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Mechanical cues sensed by tumor cells in their microenvironment can influence important mechanisms including adhesion, invasion and proliferation. However, a common mechanosensitive protein and/or pathway can be regulated in different ways among diverse types of tumors. Of particular interest are human breast epithelial cancers, which markedly exhibit a heterogeneous pattern of nuclear β-catenin localization, a protein known to be involved in both mechanotransduction and tumorigenesis. β-catenin can be aberrantly accumulated in the nucleus wherein it binds to and activates lymphoid enhancer factor/T cell factor (LEF/TCF) transcription factors. At present, little is known about how mechanical cues are integrated into breast cancer cells harboring impaired mechanisms of β-catenin's nuclear uptake and/or retention. This prompted us to investigate the influence of mechanical cues on MCF-7 human breast cancer cells which are known to fail in relocating β-catenin into the nucleus due to very low baseline levels of LEF/TCFs. Exploiting three-dimensional (3D) microscaffolds realized by two-photon lithography, we show that surrounding MCF-7 cells have not only a nuclear pool of β-catenin, but also rescue from their defective expression of TCF4 and boost invasiveness. Together with heightened amounts of vimentin, a β-catenin/TCF-target gene regulator of proliferation and invasiveness, such 3D-elicited changes indicate an epithelial-to-mesenchymal phenotypic switch of MCF-7 cells. This is also consistent with an increased in situ MCF-7 cell proliferation that can be abrogated by blocking β-catenin/TCF-transcription activity. Collectively, these data suggest that 3D microenvironments are per se sufficient to prime a TCF4-dependent rescuing of β-catenin nuclear activity in MCF-7 cells. The employed methodology could, therefore, provide a mechanism-based rationale to dissect further aspects of mechanotranscription in breast cancerogenesis, somewhat independent of β-catenin's nuclear accumulation. More importantly, by considering the heterogeneity of β-catenin signaling pathway in breast cancer patients, these data may open alternative avenues for personalized disease management and prevention. Statement of significance: Mechanical cues play a critical role in cancer pathogenesis. Little is known about their influence in breast cancer cells harboring impaired mechanisms of β-catenin's nuclear uptake and/or retention, involved in both mechanotransduction and tumorigenesis. We engineered 3D scaffold, by two-photon lithography, to study the influence of mechanical cues on MCF-7 cells which are known to fail in relocating β-catenin into the nucleus. We found that 3D microenvironments are per se sufficient to prime a TCF4-dependent rescuing of β-catenin nuclear activity that boost cell proliferation and invasiveness. Thus, let us suggest that our system could provide a mechanism-based rationale to further dissect key aspects of mechanotranscription in breast cancerogenesis and progression, somewhat independent of β-catenin's nuclear accumulation. © 2019

Fiber photometry is widely used in neuroscience labs for in vivo detection of functional fluorescence from optical indicators of neuronal activity with a simple optical fiber. The fiber is commonly placed next to the region of interest to both excite and collect the fluorescence signal. However, the path of both excitation and fluorescence photons is altered by the uneven optical properties of the brain, due to local variation of the refractive index, different cellular types, densities and shapes. Nonetheless, the effect of the local anatomy on the actual shape and extent of the volume of tissue that interfaces with the fiber has received little attention so far. To fill this gap, we measured the size and shape of fiber photometry efficiency field in the primary motor and somatosensory cortex, in the hippocampus and in the striatum of the mouse brain, highlighting how their substructures determine the detected signal and the depth at which photons can be mined. Importantly, we show that the information on the spatial expression of the fluorescent probes alone is not sufficient to account for the contribution of local subregions to the overall collected signal, and it must be combined with the optical properties of the tissue adjacent to the fiber tip.

Optogenetics offers the possibility of understanding behavior-related neuronal circuits, using light to trigger neuronal response. The exploitation of unconventional microfabrication techniques has led to the development of different optoelectronic devices to deliver light while electrically recording neural activity over small volumes even in deep brain areas.

We propose an optical method to perform fluorescence lifetime photometry with depth-resolution in brain tissue, exploiting far-field imaging of fluorescence signal collected by implantable tapered optical fibers.

