancer patients more frequently displayed LOY in CD4 + T cells and granulocytes. Moreover, bulk and single-cell RNA sequencing in leukocytes allowed scoring of LOY from mRNA data and confirmed considerable variation in the rate of LOY across individuals and cell types. LOY-associated transcriptional effect (LATE) was observed in ~ 500 autosomal genes showing dysregulation in leukocytes with LOY. The fraction of LATE genes within specific cell types was substantially larger than the fraction of LATE genes shared between different subsets of leukocytes, suggesting that LOY might have pleiotropic effects. LATE genes are involved in immune functions but also encode proteins with roles in other diverse biological processes. Our findings highlight a surprisingly broad role for chromosome Y, challenging the view of it as a "genetic wasteland", and support the hypothesis that altered immune function in leukocytes could be a mechanism linking LOY to increased risk for disease.

Role of HMOX1 Promoter Genetic Variants in Chemoresistance and Chemotherapy Induced Neutropenia in Children with Acute Lymphoblastic Leukemia.

Whilst the survival rates of childhood acute lymphoblastic leukemia (ALL) have increased remarkably over the last decades, the therapy resistance and toxicity are still the major causes of treatment failure. It was shown that overexpression of heme oxygenase-1 (HO-1) promotes proliferation and chemoresistance of cancer cells. In humans, the HO-1 gene (HMOX1) expression is modulated by two polymorphisms in the promoter region: (GT)n-length polymorphism and single-nucleotide polymorphism (SNP) A(-413)T, with short GT repeat sequences and 413-A variants linked to an increased HO-1 inducibility. We found that the short alleles are significantly more frequent in ALL patients in comparison to the control group, and that their presence may be associated with a higher risk of treatment failure, reflecting the role of HO-1 in chemoresistance. We also observed that the presence of short alleles may predispose to develop chemotherapy-induced neutropenia. In case of SNP, the 413-T variant co-segregated with short or long alleles, while 413-A almost selectively co-segregated with long alleles, hence it is not possible to determine if SNPs are actually of phenotypic significance. Our results suggest that HO-1 can be a potential target to overcome the treatment failure in ALL patients.

Gene polymorphisms influencing yield, composition and technological properties of milk from Czech Simmental and Holstein cows.

OBJECTIVE: The aim of the study was to evaluate the influence of polymorphic loci and other factors on milk performance and the technological properties of milk. METHODS: The analysis was performed on Simmental and Holstein cows in field conditions (n = 748). Milk yield in kg, fat and protein percentage and yield were evaluated. Technological properties were evaluated by milk fermentation ability, renneting, and an alcohol test. Polymorphisms in the acyl-CoA diacylgycerol transferase 1 (DGAT1), leptin (LEP), fatty acid synthase (FASN), stearoyl CoA desaturase 1 (SCD1), casein beta (CSN2), casein kappa (CSN3), and lactoglobulin beta genes were genotyped, and association analysis was performed. RESULTS: The DGAT1 AA genotype was associated with higher milk, protein and fat yields (p<0.05). The MM genotype in the LEP gene was associated with a lower protein percentage and the W allele with a higher protein percentage (p<0.05). In cows with the FASN GG genotype, the protein percentage was higher, but the A allele was associated with higher milk, protein and fat yields than the G allele. The TT genotype in SCD1 was associated with the lowest milk, protein and fat yields and with the highest milk protein percentage (p<0.01). The T allele had higher values than the C allele (p<0.05) except for fat percentage. The genotype CSN3 AA was associated with a significantly heightened milk yield; BB was associated with a high protein percentage. The effect of the alleles on the technological properties was not significant. The CSN3 BB genotype was associated with the best alcohol test (p<0.01), and the renneting order was inverse. Milk from cows with the CSN2 A1A1 genotype was best in the milk fermentation ability. CSN3 significantly affected the technological properties. CONCLUSION: The findings revealed the potential of some polymorphic loci for use in dairy cattle breeding and for the management of milk quality. In field research, the pivotal role of farms in milk yield, composition and technological properties was confirmed.

Twisting and tilting of a mechanosensitive molecular probe detects order in membranes.

