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.

Engineering tandem CD33xCD146 CAR CIK (cytokine-induced killer) cells to target the acute myeloid leukemia niche.

In acute myeloid leukemia (AML), malignant stem cells hijack the normal bone marrow niche where they are largely protected from the current therapeutic approaches. Thus, eradicating these progenitors is the ultimate challenge in the treatment of this disease. Specifically, the development of chimeric antigen receptors (CARs) against distinct mesenchymal stromal cell subpopulations involved in the maintenance of leukemic stem cells within the malignant bone marrow microenvironment could represent a new strategy to improve CAR T-cell therapy efficacy, which is still unsuccessful in AML. As a proof of concept, we generated a novel prototype of Tandem CAR, with one specificity directed against the leukemic cell marker CD33 and the other against the mesenchymal stromal cell marker CD146, demonstrating its capability of simultaneously targeting two different cell types in a 2D co-culture system. Interestingly, we could also observe an in vitro inhibition of CAR T cell functionality mediated by stromal cells, particularly in later effector functions, such as reduction of interferon-gamma and interleukin-2 release and impaired proliferation of the CAR+ effector Cytokine-Induced Killer (CIK) cells. Taken together, these data demonstrate the feasibility of a dual targeting model against two molecules, which are expressed on two different target cells, but also highlight the immunomodulatory effect on CAR CIK cells exerted by stromal cells, confirming that the niche could be an obstacle to the efficacy of CAR T cells. This aspect should be considered in the development of novel CAR T cell approaches directed against the AML bone marrow niche.

IL-3-zetakine combined with a CD33 costimulatory receptor as a dual CAR approach for safer and selective targeting of AML.

Acute myeloid leukemia (AML) still represents an unmet clinical need for adult and pediatric patients. Adoptive cell therapy by chimeric antigen receptor (CAR)-engineered T cells demonstrated a high therapeutic potential, but further development is required to ensure a safe and durable disease remission in AML, especially in elderly patients. To date, translation of CAR T-cell therapy in AML is limited by the absence of an ideal tumor-specific antigen. CD123 and CD33 are the 2 most widely overexpressed leukemic stem cell biomarkers but their shared expression with endothelial and hematopoietic stem and progenitor cells increases the risk of undesired vascular and hematologic toxicities. To counteract this issue, we established a balanced dual-CAR strategy aimed at reducing off-target toxicities while retaining full functionality against AML. Cytokine-induced killer (CIK) cells, coexpressing a first-generation low affinity anti-CD123 interleukin-3-zetakine (IL-3z) and an anti-CD33 as costimulatory receptor without activation signaling domains (CD33.CCR), demonstrated a powerful antitumor efficacy against AML targets without any relevant toxicity on hematopoietic stem and progenitor cells and endothelial cells. The proposed optimized dual-CAR cytokine-induced killer cell strategy could offer the opportunity to unleash the potential of specifically targeting CD123+/CD33+ leukemic cells while minimizing toxicity against healthy cells.

A Minimally Invasive and Highly Effective Extracorporeal CO2 Removal Device Combined With a Continuous Renal Replacement Therapy.

