Search

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.

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 β1–40 (Aβ1–40), transthyretin (TTR) variant S52P, human β2microglobulin (β2m) variants ΔN6 and D76N, Serum Amyloid A (SAA). The formation of fibrillar aggregates of the proteins was investigated by ThioflavinT fluorescence assay and validated by Atomic Force Microscopy. The results show that liposomes are preventing the transition of non-aggregated forms to the fibrillar state, with stronger effects on Aβ1–40, β2m ΔN6 and SAA. Liposomes also induce disaggregation of the amyloid aggregates of all the proteins investigated, with stronger effects on Aβ1–40, β2 D76N and TTR. SPR assays show that liposomes bind Aβ1–40 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 β2m 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 β-stranded peptide regions responsible for the amyloid formation. 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. Amyloidosis 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.

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.

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.

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.

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-β peptide (Aβ), one of the main hallmarks of AD. In turn, Aβ is a potent inducer of oxidative stress. In early stages of AD, the concomitant action of oxidative stress and Aβ 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 Aβ 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 Aβ. Moreover, we found that the extent of CNP uptake by hCMEC/D3 was +43% higher in the presence of Aβ. Scanning electron microscopy and western blot analysis suggested that changes in microvilli structures on the cell surface, under pro-oxidant stimuli (Aβ 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.

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.

Background DNA bridging promoted by the H-NS protein, combined with the compaction induced by cellular crowding, plays a major role in the structuring of the E. coli genome. However, only few studies consider the effects of the physical interplay of these two factors in a controlled environment. Methods We apply a single molecule technique (Magnetic Tweezers) to study the nanomechanics of compaction and folding kinetics of a 6 kb DNA fragment, induced by H-NS bridging and/or PEG crowding. Results In the presence of H-NS alone, the DNA shows a step-wise collapse driven by the formation of multiple bridges, and little variations in the H-NS concentration-dependent unfolding force. Conversely, the DNA collapse force observed with PEG was highly dependent on the volume fraction of the crowding agent. The two limit cases were interpreted considering the models of loop formation in a pulled chain and pulling of an equilibrium globule respectively. Conclusions We observed an evident cooperative effect between H-NS activity and the depletion of forces induced by PEG. General Significance Our data suggest a double role for H-NS in enhancing compaction while forming specific loops, which could be crucial in vivo for defining specific mesoscale domains in chromosomal regions in response to environmental changes.

ETNK1 kinase is responsible for the phosphorylation of ethanolamine to phosphoethanolamine (P-Et) (Kennedy, 1956, J Biol Chem). Recurrent somatic mutations occurring on ETNK1 were identified in about 13% of patients affected by atypical chronic myeloid leukemia (aCML), in 3-14% of chronic myelomonocytic leukemia (CMML), and in 20% of systemic mastocytosis (SM) patients with eosinophilia (Gambacorti-Passerini, 2015, Blood; Lasho, 2015, Blood Cancer J). ETNK1 mutations, encoding for H243Y, N244S/T/K, and G245V/A amino acid substitutions, cluster in a very narrow region of the ETNK1 catalytic domain and cause an impairment of ETNK1 enzymatic activity leading to a significant decrease in the intracellular concentration of P-Et (Gambacorti-Passerini, 2015, Blood). Despite this evidence, however, their oncogenic role remained largely unexplained. Here, we investigated the specific role of these mutations by using cellular CRISPR/Cas9 and ETNK1 overexpression models as well as aCML patients' samples. We showed that mutated ETNK1 causes a significant increase in mitochondrial activity (1.87 fold increase compared to WT; p=0.0002) and in ROS production (2.05 fold increase compared to WT; p Disclosures Rea: Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees. Gambacorti-Passerini:Bristol-Myers Squibb: Consultancy; Pfizer: Honoraria, Research Funding.