Recognizing the neural patterns underlying different brain functions is essential to achieve a more comprehensive view on how small sets of neurons organize in complex 3D networks to determine different behaviours. In this framework, optogenetic techniques have been successfully proven as a powerful tool to control brain functions achieving millisecond temporal resolution and cell-type specificity, by combining the use of light-gated opsins and ad-hoc light delivery optoelectronic devices. However, targeting small brain volumes with simultaneous electrical recording results in the introduction of photoelectric artefacts, in particular when light emission and recoding sites are very close one to each other. In this work we take advantage of the photonic properties of tapered fibers to present a fully integrated fibertrode to target small brain volumes with abated photoelectric noise. The device hosts a light emitting window just below a recording pad, and exploits the angled light emission from the window to achieve simultaneous activation and electrical readout of small groups of cells with no photoelectric artifacts in vivo. Despite the highly non-planar surface of the fiber taper, window’s size, shape and electrode’s impedance can be modulated by controlling the fabrication parameters during focused ion beam milling and deposition, thus resulting in a versatile, integrated and customizable optogenetic tool for neurobiology studies in closed-loop configuration over small brain volumes.

This work aims at developing a new and unconventional Sacrificial Stencil Mask (SSM) technology by exploiting Two-Photon Polymerization (2PP) in an IP-L/SU-8 double layer resist system. The process consists of the sequential deposition of two different resists, such as SU-8 and IPL, onto the same glass substrate, followed by 2PP lithography and distinct development processes. The 2PP writing process was used to polymerize structures inside the top and bottom resist layers to form, in one single exposure process, both SSM and a permanent polymeric structure, in our case a plain pedestal. The top IPL resist was developed using Isopropyl Alcohol (IPA), which does not affect either exposed or un-exposed SU-8 regions. In this way, structures written into the bottom layer remained latent, while exposed areas of the top IPL resist, including the stencil mask, were developed. The realization of 3D stencil masks, designed to be anchored inside the un-exposed bottom layer, was combined with metal evaporation to demonstrate the deposition of a plain metal line through the stencil mask. The final development of the bottom layer led to the lift off of the sacrificial stencil mask, uncovering the underlying, permanent polymer-metal structure. The combination of sacrificial polymer structures with permanent ones opens new possibilities in 3D MEMS design, enabling the integration of distributed electronic transducers in flexible polymeric structures. Keywords: Stencil lithography, Stencil mask, 3D metallization, Two-photon polymerization lithography

Optogenetic control of neural activity in deep brain regions ideally requires precise and flexible light delivery with non-invasive devices. To this end, Tapered Optical Fibers (TFs) represent a versatile tool that can deliver light over either large brain volumes or spatially confined sub-regions, while being sensibly smaller than flat-cleaved optical fibers. In this work, we report on the possibility of further extending light emission length along the taper in the range 0.4 mm-3.0 mm by increasing the numerical aperture of the TFs to NA = 0.66. We investigated the dependence between the input angle of light (θin) and the output position along the taper, finding that for θin > 10° this relationship is linear. This mode-division demultiplexing property of the taper was confirmed with a ray tracing model and characterized for 473 nm and 561 nm light in quasi-transparent solution and in brain slices, with the two wavelengths used to illuminate simultaneously two different regions of the brain using only one waveguide. The results presented in this manuscript can guide neuroscientists to design their optogenetic experiments on the base of this mode-division demultiplexing approach, providing a tool that potentially allow for dynamic targeting of regions with diverse extension, from the mouse VTA up to the macaque visual cortex.

Optogenetics promises spatiotemporal precise control of neural processes using light. However, the spatial extent of illumination within the brain is difficult to control and cannot be adjusted using standard fiber optics. We demonstrate that optical fibers with tapered tips can be used to illuminate either spatially restricted or large brain volumes. Remotely adjusting the light input angle to the fiber varies the light-emitting portion of the taper over several millimeters without movement of the implant. We use this mode to activate dorsal versus ventral striatum of individual mice and reveal different effects of each manipulation on motor behavior. Conversely, injecting light over the full numerical aperture of the fiber results in light emission from the entire taper surface, achieving broader and more efficient optogenetic activation of neurons when compared to the standard flat-faced fiber stimulation. Thus, tapered fibers permit focal or broad illumination that can be precisely and dynamically matched to experimental needs.

