The Nanomechanical Properties of CLL Cells Are Linked to the Actin Cytoskeleton and Are a Potential Target of BTK Inhibitors.

Chronic lymphocytic leukemia (CLL) is an incurable disease characterized by an intense trafficking of the leukemic cells between the peripheral blood and lymphoid tissues. It is known that the ability of lymphocytes to recirculate strongly depends on their capability to rapidly rearrange their cytoskeleton and adapt to external cues; however, little is known about the differences occurring between CLL and healthy B cells during these processes. To investigate this point, we applied a single-cell optical (super resolution microscopy) and nanomechanical approaches (atomic force microscopy, real-time deformability cytometry) to both CLL and healthy B lymphocytes and compared their behavior. We demonstrated that CLL cells have a specific actomyosin complex organization and altered mechanical properties in comparison to their healthy counterpart. To evaluate the clinical relevance of our findings, we treated the cells in vitro with the Bruton's tyrosine kinase inhibitors and we found for the first time that the drug restores the CLL cells mechanical properties to a healthy phenotype and activates the actomyosin complex. We further validated these results in vivo on CLL cells isolated from patients undergoing ibrutinib treatment. Our results suggest that CLL cells' mechanical properties are linked to their actin cytoskeleton organization and might be involved in novel mechanisms of drug resistance, thus becoming a new potential therapeutic target aiming at the normalization of the mechanical fingerprints of the leukemic cells.

Amyposomes, a nanotechnological chaperone with anti-amyloidogenic activity.

AIM: The effect of liposomes bi-functionalized with phosphatidic acid and with a synthetic peptide derived from human apolipoprotein E has been evaluated on the aggregation features of different amyloidogenic proteins: human Amyloid ß1-40 (Aß1-40), transthyretin (TTR) variant S52P, human ß2microglobulin (ß2m) variants ΔN6 and D76N, Serum Amyloid A (SAA). METHODS: The formation of fibrillar aggregates of the proteins was investigated by ThioflavinT fluorescence assay and validated by Atomic Force Microscopy. RESULTS: The results show that liposomes are preventing the transition of non-aggregated forms to the fibrillar state, with stronger effects on Aß1-40, ß2m ΔN6 and SAA. Liposomes also induce disaggregation of the amyloid aggregates of all the proteins investigated, with stronger effects on Aß1-40, ß2 D76N and TTR.SPR assays show that liposomes bind Aß1-40 and SAA aggregates with high affinity (KD in the nanomolar range) whereas binding to TTR aggregates showed a lower affinity (KD in the micromolar range). Aggregates of ß2m variants showed both high and low affinity binding sites. Computed Structural analysis of protein fibrillar aggregates and considerations on the multidentate features of liposomes allow to speculate a common mechanism of action, based on binding the ß-stranded peptide regions responsible for the amyloid formation. CONCLUSION: Thus, multifunctional liposomes perform as pharmacological chaperones with anti-amyloidogenic activity, with a promising potential for the treatment of a number of protein-misfolding diseases.Key messageAmyloidosis is a group of diseases, each due to a specific protein misfolding.Anti-amyloidogenic nanoparticles have been gaining the utmost importance as a potential treatment for protein misfolding disorders.Liposomes bi-functionalized with phosphatidic acid and with a synthetic peptide derived from human apolipoprotein E showed anti-amyloidogenic activity.

Proteinaceous microstructure in a capillary: a study of non-linear bending dynamics.

The flap of bendable structures under continuous flow impacts a variety of fields, ranging from energy harvesting to active mixing in microfluidic devices. Similar physical principles determine the flapping dynamics in a variety of systems with different sizes, but a thorough investigation of the bending dynamics at the microscale is still lacking. We employ here two-photon laser polymerization to fabricate elongated proteinaceous flexible microstructures directly within a micro-capillary and we characterize their bending dynamics. The elastic properties of the microstructures with different (circular and square) cross-sections are tested by Atomic Force Microscopy and by studying the deflection-flow dependence in microfluidic experiments at intermediate Reynolds numbers (Rey ≲ 150). The retrieved Young's modulus of the fabricated matrix (100 kPa ≤ E ≤ 4 MPa) falls in the range of most typical biological tissues and solely depends on the laser fabrication intensity. The elastic constant of the microstructures falls in the range of 0.8 nN µm-1 ≤ k ≤ 50 nN µm-1, and fully agrees with the macroscopic Euler Bernoulli theory. For soft microstructures (0.8 nN µm-1 ≤ k ≤ 8 nN µm-1) we reveal undamped bending oscillations under continuous microfluidic flow, corresponding to ∼10% of the total structure deflection. This behavior is ascribed to the coupling of the viscoelasticity and non-linear elasticity of the polymer matrix with non-linear dynamics arising from the time-dependent friction coefficient of the bendable microstructures. We envision that similar instabilities may lead to the development of promising energy conversion nanoplatforms.