The deposition of amyloid-&beta, (A&beta, ) plaques in the brain is a significant pathological signature of Alzheimer&rsquo, s disease, correlating with synaptic dysfunction and neurodegeneration. Several compounds, peptides, or drugs have been designed to redirect or stop A&beta, 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 A&beta, 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 A&beta, aggregation/disaggregation processes. The results showed that the ability of free mApoE to destroy preformed A&beta, fibrils or to hinder the A&beta, 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 A&beta, aggregation processes.

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., ETNK1 mutations are recurrent in leukemia but how they contribute to oncogenesis is still unclear. Here, the authors show that ETNK1 mutations increase mitochondrial activity, ROS and H2AX levels and that these effects can be rescued upon phosphoethanolamine supplementation.

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., Summary: The non-receptor tyrosine kinase Arg and its isoforms modulate the extra cellular matrix production that is relevant in fibrosis and tumour growth, this may open future novel therapeutic approaches.

Chronic lymphocytic leukaemia (CLL) is one of the most common and incurable B cell leukaemia. CLL cells traffic between peripheral blood, bone marrow and secondary lymphatic tissues where interact with the microenvironment. These processes are affected by the mechanical-forces present in the environment and by the capability of the cells to sense the forces. In this context we demonstrated that Hematopoietic-cell-specific Lyn-substrate-1 (HS1) protein is a cytoskeletal regulator and a prognostic factor in CLL. We proved that interfering with HS1 expression impacts on the progression and homing of CLL cells. To further study HS1 role in CLL we knocked-down HS1 expression by CRISPR/CAS9 technology in a CLL cells line (MEC1). By RNAseq and network analysis on MEC1HS1ko vs MEC1UT we found that HS1-KO significantly affects the expression of molecules involved in: cell motility, adhesion, cell-cell communication, focal adhesion formation. In particular, we found LEF1, FAK and Cortactin genes are up-regulated in MEC1HS1ko, suggesting their involvement in the mechano-signalling pathway. To study the nano-mechanical properties of those cells we are performing AFM measurements of single cell. The results provide an evaluation of the cell stiffness that is related to its deformation in response to an externally applied force. By AFM we found that MEC1HS1ko cells are less stiff if compared with MEC1UT cells. This results demonstrates a putative role for HS1 in regulating the mechanical properties of CLL cells. Due to the prognostic value of HS1 we are currently performing AFM measurement on selected patients and healthy B cells.

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.

Chronic lymphocytic leukaemia (CLL) is one of the most common and incurable B cell leukaemia. CLL cells traffic between peripheral blood, bone marrow and secondary lymphatic tissues where interact with the microenvironment. These processes are affected by the mechanical-forces present in the environment and by the capability of the cells to sense the forces. In this context we demonstrated that Hematopoietic-cell-specific Lyn-substrate-1 (HS1) protein is a cytoskeletal regulator and a prognostic factor in CLL. We proved that interfering with HS1 expression impacts on the progression and homing of CLL cells. To further study HS1 role in CLL we knocked-down HS1 expression by CRISPR/CAS9 technology in a CLL cells line (MEC1). By RNAseq and network analysis on MEC1HS1ko vs MEC1UT we found that HS1-KO significantly affects the expression of molecules involved in: cell motility, adhesion, cell-cell communication, focal adhesion formation. In particular, we found LEF1, FAK and Cortactin genes are up-regulated in MEC1HS1ko, suggesting their involvement in the mechano-signalling pathway. To study the nano-mechanical properties of those cells we are performing AFM measurements of single cell. The results provide an evaluation of the cell stiffness that is related to its deformation in response to an externally applied force. By AFM we found that MEC1HS1ko cells are less stiff if compared with MEC1UT cells. This results demonstrates a putative role for HS1 in regulating the mechanical properties of CLL cells. Due to the prognostic value of HS1 we are currently performing AFM measurement on selected patients and healthy B cells. We are planning to study in depth the role of HS1 in regulating the mechanical properties of the leukemic cells and how this contributes to leukemia development, progression and resistance to therapy.

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 &alpha, 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 &alpha, 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.