OBJECTIVES: Extracorporeal carbon dioxide removal is used to treat patients suffering from acute respiratory failure. However, the procedure is hampered by the high blood flow required to achieve a significant CO2 clearance. We aimed to develop an ultralow blood flow device to effectively remove CO2 combined with continuous renal replacement therapy (CRRT). DESIGN: Preclinical, proof-of-concept study. SETTING: An extracorporeal circuit where 200 mL/min of blood flowed through a hemofilter connected to a closed-loop dialysate circuit. An ion-exchange resin acidified the dialysate upstream, a membrane lung to increase Pco2 and promote CO2 removal. PATIENTS: Six, 38.7 ± 2.0-kg female pigs. INTERVENTIONS: Different levels of acidification were tested (from 0 to 5 mEq/min). Two l/hr of postdilution CRRT were performed continuously. The respiratory rate was modified at each step to maintain arterial Pco2 at 50 mm Hg. MEASUREMENTS AND MAIN RESULTS: Increasing acidification enhanced CO2 removal efficiency of the membrane lung from 30 ± 5 (0 mEq/min) up to 145 ± 8 mL/min (5 mEq/min), with a 483% increase, representing the 73% ± 7% of the total body CO2 production. Minute ventilation decreased accordingly from 6.5 ± 0.7 to 1.7 ± 0.5 L/min. No major side effects occurred, except for transient tachycardia episodes. As expected from the alveolar gas equation, the natural lung Pao2 dropped at increasing acidification steps, given the high dissociation between the oxygenation and CO2 removal capability of the device, thus Pao2 decreased. CONCLUSIONS: This new extracorporeal ion-exchange resin-based multiple-organ support device proved extremely high efficiency in CO2 removal and continuous renal support in a preclinical setting. Further studies are required before clinical implementation.

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.

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.

Bedside surgery in the newborn infants: survey of the Italian society of pediatric surgery.

INTRODUCTION: This is the report of the first official survey from the Italian Society of Pediatric Surgery (ISPS) to appraise the distribution and organization of bedside surgery in the neonatal intensive care units (NICU) in Italy. METHODS: A questionnaire requesting general data, staff data and workload data of the centers was developed and sent by means of an online cloud-based software instrument to all Italian pediatric surgery Units. RESULTS: The survey was answered by 34 (65%) out of 52 centers. NICU bedside surgery is reported in 81.8% of the pediatric surgery centers. A lower prevalence of bedside surgical practice in the NICU was reported for Southern Italy and the islands than for Northern Italy and Central Italy (Southern

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.

Extracorporeal Chloride Removal by Electrodialysis. A Novel Approach to Correct Acidemia.

Rationale: Acidemia is a severe condition among critically ill patients. Despite lack of evidence, sodium bicarbonate is frequently used to correct pH; however, its administration is burdened by several side effects. We hypothesized that the reduction of plasma chloride concentration could be an alternative strategy to correct acidemia.Objectives: To evaluate feasibility, safety, and effectiveness of a novel strategy to correct acidemia through extracorporeal chloride removal by electrodialysis.Methods: Ten swine (six treated and four control animals) were sedated, mechanically ventilated and connected to an extracorporeal electrodialysis device capable of selectively removing chloride. In random order, an arterial pH of 7.15 was induced either through reduction of ventilation (respiratory acidosis) or through lactic acid infusion (metabolic acidosis). Acidosis was subsequently sustained for 12-14 hours. In treatment pigs, soon after reaching target acidemia, electrodialysis was started to restore pH.Measurements and Main Results: During respiratory acidosis, electrodialysis reduced plasma chloride concentration by 26 ± 5 mEq/L within 6 hours (final pH = 7.36 ± 0.04). Control animals exhibited incomplete and slower compensatory response to respiratory acidosis (final pH = 7.29 ± 0.03; P < 0.001). During metabolic acidosis, electrodialysis reduced plasma chloride concentration by 15 ± 3 mEq/L within 4 hours (final pH = 7.34 ± 0.07). No effective compensatory response occurred in control animals (final pH = 7.11 ± 0.08; P < 0.001). No complications occurred.Conclusions: We described the first in vivo application of an extracorporeal system targeted to correct severe acidemia by lowering plasma chloride concentration. Extracorporeal chloride removal by electrodialysis proved to be feasible, safe, and effective. Further studies are warranted to assess its performance in the presence of impaired respiratory and renal functions.

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.

Not Just a Pathogen? Description of a Plant-Beneficial Pseudomonas syringae Strain.

