Native extracellular matrix probes to target patient- and tissue-specific cell-microenvironment interactions by force spectroscopy.

Atomic Force Microscopy (AFM) is successfully used for the quantitative investigation of the cellular mechanosensing of the microenvironment. To this purpose, several force spectroscopy approaches aim at measuring the adhesive forces between two living cells and also between a cell and an appropriate reproduction of the extracellular matrix (ECM), typically exploiting tips suitably functionalised with single components (e.g. collagen, fibronectin) of the ECM. However, these probes only poorly reproduce the complexity of the native cellular microenvironment and consequently of the biological interactions. We developed a novel approach to produce AFM probes that faithfully retain the structural and biochemical complexity of the ECM; this was achieved by attaching to an AFM cantilever a micrometric slice of native decellularised ECM, which was cut by laser microdissection. We demonstrate that these probes preserve the morphological, mechanical, and chemical heterogeneity of the ECM. Native ECM probes can be used in force spectroscopy experiments aimed at targeting cell-microenvironment interactions. Here, we demonstrate the feasibility of dissecting mechanotransductive cell-ECM interactions in the 10 pN range. As proof-of-principle, we tested a rat bladder ECM probe against the AY-27 rat bladder cancer cell line. On the one hand, we obtained reproducible results using different probes derived from the same ECM regions; on the other hand, we detected differences in the adhesion patterns of distinct bladder ECM regions (submucosa, detrusor, and adventitia), in line with the disparities in composition and biophysical properties of these ECM regions. Our results demonstrate that native ECM probes, produced from patient-specific regions of organs and tissues, can be used to investigate cell-microenvironment interactions and early mechanotransductive processes by force spectroscopy. This opens new possibilities in the field of personalised medicine.

Micro-mechanical fingerprints of the rat bladder change in actinic cystitis and tumor presence.

Tissue mechanics determines tissue homeostasis, disease development and progression. Bladder strongly relies on its mechanical properties to perform its physiological function, but these are poorly unveiled under normal and pathological conditions. Here we characterize the mechanical fingerprints at the micro-scale level of the three tissue layers which compose the healthy bladder wall, and identify modifications associated with the onset and progression of pathological conditions (i.e., actinic cystitis and bladder cancer). We use two indentation-based instruments (an Atomic Force Microscope and a nanoindenter) and compare the micromechanical maps with a comprehensive histological analysis. We find that the healthy bladder wall is a mechanically inhomogeneous tissue, with a gradient of increasing stiffness from the urothelium to the lamina propria, which gradually decreases when reaching the muscle outer layer. Stiffening in fibrotic tissues correlate with increased deposition of dense extracellular matrix in the lamina propria. An increase in tissue compliance is observed before the onset and invasion of the tumor. By providing high resolution micromechanical investigation of each tissue layer of the bladder, we depict the intrinsic mechanical heterogeneity of the layers of a healthy bladder as compared with the mechanical properties alterations associated with either actinic cystitis or bladder tumor.

Adhesion force spectroscopy with nanostructured colloidal probes reveals nanotopography-dependent early mechanotransductive interactions at the cell membrane level.

Mechanosensing, the ability of cells to perceive and interpret the microenvironmental biophysical cues (such as the nanotopography), impacts strongly cellular behaviour through mechanotransductive processes and signalling. These events are predominantly mediated by integrins, the principal cellular adhesion receptors located at the cell/extracellular matrix (ECM) interface. Because of the typical piconewton force range and nanometre length scale of mechanotransductive interactions, achieving a detailed understanding of the spatiotemporal dynamics occurring at the cell/microenvironment interface is challenging; sophisticated interdisciplinary methodologies are required. Moreover, an accurate control over the nanotopographical features of the microenvironment is essential, in order to systematically investigate and precisely assess the influence of the different nanotopographical motifs on the mechanotransductive process. In this framework, we were able to study and quantify the impact of microenvironmental nanotopography on early cellular adhesion events by means of adhesion force spectroscopy based on innovative colloidal probes mimicking the nanotopography of natural ECMs. These probes provided the opportunity to detect nanotopography-specific modulations of the molecular clutch force loading dynamics and integrin clustering at the level of single binding events, in the critical time window of nascent adhesion formation. Following this approach, we found that the nanotopographical features are responsible for an excessive force loading in single adhesion sites after 20-60 s of interaction, causing a drop in the number of adhesion sites. However, by manganese treatment we demonstrated that the availability of activated integrins is a critical regulatory factor for these nanotopography-dependent dynamics.

