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Acute myeloid leukemia may be characterized by a fraction of leukemia stem cells (LSCs) that sustain disease propagation eventually leading to relapse. Yet, the contribution of LSCs to early therapy resistance and AML regeneration remains controversial. We prospectively identify LSCs in AML patients and xenografts by single-cell RNA sequencing coupled with functional validation by a microRNA-126 reporter enriching for LSCs. Through nucleophosmin 1 ( NPM1 ) mutation calling or chromosomal monosomy detection in single-cell transcriptomes, we discriminate LSCs from regenerating hematopoiesis, and assess their longitudinal response to chemotherapy. Chemotherapy induced a generalized inflammatory and senescence-associated response. Moreover, we observe heterogeneity within progenitor AML cells, some of which proliferate and differentiate with expression of oxidative-phosphorylation (OxPhos) signatures, while others are OxPhos (low) miR-126 (high) and display enforced stemness and quiescence features. miR-126 (high) LSCs are enriched at diagnosis in chemotherapy-refractory AML and at relapse, and their transcriptional signature robustly stratifies patients for survival in large AML cohorts. Relapse within acute myeloid leukaemia may be driven by the presence of leukaemia stem cells. Here, the authors use single cell RNA-seq seq to characterise leukemia stem cells, and show miR-126 as a potential marker of resistance.

Late asymmetric growth arrest of the distal ulna and subsequent Madelung‐type deformity may occur following elastic stable intramedullary nailing (ESIN) performed near the distal ulnar physis. Although causality cannot be definitively proven, meticulous surgical technique and structured long‐term radiographic follow‐up are essential to minimize risk and enable early detection of growth disturbance. Limitation of the movement 9‐year operated both bone forearm fracture.

BackgroundAcute myeloid leukemia (AML) is associated with an unfavorable outcome for >50% of patients. Whereas novel immunotherapies, such as CD19-CAR-T, demonstrated striking efficacy when targeting dispensable antigens (Ag), the same approach cannot be exploited for AML, due to lack of actionable leukemia-restricted Ags. AML targets are shared with progenitors (HSPCs) or mature myeloid cells, leading to on-target/off-tumor toxicity. We reasoned that precise modification of target epitopes in donor HSPCs used in hematopoietic stem cell transplantation (HSCT) would result in loss of recognition by CAR/mAbs, without affecting protein expression and function. Epitope-editing allows targeting genes essential for leukemia survival regardless of expression in HSPC, minimizing the risk of immune-escape.1 MethodsWe selected the cytokine receptors FLT3, CD123 and KIT, found in >85% of AML cases. By library screenings, we identified substitutions in their extracellular-domain that avoid detection by therapeutic Abs. We validated the functionality of mutated receptors (ligand affinity, western-blot, proliferation, RNAseq, phospho-proteomics) and their resistance to on-target killing (mAb-affinity, CAR-T co-culture). We optimized a base-editing protocol to introduce these mutations in CD34+HSPCs. We exploited advanced in vivo models with co-engraftment of healthy HSPCs, patient-derived AML xenografts (PDX) and CAR-T to assess selective elimination of leukemia and protection of healthy hematopoiesis.ResultsEpitope variants were resistant to in vitro CAR-T killing and did not induce CAR activation. Electroporation of ABE8e mRNA+sgRNAs into CD34+HSPCs achieved 90%, 85% and 75% editing efficiency on FLT3, KIT and CD123. After xenotransplant into NBSGW mice, epitope-edited HSPC sustained long-term multi-lineage hematopoiesis, similar to AAVS1 controls. Upon treatment with FLT3-CAR-T, we observed sparing of HSPCs, granulo-mono progenitors and B-cell subsets derived from FLT3-edited HSPCs, while treatment with CD123-CAR showed protection of epitope-edited myeloid lineages, compared to AAVS1. Concomitantly, co-engrafted PDXs were eradicated by either FLT3- or CD123-CAR-T. Due to AML intra-tumoral heterogeneity and plasticity, targeting several Ags might be required to eradicate leukemia stem cells. To this end, we optimized high-efficiency multiplex-editing to enable targeting of multiple Ags without overlapping toxicities. We confirmed resistance of dual-FLT3/CD123 epitope-edited HSPCs and the superior efficacy of dual-target CAR-T in mice co-engrafted with a PDX partially resistant to FLT3-targeting alone. Additionally, our approach was able to protect HSPCs from the combination of FLT3-CARs with FLT3-tyrosine kinase inhibitor Crenolanib, while controls showed additional toxicity.ConclusionsIn conclusion, transplantation of epitope-engineered HSPCs endowed with selective resistance to multi-specifc CAR-T-cells is a novel approach to enable more effective and safer immunotherapies for difficult-to-target tumors such as AML.AcknowledgementsI would like to acknowledge my mentor, Prof. Pietro Genovese, for his kind and thorough supervision.Additionally, I would like to thank the members of the lab that have collaborated with me to generate the data for this work, among others: Adele Mucci PhD, Andrea Cosentino MD, Mohammed S. Mahmoud PhD, Iraxte Ugarte Zabala, MSc.Our close collaborators, Dan Bauer’s and Christian Brendel’s lab also deserve a special mention.Several funding agencies have provided the funding to develop this project, but I would like to specifically thank the American Society for Transplantation and Cellular Therapy and the Pediatric Transplantation and Cellular Therapy Consortium for granting me two New Investigator Awards to advance this work further and start new spin-off projects related to this abstract.ReferenceEpitope Editing Enables Targeted Immunotherapies for Acute Myeloid Leukemia, Casirati G, et al. Nature (provisionally accepted)