Cell wall modifications by α-XYLOSIDASE1 are required for control of seed and fruit size in Arabidopsis.

Cell wall modifications are of pivotal importance during plant development. Among cell wall components, xyloglucans are the major hemicellulose polysaccharide in primary cell walls of dicots and non-graminaceous monocots. They can connect the cellulose microfibril surface to affect cell wall mechanical properties. Changes in xyloglucan structure are known to play an important role in regulating cell growth. Therefore, the degradation of xyloglucan is an important modification that alters the cell wall. The α-XYLOSIDASE1 (XYL1) gene encodes the only α-xylosidase acting on xyloglucans in Arabidopsis thaliana. Here, we showed that mutation of XYL1 strongly influences seed size, seed germination, and fruit elongation. We found that the expression of XYL1 is directly regulated in developing seeds and fruit by the MADS-box transcription factor SEEDSTICK. We demonstrated that XYL1 complements the stk smaller seed phenotype. Finally, by atomic force microscopy, we investigated the role of XYL1 activity in maintaining cell stiffness and growth, confirming the importance of cell wall modulation in shaping organs.

Nanomechanics of negatively supercoiled diaminopurine-substituted DNA.

Single molecule experiments have demonstrated a progressive transition from a B- to an L-form helix as DNA is gently stretched and progressively unwound. The particular sequence of a DNA segment defines both base stacking and hydrogen bonding that affect the partitioning and conformations of the two phases. Naturally or artificially modified bases alter H-bonds and base stacking and DNA with diaminopurine (DAP) replacing adenine was synthesized to produce linear fragments with triply hydrogen-bonded DAP:T base pairs. Both unmodified and DAP-substituted DNA transitioned from a B- to an L-helix under physiological conditions of mild tension and unwinding. This transition avoids writhing and the ease of this transition may prevent cumbersome topological rearrangements in genomic DNA that would require topoisomerase activity to resolve. L-DNA displayed about tenfold lower persistence length than B-DNA. However, left-handed DAP-substituted DNA was twice as stiff as unmodified L-DNA. Unmodified DNA and DAP-substituted DNA have very distinct mechanical characteristics at physiological levels of negative supercoiling and tension.

Double-stranded flanking ends affect the folding kinetics and conformational equilibrium of G-quadruplexes forming sequences within the promoter of KIT oncogene.

G-quadruplexes embedded within promoters play a crucial role in regulating the gene expression. KIT is a widely studied oncogene, whose promoter contains three G-quadruplex forming sequences, c-kit1, c-kit2 and c-kit*. For these sequences available studies cover ensemble and single-molecule analyses, although for kit* the latter were limited to a study on a promoter domain comprising all of them. Recently, c-kit2 has been reported to fold according to a multi-step process involving folding intermediates. Here, by exploiting fluorescence resonance energy transfer, both in ensemble and at the single molecule level, we investigated the folding of expressly designed constructs in which, alike in the physiological context, either c-kit2 or c-kit* are flanked by double stranded DNA segments. To assess whether the presence of flanking ends at the borders of the G-quadruplex affects the folding, we studied under the same protocols oligonucleotides corresponding to the minimal G-quadruplex forming sequences. Data suggest that addition of flanking ends results in biasing both the final equilibrium state and the folding kinetics. A previously unconsidered aspect is thereby unravelled, which ought to be taken into account to achieve a deeper insight of the complex relationships underlying the fine tuning of the gene-regulatory properties of these fascinating DNA structures.

Nanomechanics of G-quadruplexes within the promoter of the KIT oncogene.