Non-ohmic behavior and resistive switching of Au cluster-assembled films beyond the percolation threshold.

Networks based on nanoscale resistive switching junctions are considered promising for the fabrication of neuromorphic computing architectures. To date random networks of nanowires, nanoparticles, and metal clusters embedded in a polymeric matrix or passivated by shell of ligands or oxide layers have been used to produce resistive switching systems. The strategies applied to tailor resistive switching behavior are currently based on the careful control of the volume fraction of the nanoscale conducting phase that must be fixed close to the electrical percolation threshold. Here, by blending laboratory and computer experiments, we demonstrate that metallic nanostructured Au films fabricated by bare gold nanoparticles produced in the gas phase and with thickness well beyond the electrical percolation threshold, show a non-ohmic electrical behavior and complex and reproducible resistive switching. We observe that the nanogranular structure of the Au films does not evolve with thickness: this introduces a huge number of defects and junctions affecting the electrical transport and causing a dynamic evolution of the nanoscale electrical contacts under the current flow. To uncover the origin of the resistive switching behavior in Au cluster-assembled films, we developed a simple computational model for determining the evolution of a model granular film under bias conditions. The model exploits the information provided by experimental investigation about the nanoscale granular morphology of real films. Our results show that metallic nanogranular materials have functional properties radically different from their bulk counterparts, in particular nanostructured Au films can be fabricated by assembling bare gold clusters which retain their individuality to produce an all-metal resistive switching system.

Would induction of dopamine homeostasis via coupling genetic addiction risk score (GARS®) and pro-dopamine regulation benefit benzodiazepine use disorder (BUD)?

Prescriptions for Benzodiazepines (BZDs) have risen continually. According to national statistics, the combination of BZDs with opioids has increased since 1999. BZDs (sometimes called "benzos") work to calm or sedate a person by raising the level of the inhibitory neurotransmitter GABA in the brain. In terms of neurochemistry, BZDs act at the GABAA receptors to inhibit excitatory neurons, reducing VTA glutaminergic drive to reduce dopamine release at the Nucleus accumbens. Benzodiazepine Use Disorder (BUD) is very difficult to treat, partly because BZDs are used to reduce anxiety which paradoxically induces hypodopaminergia. Considering this, we are proposing a paradigm shift. Instead of simply targeting chloride channel direct GABAA receptors for replacement or substitution therapy, we propose the induction of dopamine homeostasis. Our rationale is supported by the well-established notion that the root cause of drug and non-drug addictions (i.e. Reward Deficiency Syndrome [RDS]), at least in adults, involve dopaminergic dysfunction and heightened stress. This proposition involves coupling the Genetic Addiction Risk Score (GARS) with a subsequent polymorphic matched genetic customized Pro-Dopamine Regulator known as KB220ZPBM (Precision Behavioral Management). Induction of dopamine homeostasis will be clinically beneficial in attempts to combat BUD for at least three reasons: 1) During detoxification of alcoholism, the potential induction of dopamine regulation reduces the need for BZDs; 2) A major reason for BZD abuse is because people want to achieve stress reduction and subsequently, the potential induction of dopamine regulation acts as an anti-stress factor; and 3) BUD and OUD are known to reduce resting state functional connectivity, and as such, potential induction of dopamine regulation enhances resting state functional connectivity. Future randomized placebo-controlled studies will investigate this forward thinking proposed novel modality.