Therapeutic prime editing of hematopoietic stem and progenitor cells (HSPCs) holds great potential to remedy blood disorders. Quiescent cells have low nucleotide levels and resist retroviral infection, and it is possible that nucleotide metabolism could limit reverse transcription-mediated prime editing in HSPCs. We demonstrate that deoxynucleoside supplementation and Vpx-mediated degradation of SAMHD1 improve prime editing efficiency in HSPCs, especially when coupled with editing approaches that evade mismatch repair. Prime editing in hematopoietic cells is improved by engineering nucleotide metabolism.

An investigation aimed at recording NIS settlement by the employment of a bare artificial substratum constituted by caged lightweight expanded clay (EC) has been carried out in a confined marine basin. Packaged substratum, submitted to different organic treatments and a control was positioned in spring both in the water column and into the bottom sediment. Two months experiment provided 17 non-indigenous (NIS) versus 154 indigenous species (IS). Eleven “slow” colonizing NIS occurred at mean density of 0.2 ± 0.08 ind. L−1, whereas six species of “fast” colonizing NIS reached a mean density of 34.5 ± 13.0 ind. L−1 (individual organisms) and a coverage of 37.1 ± 31.6 cm2 L−1 (modular organisms). PCoA plots ordered assemblages according to time (from 48% up to 75% total variation) and to organic treatment (about 22%) both for individual and modular organisms. In general, organic contamination exerted negative effect both on NIS and IS, and the reference condition was preferred. Among individual NIS, the best colonizer in terms of density and biomass was primarily the sphaeromatid Paracerceis sculpta for both sediment-buried (64.78 ind-cm2 L−1 and 217.43 mg L−1) and suspended artificial substratum (143.94 ind-cm2 L−1 and 483.14 mg L−1). The amphinomid Linopherus canariensis prevailed in the buried artificial substratum (52.27 ind-cm2 L−1 and 92.81 mg L−1 respectively). Sessile and modular NIS appeared more susceptible to the initial organic contamination and the artificial substratum suspended into the water column was more suitable for these colonizers. Celleporaria brunnea (79.74 ind-cm2 L−1), Paraleucilla magna (66.69 ind-cm2 L−1), Hydroides elegans (12.49 ind-cm2 L−1) prevailed in the mean surface coverage among other NIS and IS species. The organic enrichment reduced to four up to five times the colonization rate of the first two species.Experimental data showed the great competitive capacity of NIS in the early stages of fouling, both under control and organically-enriched conditions, although this type of disturbance affected the whole fouling assemblage.