We exploit mode-divison multiplexing and demultiplexing in tapered optical fibers to optically interface with the brain, enabling depth-resolution in sub-cortical structures with only one and minimally invasive waveguide.

Two-photon lithography is a laser writing technique that can produce 3D microstructures with resolutions below the diffraction limit. This review focuses on its applications to study mechanical properties of cells, an emerging field known as mechanobiology. We review 3D structural designs and materials in the context of new experimental designs, including estimating forces exerted by single cells, studying selective adhesion on substrates, and creating 3D networks of cells. We then focus on emerging applications, including structures for assessing cancer cell invasiveness, whose migration properties depend on the cell mechanical response to the environment, and 3D architectures and materials to study stem cell differentiation, as 3D structure shape and patterning play a key role in defining cell fates. © 2018 Elsevier Ltd

Techniques to monitor functional fluorescence signal from the brain are increasingly popular in the neuroscience community. However, most implementations are based on flat cleaved optical fibers (FFs) that can only interface with shallow tissue volumes adjacent to the fiber opening. To circumvent this limitation, we exploit modal properties of tapered optical fibers (TFs) to structure light collection over the wide optically active area of the fiber taper, providing an approach to efficiently and selectively collect light from the region(s) of interest. While being less invasive than FFs, TF probes can uniformly collect light over up to 2 mm of tissue and allow for multisite photometry along the taper. Furthermore, by micro-structuring the non-planar surface of the fiber taper, collection volumes from TFs can also be engineered arbitrarily in both shape and size. Owing to the abilities offered by these probes, we envision that TFs can set a novel, powerful paradigm in optically targeting not only the deep brain, but, more in general, any biological system or organ where light collection from the deep tissues is beneficial but challenging because of tissue scattering and absorption.

Physical and mechanical properties of extracellular matrix (ECM) have been proved to be crucial in the metastatic process. However, currently available studies on the interplay between ECM stiffness and cancer cell invasive behaviour are performed on planar assays, while the in vivo interaction takes place in three-dimensions. To take into consideration the ECM structural and mechanical complexity in the cell/structure interactions, we fabricated 3D microscaffolds through two-photon lithography (2PL) and tested how they are invaded by human colorectal adenocarcinoma (LS-174T) tumor cells, showing that it is possible to detect significant differences in cells/structure interaction when structural parameters are modified. In particular, both scaffold geometry and 2PL fabrication parameters were optimized to obtain 3D polymeric cylindrical structures with controlled Young's modulus and with linear stiffness gradients. The ability of LS-174T to migrate in the scaffolds was tested in different experimental conditions, including scaffolds functionalization and under β-catenin downregulation. It was observed that high Young's modulus scaffolds are always less invaded than softer ones, confirming the role of the 3D micro-environmental stiffness in mediating cells migration, including when specific functionalization or pharmacological treatments are performed.

For more than 50 years, carotid endarterectomy has been considered the gold standard therapy to prevent stroke in patients with carotid artery disease. Carotid artery stenting (CAS) has emerged in the last 15 years as minimally invasive alternative to surgery, especially in patients at high surgical risk. Recent randomized trials have challenged this concept reporting a relatively high incidence rate (4–6%) of post-CAS neurological adverse events. The aim of this study is to evaluate the current role of CAS in all-comers with advanced carotid disease, managed with mandatory proximal neuroprotection in a high-volume centre. From our database, the last 393 consecutive patients, from January 2011 to August 2013, undergoing CAS under proximal neuroprotection were analysed (mean age 72 ± 8 years). Indications included symptomatic stenoses ≥60% ( n = 87; 22%) and asymptomatic stenoses ≥80% ( n = 306; 78%). In an intention-to-treat approach, CAS success rate was 100%. The in-hospital all stroke/death rate was 1% (minor stroke 1%, major stroke 0%, and death 0%), and the rate of myocardial infarction was 0.3%. The minor stroke rate was 2.3% in symptomatic patients and 0.7% in asymptomatic patients. Carotid artery stenting performed in a high experience centre with the use of proximal embolic protection device is safe and has a low incidence rate of stroke.