Chronic lymphocytic leukaemia (CLL) is one of the most common and incurable B cell leukaemia. CLL cells traffic between peripheral blood, bone marrow and secondary lymphatic tissues where interact with a supportive microenvironment. These processes are affected by the mechanical forces present in the environment and by the capability of the cells to sense the forces. In order to migrate from a fluid environment like the blood to a significantly more viscous surrounding, B lymphocytes need to modify their cytoskeleton and consequently their rigidity. Those processes are known to be regulated by Hematopoietic-cell-specific Lyn-substrate-1 (HS1) protein, which promotes the homing processes and is involved in cell-cell communication and focal adhesions formation. From a nanomechanical point of view, cytoskeleton rearrangements can be observed as a difference in the force distribution along the cell and, ultimately, as a change of the overall rigidity of the cell. To measure this change, we used Atomic Force Microscopy (AFM) as an external pressure stimulus and we observed the cell deformation, which finally determines the value of its stiffness. By AFM we measured primary B lymphocytes from selected CLL patients and healthy donors, and we found a significant decrease of stiffness in leukaemia cells compared to healthy ones. We also performed Real-Time Deformability Cytometry (RT-DC) to test the rigidity of cells in flow condition. Finally, we tested the effect of a first line drug (Ibrutinib) on the mechanical properties of leukemic and healthy cells with both techniques in order to understand its effect on the mechanics of cells along their path to development of the disease.

Background Amyloid β (Aβ) 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 Aβ oligomers rather than formation of the senile plaques created by the deposition of large fibrillary aggregates of Aβ. However, several questions remain about the preliminary steps and the progression of Aβ oligomerization. Methods We show that the initial stages of the aggregation of fluorescently labeled Aβ 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 Aβ concentration. We determine the chemical binding affinity of fluorescently labeled Aβ for liposomes that have been recently shown to be pharmacologically active in vivo, reducing the Aβ burden within the brain. We also probe their capacity to hinder the Aβ oligomerization process in vitro. Conclusions We introduced a fluorescence assay allowing investigation of the earliest steps of Aβ oligomerization, the peptide involved in Alzheimer's disease. The assay proved to be sensitive even at Aβ 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 Aβ 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.

Methylation is one of the most important epigenetic mechanisms in eukaryotes. As a consequence of cytosine methylation, the binding of proteins that are implicated in transcription to gene promoters is severely hindered, which results in gene regulation and, eventually, gene silencing. To date, the mechanisms by which methylation biases the binding affinities of proteins to DNA are not fully understood; however, it has been proposed that changes in double-strand conformations, such as stretching, bending, and over-twisting, as well as local variations in DNA stiffness/flexibility may play a role. The present work investigates, at the single molecule level, the morphological consequences of DNA methylation in vitro. By tracking the atomic force microscopy images of single DNA molecules, we characterize DNA conformations pertaining to two different degrees of methylation. In particular, we observe that methylation induces no relevant variations in DNA contour lengths, but produces measurable incremental changes in persistence lengths. Furthermore, we observe that for the methylated chains, the statistical distribution of angles along the DNA coordinate length is characterized by a double exponential decay, in agreement with what is predicted for polyelectrolytes. The results reported herein support the claim that the biological consequences of the methylation process, specifically difficulties in protein-DNA binding, are at least partially due to DNA conformation modifications.

Two‐photon laser writing is used here to fabricate 3D proteinaceous microstructures with photothermal functionality in the near‐infrared spectral region and tunable elasticity. The photo‐cross‐linking is initiated in bovine serum albumin (BSA) by rose bengal or methylene blue and the photo‐thermal effect arises from gold non‐spherically symmetric nanoparticles dispersed in the ink. Massive energy transfer of the plasmonic resonances of the gold nanoparticles to methylene blue prevents effective photo‐crosslinking of BSA. However, stable microstructures with photo‐thermal functionality can be fabricated in the rose bengal proteinaceous inks. On these microstructures, with a gold atom concentration as low as 1% w/w, a highly localized temperature increase can be quickly (≅1 s) reached and maintained under continuous wave laser irradiation at 800 nm. The photothermal efficiency under continuous wave laser irradiation depends on the thickness of the microstructure and can reach 12.2 ± 0.4 °C W−1 These proteinaceous microstructures represent therefore a promising platform for future applications in the fields like physical stimulation of cells for regenerative nanomedicine.