Evaluación de factores que inciden en el promedio de permanencia en UCIP por pacientes crónicos dependientes de tecnología del subsector publico / Factors that affect extension of stay in the PICU chronic technology dependent in the public sector

En los últimos años, el avance tecnológico permitió disminuir la mortalidaden Unidades de Cuidados Intensivos Pediátricos (UCIP) e incrementar lasupervivencia con secuelas dependientes de tecnología. Al alcanzar laestabilidad clínica, los pacientes deben ser externados con internacióndomiciliaria; muchos de ellos carecen de cobertura social o financiamiento,quedan internados y ocupan así camas críticas para pacientes agudos.ObjetivosIdentificar los factores generales que prolongan la estadía de pacientes crónicosdependientes de tecnología (CDT) internados en las UCIP del subsectorpúblico. Determinar la frecuencia de factores relacionados con el retrasoen la externación de esos niños. Describir las características, la evolución, laincidencia y la prevalencia de la población de pacientes CDT. Especificar lascausas médicas y no médicas que prolongan la estadía de pacientes CDT.MétodosSe realizó un estudio descriptivo multicéntrico observacional en cinco UCIPpolivalentes: tres de la Ciudad Autónoma de Buenos Aires y dos de la Provinciade Buenos Aires. Se incluyó a los pacientes CDT internados en las UCIP entre el1/6/2014 y el 31/3/2015. Se registraron datos poblacionales globales de incidencia,de prevalencia e individuales para el análisis de las dificultades del egreso.ResultadosSe estudió a 76 pacientes CDT, que representaron un 3,36% de los ingresos y el28% de la estadía global. La incidencia global de pacientes CDT fue del 2,12%. Laprevalencia inicial fue del 25%; la segunda, post IRAB (infecciones respiratoriasagudas bajas), del 26%; y la tercera, del 31% sin diferencias significativas.ConclusionesLas causas financieras, los problemas de vivienda y la situación social complejason los obstáculos que más demoran la externación al domicilio de pacientesCDT. Debido a la prolongación de estadía en UCIP a la espera de externación,las camas críticas ocupadas no permitieron el ingreso de 132 pacientes agudosen las cinco unidades.

Bottom-up engineering of the surface roughness of nanostructured cubic zirconia to control cell adhesion.

Nanostructured cubic zirconia is a strategic material for biomedical applications since it combines superior structural and optical properties with a nanoscale morphology able to control cell adhesion and proliferation. We produced nanostructured cubic zirconia thin films at room temperature by supersonic cluster beam deposition of nanoparticles produced in the gas phase. Precise control of film roughness at the nanoscale is obtained by operating in a ballistic deposition regime. This allows one to study the influence of nanoroughness on cell adhesion, while keeping the surface chemistry constant. We evaluated cell adhesion on nanostructured zirconia with an osteoblast-like cell line using confocal laser scanning microscopy for detailed morphological and cytoskeleton studies. We demonstrated that the organization of cytoskeleton and focal adhesion formation can be controlled by varying the evolution of surface nanoroughness.

Nano-indentation of a room-temperature ionic liquid film on silica: a computational experiment.

We investigate the structure of the [bmim][Tf(2)N]/silica interface by simulating the indentation of a thin (4 nm) [bmim][Tf(2)N] film by a hard nanometric tip. The ionic liquid/silica interface is represented in atomistic detail, while the tip is modelled by a spherical mesoscopic particle interacting via an effective short-range potential. Plots of the normal force (F(z)) on the tip as a function of its distance from the silica surface highlight the effect of weak layering in the ionic liquid structure, as well as the progressive loss of fluidity in approaching the silica surface. The simulation results for F(z) are in near-quantitative agreement with new AFM data measured on the same [bmim][Tf(2)N]/silica interface under comparable thermodynamic conditions.

Adhesive-free colloidal probes for nanoscale force measurements: production and characterization.

We describe novel approaches for the production and characterization of epoxy- and adhesive-free colloidal probes for atomic force microscopy (AFM). Borosilicate glass microspheres are strongly attached to commercial AFM cantilevers exploiting the capillary adhesion force due to the formation of a water meniscus, and then a thermal annealing of the sphere-cantilever system at a temperature slightly below the softening point of borosilicate glass. Controlling the wettability of the surfaces involved turned out to be a crucial element for the control of surface adhesion and for the implementation of a completely adhesive-free production method of colloidal probes. Moreover, we present a statistical characterization protocol of the probe dimensions and roughness based on the AFM inverse imaging of colloidal probes on spiked gratings. We have assessed the influence of defects of the grating on the characterization of the probe, and discussed the accuracy of our characterization technique in comparison to the methods based on scanning electron or optical microscopy, or on the manual analysis of AFM inverse images.