Despite the considerable efficacy observed when targeting a dispensable lineage antigen, such as CD19 in B cell acute lymphoblastic leukaemia 1 , 2 , the broader applicability of adoptive immunotherapies is hampered by the absence of tumour-restricted antigens 3 – 5 . Acute myeloid leukaemia immunotherapies target genes expressed by haematopoietic stem/progenitor cells (HSPCs) or differentiated myeloid cells, resulting in intolerable on-target/off-tumour toxicity. Here we show that epitope engineering of donor HSPCs used for bone marrow transplantation endows haematopoietic lineages with selective resistance to chimeric antigen receptor (CAR) T cells or monoclonal antibodies, without affecting protein function or regulation. This strategy enables the targeting of genes that are essential for leukaemia survival regardless of shared expression on HSPCs, reducing the risk of tumour immune escape. By performing epitope mapping and library screenings, we identified amino acid changes that abrogate the binding of therapeutic monoclonal antibodies targeting FLT3, CD123 and KIT, and optimized a base-editing approach to introduce them into CD34 + HSPCs, which retain long-term engraftment and multilineage differentiation ability. After CAR T cell treatment, we confirmed resistance of epitope-edited haematopoiesis and concomitant eradication of patient-derived acute myeloid leukaemia xenografts. Furthermore, we show that multiplex epitope engineering of HSPCs is feasible and enables more effective immunotherapies against multiple targets without incurring overlapping off-tumour toxicities. We envision that this approach will provide opportunities to treat relapsed/refractory acute myeloid leukaemia and enable safer non-genotoxic conditioning. Epitope engineering of donor haematopoietic stem/progenitor cells endows haematopoietic lineages with selective resistance to CAR T cells or monoclonal antibodies, without affecting protein function or regulation, enabling the targeting of genes that are essential for leukaemia survival and reducing the risk of tumour immune escape.

Therapeutic prime editing of hematopoietic stem and progenitor cells (HSPCs) holds great potential to remedy blood disorders. Since quiescent cells have low nucleotide levels and resist retroviral infection, we hypothesized that nucleotide metabolism could limit reverse transcription mediated prime editing in HSPCs. We demonstrate that deoxynucleoside supplementation and Vpx-mediated degradation of SAMHD1 improve prime editing efficiency in HSPCs, especially when coupled with editing approaches that evade mismatch repair.

An assemblage of cirratulids in a confined marine basin (Mediterranean Sea) was investigated at small spatio-temporal scales in an experiment in which artificial granules of expanded fire-clay were used as a bare substratum for colonization. Analysis of core samples of 3.5 l undisturbed natural sediments (source area) and equal volumes of artificial substratum (new settlement area), plunged into the sea bottom were carried out from May to July 2008 after 0, 15, 30 and 60 days. Caulleriella bioculata showed the highest total mean density of 10.1 ± 5.8 N l−1 in the natural sediment, and the lowest at 0.13 ± 0.08 N l−1in the artificial substratum, with a marked temporal decrease. Individuals were not strongly aggregated and were found in a deeper sediment layer. The density of large Cirriformia tentaculata was 0.5 ± 0.3 N l−1, with individuals more aggregated and confined to shallower sediments, versus 0.10 ± 0.06 N l−1 but found deeper in the artificial substratum; temporal trends were not straightforward for this species. The adult stage of sedentary cirratulids appeared to actively move into the new available substratum from the neighbouring sediments. An early cirratulid juvenile stage was observed in both microhabitats at a comparable density of 1.8 ± 2.8 N l−1 with highly aggregated individuals (cohorts). The peculiar morphology of branchiae, the threadlike body shape and the multivariate morphological differences between two sub-populations settled in the different substrata are reported for these problematic specimens.

The article investigates the intellectual capital disclosure of Italian banks over the years 2016–2017, applying the specific lens of healthy and distressed banks. To this end, we used content analysis and encoding techniques. The main results point out that intellectual capital (IC) disclosure is generally poor and that the intensity of disclosure varies slightly between healthy and distressed banks. Regarding the quality of disclosure, healthy banks present a higher, albeit modest, tendency to disclose non-qualitative and forward-looking information, maybe due to the fact that they are more focused on the strategies and the relationships with stakeholders as opposed to a more short-term approach of the distressed banks. To complement our study on healthy and distressed banks, we repeated the analysis focusing on bank size and independent directors. In this case, results do not show relevant differences in terms of IC disclosure. Hence, our findings suggest the need to consider banks’ IC disclosure as a strategic asset for increasing, among others, transparency and reputation.

A new species of Abyssoninoe (Polychaeta: Lumbrineridae) has been found on sublittoral soft bottoms in the Gulf of Milazzo (central Mediterranean Sea). The description is based on eleven specimens recorded in six grab samples carried out by Van Veen grab from 20 to 50 m depth. Abyssoninoe bidentata sp. nov. differs from the six hitherto known species of Abyssoninoe by having the maxillae III with two subequal teeth instead of one, the shape of the parapodial lobes and chaetal distribution.