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.

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-1 alpha transcriptional activity is the LOX gene, which codes for the inactive proenzyme (Pro-Lox) from which, after extra cellular 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-1 alpha 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-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.

Binding of ligands to DNA gives rise to several relevant biological and biomedical effects. Here, through the use of atomic force microscopy (AFM), we studied the consequences of drug binding on the morphology of single DNA molecules. In particular, we quantitatively analyzed the effects of three different DNA-binding molecules (doxorubicin, ethidium bromide, and netropsin) that exert various pharmacologic and therapeutic effects. The results of this study show the consequences of intercalation and groove molecular binding on DNA conformation. These single-molecule measurements demonstrate morphological features that reflect the specific modes of drug-DNA interaction. This experimental approach may have implications in the design of therapeutically effective agents.

Herein, we study the nanomechanical characteristics of single DNA molecules in the presence of DNA binders, including intercalating agents (ethidium bromide and doxorubicin), a minor groove binder (netropsin) and a typical alkylating damaging agent (cisplatin). We have used magnetic tweezers manipulation techniques, which allow us to measure the contour and persistence lengths together with the bending and torsional properties of DNA. For each drug, the specific variations of the nanomechanical properties induced in the DNA have been compared. We observed that the presence of drugs causes a specific variation in the DNA extension, a shift in the natural twist and a modification of bending dependence on the imposed twist. By introducing a naive model, we have justified an anomalous correlation of torsion data observed in the presence of intercalators. Finally, a data analysis criterion for discriminating between different molecular interactions among DNA and drugs has been suggested.

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.

We have investigated the influence on adhesion and proliferation of NIH 3T3 fibroblasts of the surface morphology of cluster assembled carbon films deposited by Supersonic Cluster Beam Deposition. Nanostructured carbon films exhibit a multi-scale morphology, which resembles the surface structure of the extracellular matrix, and possess a high specific area, while being relatively smooth at all scales. Correlations between measured morphological parameters and adaptive cell response have been brought out. High specific area and smoothness appear to conceivably favour both the early attachment of plated cells and the long-term survival of adherent cells. Moreover, nano-structured carbon films affect the cells morphology as well as the extension and the number of the focal contacts.

The ability of detecting the subtle variations occurring, among different individuals, within specific DNA sequences encompassed in highly polymorphic genes discloses new applications in genomics and diagnostics. DQB1 is a gene of the HLA-II DQ locus of the Human Leukocyte Antigens (HLA) system. The polymorphisms of the trait of the DQB1 gene including codons 52-57 modulate the susceptibility to a number of severe pathologies. Moreover, the donor-receiver tissue compatibility in bone marrow transplantations is routinely assessed through crossed genotyping of DQB and DQA. For the above reasons, the development of rapid, reliable and cost-effective typing technologies of DQB1 in general, and more specifically of the codons 52-57, is a relevant although challenging task. Quantitative assessment of the fluorescence resonance energy transfer (FRET) efficiency between chromophores labelling the opposite ends of gene-specific oligonucleotide probes has proven to be a powerful tool to type DNA polymorphisms with single-nucleotide resolution. The FRET efficiency can be most conveniently quantified by applying a time-resolved fluorescence analysis methodology, i.e. time-correlated single-photon counting, which allows working on very diluted template specimens and in the presence of fluorescent contaminants. Here we present a full in-vitro characterization of the fluorescence responses of two probes when hybridized to oligonucleotide mixtures mimicking all the possible genotypes of the codons 52-57 trait of DQB1 (8 homozygous and 28 heterozygous). We show that each genotype can be effectively tagged by the combination of the fluorescence decay constants extrapolated from the data obtained with such probes.