Antibodies for therapeutic uses and the evolution of biotechniques.

Protein therapeutics are playing an expanding role in modern medicinal chemistry. Among them, native or engineered molecules exploiting the binding and catalytic potential of the immune repertoire form an extremely exciting and emerging business area. They represent by far the single largest category of biopharmaceutical substances under investigation. The fast increase of this pharmaceutical category paralleled the scientific and technical progress from murine to chimeric, humanized and, finally, human engineered antibodies. Indeed, the development of the phage display technology, allowing libraries of shuffled murine or human antibody binding domains to be screened for affinity against a selected target antigen or activity against a specific reaction substrate, open new perspectives, disclosing the opportunity to circumvent restrictions inherent to the in vivo immunisation. Transgenic technology represents another powerful method for generating fully human monoclonal antibodies against a wide variety of drug targets, while recombinant technology continues to evolve, improving the pharmacodynamic and pharmacokinetic properties of antibody therapeutics, with the production of different antibody constructs or formats, such as bispecific antibodies, diabodies and others, and different functional activities, such as catalysis, cellular internalisation and antigen-mimicking. The aim of the present review is to overview native or recombinant antibodies while discussing the underlying antibody technology, with the aim to favour understanding of the antibody therapeutics that are in use or will enter market in the near future.

Nanometric ionic-liquid films on silica: a joint experimental and computational study.

Atomic force microscopy images for [bmim][Tf(2)N] films deposited at ambient conditions by drop-casting show a population of terraced islands of mesoscopic area (1-100 µ(2)) and ∼50 nm height. The regularity of terraces and steps, stiff mechanical properties and a fragile fracture mode all suggest that the islands are solid-like, even though bulk [bmim][Tf(2)N] is liquid at the temperature of the experiment. Molecular dynamics simulations for a homogeneous [bmim][Tf(2)N] film 4 nm thick on silica also display marked layering in proximity to silica of periodicity closely matching the experimental estimate of the step height. The density modulation of the simulated sample, however, decays into an approximatively homogeneous and fluid-like density distribution ∼2 nm from the solid surface. The detailed comparison of experiments and simulations is contained in the closing section of the paper.

[Adoptive parental couple: a pilot study]. / La coppia genitoriale adottiva: uno studio pilota.

International adoption is a phenomenon in constant growth, involving over one hundred countries. In Italy there are approximately two thousand adoptions of foreign minors per year. The needs demonstrated by the families adopting these children, sometimes bearing complex problems, have led to the offer of a combined medical and psychological intervention oriented towards a global and integrated approach to the needs of a family that is changing with the arrival of a new member. Therefore, a clinical evaluation of the health status of the child recently arrived in Italy, targeted at identifying the presence of medical or psychological conditions, is performed during the day spent at the day hospital. The authors present data and considerations emerging from their ongoing experience, which has already involved 113 children and 89 families. The majority of the children evaluated so far show acute physical conditions, requiring major attention and support from their parents. The authors believe that in order to organize an adequate intervention it is useful to consider the parents of these children as caregivers. The function of caregiver is identified at three levels: the care of the child as son/daughter (typical of the parental function), the care of the problems deriving from the specific preadoption experience that accompany the child in the new family, and the care of the sick child. The authors deem that the combined medical and psychological intervention adopted at present with these families constitutes a useful support to the caregiver function.

Adhesion and proliferation of fibroblasts on cluster-assembled nanostructured carbon films: the role of surface morphology.

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.

Micro- and nanoscale modification of poly(2-hydroxyethyl methacrylate) hydrogels by AFM lithography and nanoparticle incorporation.

Poly(2-hydroxyethyl methacrylate) (PHEMA) hydrogels are widely used as biomaterials. Due to their unique combination of biocompatibility and good mechanical properties, they have potential as scaffolds for tissue engineering applications. To this purpose, topographic and chemical patterning at the nano- to the mesoscale is crucial in order to favor and to characterize cell adhesion and proliferation. Here we report the characterization of as-prepared and patterned PHEMA hydrogels, produced by conventional radical polymerization in water and dimethylformamide. We have obtained chemical and morphological micro- and nanoscale patterning by atomic force microscopy based lithography. We also demonstrate that it is possible to incorporate carbon nanoparticles in the hydrogel matrix by supersonic cluster beam deposition.