Plants develop in a microbe-rich environment and must interact with a plethora of microorganisms, both pathogenic and beneficial. Indeed, such is the case of Pseudomonas, and its model organisms P. fluorescens and P. syringae, a bacterial genus that has received particular attention because of its beneficial effect on plants and its pathogenic strains. The present study aims to compare plant-beneficial and pathogenic strains belonging to the P. syringae species to get new insights into the distinction between the two types of plant-microbe interactions. In assays carried out under greenhouse conditions, P. syringae pv. syringae strain 260-02 was shown to promote plant-growth and to exert biocontrol of P. syringae pv. tomato strain DC3000, against the Botrytis cinerea fungus and the Cymbidium Ringspot Virus. This P. syringae strain also had a distinct volatile emission profile, as well as a different plant-colonization pattern, visualized by confocal microscopy and gfp labeled strains, compared to strain DC3000. Despite the different behavior, the P. syringae strain 260-02 showed great similarity to pathogenic strains at a genomic level. However, genome analyses highlighted a few differences that form the basis for the following hypotheses regarding strain 260-02. P. syringae strain 260-02: (i) possesses non-functional virulence genes, like the mangotoxin-producing operon Mbo; (ii) has different regulation pathways, suggested by the difference in the autoinducer system and the lack of a virulence activator gene; (iii) has genes encoding DNA methylases different from those found in other P. syringae strains, suggested by the presence of horizontal-gene-transfer-obtained methylases that could affect gene expression.

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.

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.

Acute abdominal pain in an adolescent girl with an ovarian yolk sac tumor.

Yolk sac tumor (YST) is a rare tumor that usually occurs in the first two decades of life. It is considered the second most common malignant germ cell tumor of the ovary, characterized by a rapid growth and a bad prognosis due to the frequent metastasis. We report the case of a 12-year-old girl who came to our observation for an acute abdominal pain. Clinical examination evidenced a vague mass in the suprapubic region and a lower abdomen tenderness, the US imaging revealed a complex lesion of the left ovary (19 x 13 cm) and the alpha-fetoprotein (AFP) resulted high (5858 ng/mL). Computed tomography (CT) revealed a large pelvic mass. The treatment consisted of debulking surgery of yolk sac tumor followed by 4 cycles of BEP protocol (Bleomycin, Etoposide, Cisplatin). After 3 years of follow-up there was no evidence of disease recurrence. (www.actabiomedica.it).

Ischemic Conditions Affect Rerouting of Tau Protein Levels: Evidences for Alteration in Tau Processing and Secretion in Hippocampal Neurons.

The spreading of misfolded protein species contributes to the propagation of harmful mediators in proteinopathies, including Alzheimer's disease (AD). Cellular stress circumstances, such as abnormal protein accumulation or nutrient deprivation, elicit the secretion of soluble misprocessed proteins and insoluble aggregates via multiple mechanisms of unconventional secretion. One of them consists in the rerouting of autophagic vacuoles towards exocytosis, an unconventional type of autophagy mediated by caspase-3 activation under starvation. Ischemic injury is a starvation condition characterized by oxygen/nutrient deprivation, whose contribution in AD onset has definitely been endorsed. Thus, we investigated the effect of oxygen-glucose deprivation (OGD), an experimental condition mimicking cerebral ischemia, in search of alteration in Tau processing and secretion in hippocampal neurons primary cultures. Our results showed that OGD caused alterations in Tau phosphorylation and processing, paralleled by an induction of its secretion. Interestingly, together with caspase-3 activation, full-length (FL) and fragmented Tau forms were secreted by their own or through a heterogeneous population of microvesicles (MVs), including autophagosome marker LC3-positive vesicles. Accordingly, confocal microscopy revealed a partial colocalization of intracellular Tau and LC3. Summarizing, our findings indicate that OGD alters Tau intracellular levels and protein processing. Consequently, Tau clearance was stimulated through multiple mechanisms related to unconventional Tau secretion, including exophagy. However, the activation of this response represent a double edge sword, because it could contribute to the spreading of misfolded Tau, a neurodegeneration pathway in AD and other tauopathies.