We use the "magnetic tweezers" technique to reveal the structural transitions that DNA undergoes in the force-torsion space. In particular, we focus on regions corresponding to negative supercoiling. These regions are characterized by the formation of so-called denaturation bubbles, which have an essential role in the replication and transcription of DNA. We experimentally map the region of the force-torsion space where the denaturation takes place. We observe that large fluctuations in DNA extension occur at one of the boundaries of this region, i.e., when the formation of denaturation bubbles and of plectonemes are competing. To describe the experiments, we introduce a suitable extension of the classical model. The model correctly describes the position of the denaturation regions, the transition boundaries, and the measured values of the DNA extension fluctuations., Comment: 5 pages and 4 figure

Alzheimer disease (AD) is the most prevalent neurodegenerative disease, characterized by an increased deposition of β-amyloid (Abeta) within the central nervous system, leading to neuronal death. The availability of effective models, in which confirming novel pathogenic hypotheses and developing therapeutic targets, represents a very important goal for the field of AD. Fibroblasts from these patients may be relevant models in which addressing these issues, as they display biochemical alterations mirroring SNC ones. In this work, fibroblasts obtained from controls were studied after exposure to nonfibrillar Abeta 1-42, showing decreased glutamate uptake, similar to that observed in AD cells, in absence of transporters modifications. Nonfibrillar Abeta 1-42 was able to induce in control cells mitochondrial alterations and p38-phosphorylation, mirroring similar alterations found in AD fibroblasts. Under our experimental conditions, this treatment induced neither apoptosis nor necrosis. To investigate a putative role of p38-modulation in mediating nonfibrillar Abeta 1-42 toxicity, fibroblasts from controls were pretreated with retinoic-acid, and SB203580, a p38-inhibitor. These pretreatments prevented both p38-phosphorylation and glutamate uptake inhibition. Our results suggest that nonfibrillar Abeta 1-42 downregulates glutamate transporters activity interfering with p38-activation and mitochondrial stress. Thus, modulating complex kinase signaling pathway might represent a future therapeutic target in AD.

β-Amyloid peptide (Aβ) plays a key role in the pathogenesis of Alzheimer disease (AD). Monomeric Aβ undergoes aggregation, forming oligomers and fibrils, resulting in the deposition of plaques in the brain of AD patients. A widely used protocol for fibril formation in vitro is based on incubation of the peptide at low pH and ionic strength, which generates Aβ fibrils several microns long. What happens to such fibrils once they are brought to physiological pH and ionic strength for biological studies is not fully understood. In this investigation, we show that these changes strongly affect the morphology of fibrils, causing their fragmentation into smaller ones followed by their aggregation into disordered structures. We show that an increase in pH is responsible for fibril fragmentation, while increased ionic strength is responsible for the aggregation of fibril fragments. This behavior was confirmed on different batches of peptide either produced by the same company or of different origin. Similar aggregates of short fibrils are obtained when monomeric peptide is incubated under physiological conditions of pH and ionic strength, suggesting that fibril morphology is independent of the fibrillation protocol but depends on the final chemical environment. This was also confirmed by experiments with cell cultures showing that the toxicity of fibrils with different initial morphology is the same after addition to the medium. This information is of fundamental importance when Aβ fibrils are prepared in vitro at acidic pH and then diluted into physiological buffer for biological investigations.

The electrical properties of cluster-assembled nanostructured palladium oxide $({\text{ns-PdO}}_{x})$ thin films grown by supersonic cluster beam deposition have been characterized by means of a customized ac current-sensing atomic force microscope. Scanning impedance microscopy is shown to provide a deep picture of the electrical properties of thin nanostructured interfaces even in the case of very soft and poorly adherent films. In particular, the dielectric constant of ${\text{ns-PdO}}_{x}$ can be quantitatively determined as well as its $I\text{\ensuremath{-}}V$ characteristics. Moreover, the measurement of the tip-sample parasitic capacitance can be exploited to probe the overall mesoscale conductive character of thin films and to give a complementary and more precise view of the oxidation of ${\text{ns-PdO}}_{x}$ obtained by x-ray photoemission spectroscopy.

