Characterization of the conformational space of the murine prion protein using single-molecule force spectroscopy techniques

The conversion of the cellular prion protein (PrPC) to its infectious counterpart (PrPSc) is the initial step of prion diseases. These neurodegenerative disorders are characterized by different incubation times, sympthoms and disease phenotypes. Structural heterogenity of PrP aggregates is responsible for this biological diversity. Understanding the structural rearrangements of PrP at the monomeric and oligomeric level is essential to gain insights into its aggregation processes. However traditional \u201cin-bulk\u201d techniques can only provide ensemble-averaged information for monomer and oligomer structures. We applied single-molecule force spectroscopy to characterize the heterogeneous structural ensemble of the murine PrP at the monomeric and at the oligomeric level. By stretching chimeric protein construct carrying one MoPrP molecule we found that the protein folds with a two state mechanism. Less frequently the protein can adopt more extended conformations that encompass also the N-terminal domain. These structures might be involved in subsequent aggregation processes. We also developed an assay to characterize the oligomerization processes using multiple PrP constructs. By analyzing the extension of these constructs under tension we characterized the structure between different PrP moieties, under different conditions. We found that reciprocal PrP orientation affects the length and mechanical resistance of these structures but their events frequency. Comparing the structures observed from monomers, dimers, trimers and tetramers we found that their frequency of events and their average length increased by increasing the number of PrP moieties. Remarkably, decreasing pH to more acidic values resulted in a higher frequency of events that involved structures between PrP moieties only in multimeric constructs. Instead, increasing the ionic strength significantly diminished their frequency, indicating how solution conditions can strongly alter the conformational transitions. These results provide a new scenario on PrP misfolding and aggregation processes, characterizing their early aggregation steps under different reaction conditions

Drug resistance and minimal residual disease in multiple myeloma

Great progress has been made in improving survival in multiple myeloma (MM) patients over the last 30 years. New drugs have been introduced and complete responses are frequently seen. However, the majority of MM patients do experience a relapse at a variable time after treatment, and ultimately the disease becomes drug-resistant following therapies. Recently, minimal residual disease (MRD) detection has been introduced in clinical trials utilizing novel therapeutic agents to measure the depth of response. MRD can be considered as a surrogate for both progression-free and overall survival. In this perspective, the persistence of a residual therapy-resistant myeloma plasma cell clone can be associated with inferior survivals. The present review gives an overview of drug resistance in MM, i.e., mutation of β5 subunit of the proteasome; upregulation of pumps of efflux; heat shock protein induction for proteasome inhibitors; downregulation of CRBN expression; deregulation of IRF4 expression; mutation of CRBN, IKZF1, and IKZF3 for immunomodulatory drugs and decreased target expression; complement protein increase; sBCMA increase; and BCMA down expression for monoclonal antibodies. Multicolor flow cytometry, or next-generation flow, and next-generation sequencing are currently the techniques available to measure MRD with sensitivity at 10-5. Sustained MRD negativity is related to prolonged survival, and it is evaluated in all recent clinical trials as a surrogate of drug efficacy

Evidence of Orientation-Dependent Early States of Prion Protein Misfolded Structures from Single Molecule Force Spectroscopy

Prion diseases are neurodegenerative disorders characterized by the presence of oligomers and amyloid fibrils. These are the result of protein aggregation processes of the cellular prion protein (PrPC) into amyloidal forms denoted as prions or PrPSc. We employed atomic force microscopy (AFM) for single molecule pulling (single molecule force spectroscopy, SMFS) experiments on the recombinant truncated murine prion protein (PrP) domain to characterize its conformations and potential initial oligomerization processes. Our AFM-SMFS results point to a complex scenario of structural heterogeneity of PrP at the monomeric and dimer level, like other amyloid proteins involved in similar pathologies. By applying this technique, we revealed that the PrP C-terminal domain unfolds in a two-state process. We used two dimeric constructs with different PrP reciprocal orientations: one construct with two sequential PrP in the N- to C-terminal orientation (N-C dimer) and a second one in the C- to C-terminal orientation (C-C dimer). The analysis revealed that the different behavior in terms of unfolding force, whereby the dimer placed C-C dimer unfolds at a higher force compared to the N-C orientation. We propose that the C-C dimer orientation may represent a building block of amyloid fibril formation

Venetoclax in association with decitabine as effective bridge to transplant in a case of relapsed early T‐cell lymphoblastic leukemia

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A BRAF Negative Classic Hairy Cell Leukemia Patient in Long Lasting Complete Remission after Rituximab and Pentostatin

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Circulating CD34+/CD38-/CD26+ Leukemia Stem Cells along Chronic Myeloid Leukemia progression: differences between Chronic, Accelerated and Blast Phase

In Chronic Myeloid Leukemia (CML) patients, CD34+/CD38-/CD26+ cell population represents a “CML specific” Leukemia Stem Cell (LSC) compartment. Indeed, preliminary studies showed that the expression of CD26 discriminates bone marrow CML Leukemic Stem Cells (LSCs) from nor-mal Hematopoietic Stem Cells (HSCs) or from LSCs of other myeloid neoplasms. We were first to demonstrate that at diagnosis CD34+/CD38-/CD26+ cells are easily measurable also in Peripheral Blood (PB) and that residual circulating CD26+LSCs persist, with a fluctuating trend, in most pa-tients in optimal response during treatment with Tyrosine Kinase Inhibitors (TKIs) and even after successful TKI discontinuation. These data corroborate and confirm the possibility of using flow-cytometry CD26+ evaluation as an important diagnostic tool that, combined with molecular biology and cytogenetic, could provide a rapid diagnosis of Chronic Phase (CP) CML starting from a simple PB sample. Yet, few data are available regarding the behavior of CD26+LSCs during Accelerated Phase (AP) or Blast Phase (BP) CML and the role, if any, this peculiar staminal cell compartment may play in disease progression. In the present study we compared the presence and phenotypic characteristics of circulating CD26+LSCs in CP CML patients at diagnosis, during AP and in cases of progression to lymphoid BP, inquiring a possible role of these cells during dis-ease evolution

PATHOGENETIC ASPECTS OF MYELODISPLASTIC SYNDROMES

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PATHOGENETIC ASPECTS OF MYELODISPLASTIC SYNDROMES

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