G-quadruplexes (G4s) are tetrahelical DNA structures stabilized by four guanines paired via Hoogsteen hydrogen bonds into quartets. While their presence within eukaryotic DNA is known to play a key role in regulatory processes, their functional mechanisms are still under investigation. In the present work, we analysed the nanomechanical properties of three G4s present within the promoter of the KIT proto-oncogene from a single-molecule point of view through the use of magnetic tweezers (MTs). The study of DNA extension fluctuations under negative supercoiling allowed us to identify a characteristic fingerprint of G4 folding. We further analysed the energetic contribution of G4 to the double-strand denaturation process in the presence of negative supercoiling, and we observed a reduction in the energy required for strands separation.

Oxidative Stress Boosts the Uptake of Cerium Oxide Nanoparticles by Changing Brain Endothelium Microvilli Pattern.

Vascular oxidative stress is considered a worsening factor in the progression of Alzheimer's disease (AD). Increased reactive oxygen species (ROS) levels promote the accumulation of amyloid-ß peptide (Aß), one of the main hallmarks of AD. In turn, Aß is a potent inducer of oxidative stress. In early stages of AD, the concomitant action of oxidative stress and Aß on brain capillary endothelial cells was observed to compromise the blood-brain barrier functionality. In this context, antioxidant compounds might provide therapeutic benefits. To this aim, we investigated the antioxidant activity of cerium oxide nanoparticles (CNP) in human cerebral microvascular endothelial cells (hCMEC/D3) exposed to Aß oligomers. Treatment with CNP (13.9 ± 0.7 nm in diameter) restored basal ROS levels in hCMEC/D3 cells, both after acute or prolonged exposure to Aß. Moreover, we found that the extent of CNP uptake by hCMEC/D3 was +43% higher in the presence of Aß. Scanning electron microscopy and western blot analysis suggested that changes in microvilli structures on the cell surface, under pro-oxidant stimuli (Aß or H2O2), might be involved in the enhancement of CNP uptake. This finding opens the possibility to exploit the modulation of endothelial microvilli pattern to improve the uptake of anti-oxidant particles designed to counteract ROS-mediated cerebrovascular dysfunctions.

ETNK1 mutations induce a mutator phenotype that can be reverted with phosphoethanolamine.

Recurrent somatic mutations in ETNK1 (Ethanolamine-Kinase-1) were identified in several myeloid malignancies and are responsible for a reduced enzymatic activity. Here, we demonstrate in primary leukemic cells and in cell lines that mutated ETNK1 causes a significant increase in mitochondrial activity, ROS production, and Histone H2AX phosphorylation, ultimately driving the increased accumulation of new mutations. We also show that phosphoethanolamine, the metabolic product of ETNK1, negatively controls mitochondrial activity through a direct competition with succinate at mitochondrial complex II. Hence, reduced intracellular phosphoethanolamine causes mitochondria hyperactivation, ROS production, and DNA damage. Treatment with phosphoethanolamine is able to counteract complex II hyperactivation and to restore a normal phenotype.

The Clustering of mApoE Anti-Amyloidogenic Peptide on Nanoparticle Surface Does Not Alter Its Performance in Controlling Beta-Amyloid Aggregation.

The deposition of amyloid-ß (Aß) plaques in the brain is a significant pathological signature of Alzheimer's disease, correlating with synaptic dysfunction and neurodegeneration. Several compounds, peptides, or drugs have been designed to redirect or stop Aß aggregation. Among them, the trideca-peptide CWG-LRKLRKRLLR (mApoE), which is derived from the receptor binding sequence of apolipoprotein E, is effectively able to inhibit Aß aggregation and to promote fibril disaggregation. Taking advantage of Atomic Force Microscopy (AFM) imaging and fluorescence techniques, we investigate if the clustering of mApoE on gold nanoparticles (AuNP) surface may affect its performance in controlling Aß aggregation/disaggregation processes. The results showed that the ability of free mApoE to destroy preformed Aß fibrils or to hinder the Aß aggregation process is preserved after its clustering on AuNP. This allows the possibility to design multifunctional drug delivery systems with clustering of anti-amyloidogenic molecules on any NP surface without affecting their performance in controlling Aß aggregation processes.

Depicting Conformational Ensembles of α-Synuclein by Single Molecule Force Spectroscopy and Native Mass Spectroscopy.