We report the structural and tribological characterization of nanostructured CNx thin films produced by the deposition of a supersonic carbon cluster beam assisted by nitrogen ion bombardment. The influence of the deposition parameters on the chemical composition and structure of the films has been systematically studied by X-ray photoelectron spectroscopy, elastic recoil detection analysis, transmission electron microscopy and atomic force microscopy. Depending on the deposition parameters, the films show a structure ranging from amorphous to disordered graphitic with interlinked planes. Nitrogen content depends on the nitrogen ion kinetic energy. The films have a very low density with a high surface roughness. Friction measurements at the nanoscale show a correlation between nitrogen content and mechanical properties of the system.

Light scattering techniques are widely used in many fields of condensed and sof t matter physics. Usually these methods are based on the study of the scattered light in the far field. Recently, a new family of near field detection schemes has been developed, mainly for the study of small angle light scattering. These techniques are based on the detection of the light intensity near to the sample, where light scattered at different directions overlaps but can be distinguished by Fourier transform analysis. Here we report for the first time data obtained with a dynamic near field scattering instrument, measuring both polarized and depolarized scattered light. Advantages of this procedure over the traditional far field detection include the immunity to stray light problems and the possibility to obtain a large number of statistical samples for many different wave vectors in a single instantaneous measurement. By using the proposed technique we have measured the translational and rotational diffusion coefficients of rod-like colloidal particles. The obtained data are in very good agreement with the data acquired with a traditional light scattering apparatus., Comment: Published in Optics Express. This version has changes in bibliography

Light scattering detection in the near field, a rapidly expanding family of scattering techniques, has recently proved to be an appropriate procedure for performing dynamic measurements. Here we report an algorithm, first suggested by Oh et al. (Phys. Rev. E 69, 21106 (2004)), based on the evaluation of the Exposure Time Dependent Spectrum (ETDS), which makes it possible to measure the fast dynamics of a colloidal suspension with the aid of a simple near field scattering apparatus and a CCD camera. The algorithm consists in acquiring static spectra in the near field at different exposure times, so that the measured decay times are limited only by the exposure time of the camera and not by its frame rate. The experimental set-up is based on a modified microscope, where the light scattered in the near field is collected by a commercial objective, but (unlike in standard microscopes) the light source is a He-Ne laser which increases the instrument sensitivity. The apparatus and the algorithm have been validated by considering model systems of standard spherical nano-particle.

We investigated the electrical transport properties of nanostructured carbon and carbon/titanium oxide nanocomposite films produced by supersonic cluster beam deposition and irradiated by highly focused vacuum UV photon beam. We have observed a relevant increase of the density of states at Fermi level, suggesting that the films acquire a ¿metallic¿ character. This is confirmed by the increment of the conductivity of four orders of magnitude for pure nanostructured carbon films and at least eight orders of magnitude for films containing 9 at. % of titanium. A partial reversibility of the process is observed by exposing the modified films to molecular oxygen or directly to air. We demonstrate the capability of writing micrometric conductive strips (2-3 µm width and 60 µm length) and controlling the variation of the conductivity as a function of the titanium concentration.

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 β-amyloid (Aβ) 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 Aβ aggregates, and to affect Aβ efflux across the BBB was assessed in vitro using a transwell system. The results showed that discoidal HDL displayed a superior capability to promote Aβ efflux in vitro (9 × 10-5 cm/min), when compared to apoA-I in other lipidation states. In particular, no effect on Aβ efflux was detected when apoA-I was in mature spherical HDL, suggesting that apoA-I conformation, and lipidation could play a role in Aβ 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 Aβ fibrils by decreasing the order of the fibril structure (-24%) and the β-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.