[Angiomatous meningioma and epilepsy in a 2.6-years-old girl]. / Meningioangiomatosi ed epilessia in una bambina di 2 anni e sei mesi.

A case of meningo-angiomatosis (MA), in a 2.6 -years-old girl with refractory focal and secondary generalized seizures, starting at the age of 2 years, is presented. MRI evaluation revealed a lesion located at the left temporal lobe; the patient underwent surgical intervention. Histology revealed the lesion to have the features of MA.

Atomic force microscopy study of DNA deposited on poly L-ornithine-coated mica.

Analyses of individual biomolecules, like DNA, or DNA-protein complexes, via atomic force microscopy, require 'gentle' methods to immobilize DNA on surfaces, which allow the ensemble of molecules to adopt conformations dictated primarily by their physical characteristics, and which possibly permit the use of a wide selection of buffers. We show that poly-L-ornithine-coated mica is a good substrate for fast, reliable deposition of DNA for wet or dry imaging. The surface firmly secures DNA, which retains the B-form helical rise (0.34 nm bp(-1)). The conformations of DNA that result are reminiscent of three-dimensional random coils projected on to a plane. The contrast is good, especially in solution, and buffers with physiological concentrations of salt with or without divalent cations may be used. This is important for comparison of scanning probe microscopy results with those obtained by different techniques.

[Sudden infant death syndrome (SIDS)]. / La sindrome della morte improvvisa del lattante (SIDS).

Sudden Infant Death Syndrome (SIDS) is the term first proposed in 1969 for a distinctive subgroup of unexpected infant deaths occurring during the first months of life, with relatively consistent clinical, epidemiological and pathological features, which remain unexplained after a thorough case investigation, including a complete autopsy, examination of death scene and review of clinical history. Sudden infant death unnecessary means SIDS. According to definition, SIDS remains a diagnosis of exclusion, distinguished from others only by subjective and permissive variables. Despite the vague and permissive nature of the definition, epidemiological studies identified some risk factors as prematurity and social disadvantage. Nevertheless, the most interesting findings are those related to environmental and care features, as drug addiction and/or smoke exposition during pregnancy, sleep position of the infant, environmental temperature, parental bed sharing and breast feeding. Those factors play a variable role, but their correction reduced SIDS incidence. Sudden infant death is a diagnosis made by expert pathologists with pediatrician's and investigator's advice, based primarily on autopsy findings and death scene investigation performed through the severe application of investigative protocols.

Epoetin alpha in elective coronary and valve surgery in Jehovah's Witnesses patients. Experience in 45 patients.

BACKGROUND: The religious beliefs of Jehovah's Witnesses who refuse homologous and autologous blood transfusion poses serious problems for surgeons when operating on patients requiring a mean transfusion requirement of =/>2 units of blood. METHODS: After a number of encouraging studies in a randomised sample of patients 2-3 and after the treatment of some Jehovah's Witnesses 1, a group of 45 patients (23 females and 22 males) underwent elective heart surgery between June 1998 and December 2000. The patients, who were all Jehovah's Witnesses, received pre-treatment with epoetin alpha and ferrous sulphate. In the light of recent studies, it was also decided to repeat medullary preconditioning using the same intervals but with a higher dose. The patients underwent surgery involving myocardial revascularisation, mitral and/or aortic valve replacement, associated interventions, valvuloplasty and ascending aortic aneurysms. After obtaining informed and signed consent, the treatment protocol comprised the administration of 140 IU/kg epoetin alpha three times a week for 3 weeks associated with oral ferrous sulphate 3 times a day. Hematochemical levels (hemoglobin, free hemoglobin, hematocrit, ferritin, transferrin, haptoglobin, reticulocytes, iron levels) were monitored from admission to Day Hospital to discharge. RESULTS: No patient in the study required blood transfusion. CONCLUSIONS: The short, medium and long-term follow-up reconfirmed the substantial reliability of this drug linked to the absence of collateral effects.