Description of heterogeneous molecular ensembles, such as intrinsically disordered proteins, represents a challenge in structural biology and an urgent question posed by biochemistry to interpret many physiologically important, regulatory mechanisms. Single-molecule techniques can provide a unique contribution to this field. This work applies single molecule force spectroscopy to probe conformational properties of α-synuclein in solution and its conformational changes induced by ligand binding. The goal is to compare data from such an approach with those obtained by native mass spectrometry. These two orthogonal, biophysical methods are found to deliver a complex picture, in which monomeric α-synuclein in solution spontaneously populates compact and partially compacted states, which are differently stabilized by binding to aggregation inhibitors, such as dopamine and epigallocatechin-3-gallate. Analyses by circular dichroism and Fourier-transform infrared spectroscopy show that these transitions do not involve formation of secondary structure. This comparative analysis provides support to structural interpretation of charge-state distributions obtained by native mass spectrometry and helps, in turn, defining the conformational components detected by single molecule force spectroscopy.

The Extent of Human Apolipoprotein A-I Lipidation Strongly Affects the ß-Amyloid Efflux Across the Blood-Brain Barrier in vitro.

Much evidence suggests a protective role of high-density lipoprotein (HDL) and its major apolipoprotein apoA-I, in Alzheimer's disease (AD). The biogenesis of nascent HDL derived from a first lipidation of apoA-I, which is synthesized by the liver and intestine but not in the brain, in a process mediated by ABCA1. The maturation of nascent HDL in mature spherical HDL is due to a subsequent lipidation step, LCAT-mediated cholesterol esterification, and the change of apoA-I conformation. Therefore, different subclasses of apoA-I-HDL simultaneously exist in the blood circulation. Here, we investigated if and how the lipidation state affects the ability of apoA-I-HDL to target and modulate the cerebral ß-amyloid (Aß) content from the periphery, that is thus far unclear. In particular, different subclasses of HDL, each with different apoA-I lipidation state, were purified from human plasma and their ability to cross the blood-brain barrier (BBB), to interact with Aß aggregates, and to affect Aß efflux across the BBB was assessed in vitro using a transwell system. The results showed that discoidal HDL displayed a superior capability to promote Aß efflux in vitro (9 × 10-5 cm/min), when compared to apoA-I in other lipidation states. In particular, no effect on Aß efflux was detected when apoA-I was in mature spherical HDL, suggesting that apoA-I conformation, and lipidation could play a role in Aß clearance from the brain. Finally, when apoA-I folded its structure in discoidal HDL, rather than in spherical ones, it was able to cross the BBB in vitro and strongly destabilize the conformation of Aß fibrils by decreasing the order of the fibril structure (-24%) and the ß-sheet content (-14%). These data suggest that the extent of apoA-I lipidation, and consequently its conformation, may represent crucial features that could exert their protective role in AD pathogenesis.

A New Approach for Glyco-Functionalization of Collagen-Based Biomaterials.

The cell microenvironment plays a pivotal role in mediating cell adhesion, survival, and proliferation in physiological and pathological states. The relevance of extracellular matrix (ECM) proteins in cell fate control is an important issue to take into consideration for both tissue engineering and cell biology studies. The glycosylation of ECM proteins remains, however, largely unexplored. In order to investigate the physio-pathological effects of differential ECM glycosylation, the design of affordable chemoselective methods for ECM components glycosylation is desirable. We will describe a new chemoselective glycosylation approach exploitable in aqueous media and on non-protected substrates, allowing rapid access to glyco-functionalized biomaterials.

The 1ALCTL and 1BLCTL isoforms of Arg/Abl2 induce fibroblast activation and extra cellular matrix remodelling differently.

The fibrotic tissue and the stroma adjacent to cancer cells are characterised by the presence of activated fibroblasts (myofibroblasts) which play a role in creating a supportive tissue characterised by abundant extracellular matrix (ECM) secretion. The myofibroblasts remodel this tissue through secreted molecules and modulation of their cytoskeleton and specialized contractile structures. The non-receptor protein tyrosine kinase Arg (also called Abl2) has the unique ability to bind directly to the actin cytoskeleton, transducing diverse extracellular signals into cytoskeletal rearrangements. In this study we analysed the 1ALCTL and 1BLCTL Arg isoforms in Arg-/- murine embryonal fibroblasts (MEF) cell line, focusing on their capacity to activate fibroblasts and to remodel ECM. The results obtained showed that Arg isoform 1BLCTL has a major role in proliferation, migration/invasion of MEF and in inducing a milieu able to modulate tumour cell morphology, while 1ALCTL isoform has a role in MEF adhesion maintaining active focal adhesions. On the whole, the presence of Arg in MEF supports the proliferation, activation, adhesion, ECM contraction and stiffness, while the absence of Arg affected these myofibroblast features.This article has an associated First Person interview with the first author of the paper.