Lateral forces in biological membranes affect a variety of dynamic cellular processes. Recent synthetic efforts have introduced fluorescent "flippers" as environment-sensitive planarizable push-pull probes that can detect lipid packing and membrane tension, and respond to lipid-induced mechanical forces by a shift in their spectroscopic properties. Herein, we investigate the molecular origin of the mechanosensitivity of the best known flipper, Flipper-TR, by an extended set of molecular dynamics (MD) simulations in membranes of increasing complexity and under different physicochemical conditions, revealing unprecedented details of the sensing process. Simulations enabled by accurate refinement of Flipper-TR force field using quantum mechanical calculations allowed us to unambiguously correlate the planarization of the two fluorescent flippers to spectroscopic response. In particular, Flipper-TR conformation exhibits bimodal distribution in disordered membranes and a unimodal distribution in highly ordered membranes. Such dramatic change was associated with a shift in Flipper-TR excitation spectra, as supported both by our simulated and experimentally-measured spectra. Flipper-TR sensitivity to phase-transition is confirmed by a temperature-jump protocol that alters the lipid phase of an ordered membrane, triggering an instantaneous mechanical twisting of the probe. Simulations show that the probe is also sensitive to surface tension, since even in a naturally disordered membrane, the unimodal distribution of coplanar flippers can be achieved if a sufficiently negative surface tension is applied to the membrane. MD simulations in ternary mixtures containing raft-like nanodomains show that the probe can discriminate lipid domains in phase-separated complex bilayers. A histogram-based approach, called DOB-phase classification, is introduced that can differentiate regions of disordered and ordered lipid phases by comparing dihedral distributions of Flipper-TR. Moreover, a new sensing mechanism involving the orientation of Flipper-TR is elucidated, corroborating experimental evidence that the probe tilt angle is strongly dependent on lipid ordering. The obtained atomic-resolution description of Flipper-TR mechanosensitivity is key to the interpretation of experimental data and to the design of novel mechanosensors with improved spectroscopic properties.

HaloFlippers: A General Tool for the Fluorescence Imaging of Precisely Localized Membrane Tension Changes in Living Cells.

Tools to image membrane tension in response to mechanical stimuli are badly needed in mechanobiology. We have recently introduced mechanosensitive flipper probes to report quantitatively global membrane tension changes in fluorescence lifetime imaging microscopy (FLIM) images of living cells. However, to address specific questions on physical forces in biology, the probes need to be localized precisely in the membrane of interest (MOI). Herein we present a general strategy to image the tension of the MOI by tagging our newly introduced HaloFlippers to self-labeling HaloTags fused to proteins in this membrane. The critical challenge in the construction of operational HaloFlippers is the tether linking the flipper and the HaloTag: It must be neither too taut nor too loose, be hydrophilic but lipophilic enough to passively diffuse across membranes to reach the HaloTags, and allow partitioning of flippers into the MOI after the reaction. HaloFlippers with the best tether show localized and selective fluorescence after reacting with HaloTags that are close enough to the MOI but remain nonemissive if the MOI cannot be reached. Their fluorescence lifetime in FLIM images varies depending on the nature of the MOI and responds to myriocin-mediated sphingomyelin depletion as well as to osmotic stress. The response to changes in such precisely localized membrane tension follows the validated principles, thus confirming intact mechanosensitivity. Examples covered include HaloTags in the Golgi apparatus, peroxisomes, endolysosomes, and the ER, all thus becoming accessible to the selective fluorescence imaging of membrane tension.

Disseminated Juvenile Xanthogranuloma and Hemophagocytic Lymphohistiocytosis Developed During Treatment of Acute Lymphoblastic Leukemia: Case Report.

The association between acute lymphoblastic leukemia (ALL), non-Langerhans cell histiocytosis (non-LCH), and hemophagocytic lymphohistiocytosis (HLH), to the best of our knowledge, has not been published to date. Juvenile xanthogranuloma (JXG), as a type of non-LCH, is usually a benign disease limited to the skin. Systemic involvement is rarely reported. The present case report describes a 15-year-old boy diagnosed with disseminated JXG involving skin and bone marrow concurrent with severe symptoms of HLH during ALL therapy. Examination of immunoglobulin heavy chain genes in B-cell precursor leukemic blasts and histiocytes in the skin and bone marrow revealed identical rearrangements, confirming clonal relationship between both diseases. Implementation of corticosteroids, vinblastine, etoposide, cyclosporine, and tocilizumab resulted in partial skin lesion resolution with no improvement of bone marrow function; therefore, hematopoietic stem cell transplantation (HSCT) was eventually performed. The patient's hematological and general status has improved gradually; however, remarkable recovery of skin lesions was observed after empirical antitubercular therapy. Mycobacterium spp. infection should be considered as a possible secondary HLH trigger. Triple association of ALL, non-LCH, and HLH highlights heterogeneity of histiocytic disorders and possible common origin of dendritic and lymphoid cells.

Fluorescent Membrane Tension Probes for Super-Resolution Microscopy: Combining Mechanosensitive Cascade Switching with Dynamic-Covalent Ketone Chemistry.

We report the design, synthesis, and evaluation of fluorescent flipper probes for single-molecule super-resolution imaging of membrane tension in living cells. Reversible switching from bright-state ketones to dark-state hydrates, hemiacetals, and hemithioacetals is demonstrated for twisted and planarized mechanophores in solution and membranes. Broadband femtosecond fluorescence up-conversion spectroscopy evinces ultrafast chalcogen-bonding cascade switching in the excited state in solution. According to fluorescence lifetime imaging microscopy, the new flippers image membrane tension in live cells with record red shifts and photostability. Single-molecule localization microscopy with the new tension probes resolves membranes well below the diffraction limit.