Nanomechanics of Diaminopurine-Substituted DNA.

2,6-diaminopurine (DAP) is a nucleobase analog of adenine. When incorporated into double-stranded DNA (dsDNA), it forms three hydrogen bonds with thymine. Rare in nature, DAP substitution alters the physical characteristics of a DNA molecule without sacrificing sequence specificity. Here, we show that in addition to stabilizing double-strand hybridization, DAP substitution also changes the mechanical and conformational properties of dsDNA. Thermal melting experiments reveal that DAP substitution raises melting temperatures without diminishing sequence-dependent effects. Using a combination of atomic force microscopy (AFM), magnetic tweezer (MT) nanomechanical assays, and circular dichroism spectroscopy, we demonstrate that DAP substitution increases the flexural rigidity of dsDNA yet also facilitates conformational shifts, which manifest as changes in molecule length. DAP substitution increases both the static and dynamic persistence length of DNA (measured by AFM and MT, respectively). In the static case (AFM), in which tension is not applied to the molecule, the contour length of DAP-DNA appears shorter than wild-type (WT)-DNA; under tension (MT), they have similar dynamic contour lengths. At tensions above 60 pN, WT-DNA undergoes characteristic overstretching because of strand separation (tension-induced melting) and spontaneous adoption of a conformation termed S-DNA. Cyclic overstretching and relaxation of WT-DNA at near-zero loading rates typically yields hysteresis, indicative of tension-induced melting; conversely, cyclic stretching of DAP-DNA showed little or no hysteresis, consistent with the adoption of the S-form, similar to what has been reported for GC-rich sequences. However, DAP-DNA overstretching is distinct from GC-rich overstretching in that it happens at a significantly lower tension. In physiological salt conditions, evenly mixed AT/GC DNA typically overstretches around 60 pN. GC-rich sequences overstretch at similar if not slightly higher tensions. Here, we show that DAP-DNA overstretches at 52 pN. In summary, DAP substitution decreases the overall stability of the B-form double helix, biasing toward non-B-form DNA helix conformations at zero tension and facilitating the B-to-S transition at high tension.

Major Action of Endogenous Lysyl Oxidase in Clear Cell Renal Cell Carcinoma Progression and Collagen Stiffness Revealed by Primary Cell Cultures.

Human clear cell renal cell carcinoma (ccRCC) is therapy resistant; therefore, it is worthwhile studying in depth the molecular aspects of its progression. In ccRCC the biallelic inactivation of the VHL gene leads to stabilization of hypoxia-inducible factors (HIFs). Among the targets of HIF-1α transcriptional activity is the LOX gene, which codes for the inactive proenzyme (Pro-Lox) from which, after extracellular secretion and proteolysis, derives the active enzyme (Lox) and the propeptide (Lox-PP). By increasing stiffness of extracellular matrix by collagen crosslinking, Lox promotes tumor progression and metastasis. Lox and Lox-PP can reenter the cells where Lox promotes cell proliferation and invasion, whereas Lox-PP acts as tumor suppressor because of its Ras recision and apoptotic activity. Few data are available concerning LOX in ccRCC. Using an in vitro model of ccRCC primary cell cultures, we performed, for the first time in ccRCC, a detailed study of endogenous LOX and also investigated their transcriptomic profile. We found that endogenous LOX is overexpressed in ccRCC, is involved in a positive-regulative loop with HIF-1α, and has a major action on ccRCC progression through cellular adhesion, migration, and collagen matrix stiffness increment; however, the oncosuppressive action of Lox-PP was not found to prevail. These findings may suggest translational approaches for new therapeutic strategies in ccRCC.

Platinum-Based Drugs and DNA Interactions Studied by Single-Molecule and Bulk Measurements.

Platinum-containing molecules are widely used as anticancer drugs. These molecules exert cytotoxic effects by binding to DNA through various mechanisms. The binding between DNA and platinum-based drugs hinders the opening of DNA, and therefore, DNA duplication and transcription are severely hampered. Overall, impeding the above-mentioned important DNA mechanisms results in irreversible DNA damage and the induction of apoptosis. Several molecules, including multinuclear platinum compounds, belong to the family of platinum drugs, and there is a body of research devoted to developing more efficient and less toxic versions of these compounds. In this study, we combined different biophysical methods, including single-molecule assays (magnetic tweezers) and bulk experiments (ultraviolet absorption for thermal denaturation) to analyze the differential stability of double-stranded DNA in complex with either cisplatin or multinuclear platinum agents. Specifically, we analyzed how the binding of BBR3005 and BBR3464, two representative multinuclear platinum-based compounds, to DNA affects its stability as compared with cisplatin binding. Our results suggest that single-molecule approaches can provide insights into the drug-DNA interactions that underlie drug potency and provide information that is complementary to that generated from bulk analysis; thus, single-molecule approaches have the potential to facilitate the selection and design of optimized drug compounds. In particular, relevant differences in DNA stability at the single-molecule level are demonstrated by analyzing nanomechanically induced DNA denaturation. On the basis of the comparison between the single-molecule and bulk analyses, we suggest that transplatinated drugs are able to locally destabilize small portions of the DNA chain, whereas other regions are stabilized.

Fluorimetric detection of the earliest events in amyloid ß oligomerization and its inhibition by pharmacologically active liposomes.

BACKGROUND: Amyloid ß (Aß) peptide aggregation is the main molecular mechanism underlying the development of Alzheimer's disease, the most widespread form of senile dementia worldwide. Increasing evidence suggests that the key factor leading to impaired neuronal function is accumulation of water-soluble Aß oligomers rather than formation of the senile plaques created by the deposition of large fibrillary aggregates of Aß. However, several questions remain about the preliminary steps and the progression of Aß oligomerization. METHODS: We show that the initial stages of the aggregation of fluorescently labeled Aß can be determined with a high degree of precision and at physiological (i.e., nanomolar) concentrations by using either steady-state fluorimetry or time-correlated single-photon counting. RESULTS: We study the dependence of the oligomerization extent and rate on the Aß concentration. We determine the chemical binding affinity of fluorescently labeled Aß for liposomes that have been recently shown to be pharmacologically active in vivo, reducing the Aß burden within the brain. We also probe their capacity to hinder the Aß oligomerization process in vitro. CONCLUSIONS: We introduced a fluorescence assay allowing investigation of the earliest steps of Aß oligomerization, the peptide involved in Alzheimer's disease. The assay proved to be sensitive even at Aß concentrations as low as those physiologically observed in the cerebrospinal fluid. GENERAL SIGNIFICANCE: This work represents an extensive and quantitative study on the initial events of Aß oligomerization at physiological concentration. It may enhance our comprehension of the molecular mechanisms leading to Alzheimer's disease, thus paving the way to novel therapeutic strategies.

Effects of non-CpG site methylation on DNA thermal stability: a fluorescence study.

Cytosine methylation is a widespread epigenetic regulation mechanism. In healthy mature cells, methylation occurs at CpG dinucleotides within promoters, where it primarily silences gene expression by modifying the binding affinity of transcription factors to the promoters. Conversely, a recent study showed that in stem cells and cancer cell precursors, methylation also occurs at non-CpG pairs and involves introns and even gene bodies. The epigenetic role of such methylations and the molecular mechanisms by which they induce gene regulation remain elusive. The topology of both physiological and aberrant non-CpG methylation patterns still has to be detailed and could be revealed by using the differential stability of the duplexes formed between site-specific oligonucleotide probes and the corresponding methylated regions of genomic DNA. Here, we present a systematic study of the thermal stability of a DNA oligonucleotide sequence as a function of the number and position of non-CpG methylation sites. The melting temperatures were determined by monitoring the fluorescence of donor-acceptor dual-labelled oligonucleotides at various temperatures. An empirical model that estimates the methylation-induced variations in the standard values of hybridization entropy and enthalpy was developed.