Optogenetic control of neural activity in deep brain regions requires precise and flexible light delivery with non-invasive devices. To this end, Tapered Optical Fibers (TFs) represent a minimally-invasive tool that can deliver light over either large brain volumes or spatially confined subregions. This work links the emission properties of TFs with the modal content injected into the fiber, finding that the maximum transversal propagation constant (kt) and the total number of guided modes sustained by the waveguide are key parameters for engineering the mode demultiplexing properties of TFs. Intrinsic features of the optical fiber (numerical aperture and core/cladding diameter) define the optically active segment of the taper (up to ∼3mm), along which a linear relation between the propagating set of kt values and the emission position exists. These site-selective light-delivery properties are preserved at multiple wavelengths, further extending the range of applications expected for tapered fibers for optical control of neural activity.

Sperm cells progressive motility is the most important parameter involved in the fertilization process. Sperm middle piece contains mitochondria, which play a critical role in energy production and whose proper operation ensures the reproductive success. Notably, sperm progressive motility is strictly related to mitochondrial membrane potential (MMP) and consequently to mitochondrial functionality. Although previous studies presented an evaluation of mitochondrial function through MMP assessment in entire sperm cells samples, a quantitative approach at single-cell level could provide more insights in the analysis of semen quality. Here we combine laser scanning confocal microscopy and functional fluorescent staining of mitochondrial membrane to assess MMP distribution among isolated spermatozoa. We found that the sperm fluorescence value increases as a function of growing progressive motility and that such fluorescence is influenced by MMP disruptors, potentially allowing for the discrimination of different quality classes of sperm cells in heterogeneous populations.

Cells are highly dynamic elements, continuously interacting with the extracellular environment. Mechanical forces sensed and applied by cells are responsible for cellular adhesion, motility, and deformation, and are heavily involved in determining cancer spreading and metastasis formation. Cell/extracellular matrix interactions are commonly analyzed with the use of hydrogels and 3D microfabricated scaffolds. However, currently available techniques have a limited control over the stiffness of microscaffolds and do not allow for separating environmental properties from biological processes in driving cell mechanical behavior, including nuclear deformability and cell invasiveness. Herein, a new approach is presented to study tumor cell invasiveness by exploiting an innovative class of polymeric scaffolds based on two-photon lithography to control the stiffness of deterministic microenvironments in 3D. This is obtained by fine-tuning of the laser power during the lithography, thus locally modifying both structural and mechanical properties in the same fabrication process. Cage-like structures and cylindric stent-like microscaffolds are fabricated with different Young's modulus and stiffness gradients, allowing obtaining new insights on the mechanical interplay between tumor cells and the surrounding environments. In particular, cell invasion is mostly driven by softer architectures, and the introduction of 3D stiffness “weak spots” is shown to boost the rate at which cancer cells invade the scaffolds. The possibility to modulate structural compliance also allowed estimating the force distribution exerted by a single cell on the scaffold, revealing that both pushing and pulling forces are involved in the cell–structure interaction. Overall, exploiting this method to obtain a wide range of 3D architectures with locally engineered stiffness can pave the way for unique applications to study tumor cell dynamics. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Aims: To assess the 30-day clinical outcome of endovascular and surgical revascularisation procedures in patients with carotid obstructive disease (COD) and concomitant coronary artery disease (CAD). Methods and results: Between January 2006 and December 2009, 659 patients with COD and concomitant CAD were treated. The incidence of the primary endpoint (composite of death, MI and stroke) was 4.25% (0.9%, 1.1% and 2.3%, respectively). Acute renal insufficiency occurred in 2.4% and major bleedings in 4.4% of patients. According to the treatment forms patients were divided into three groups: surgical, 185 patients (28.1%), endovascular, 378 (57.4%), and hybrid, 89 (13.5%). Seven patients (1%) were managed medically only. The primary endpoint of the study occurred in 4.8%, 2.4% and 8.6%, respectively, p=0.01. The secondary endpoint, that included the occurrence of renal or respiratory insufficiency and major bleedings occurred in 10.1%, 6.5% and 23.8%, respectively, p

Aims: We aimed to determine the success, safety and long-term durability of carotid artery stenting (CAS) in stroke prevention for all-comers managed with mandatory neuroprotection and a tailored-approach to intervention. Methods and results: From our CAS registry (beginning July 1997) all procedures up to September 2007 with intention-to-treat by stenting under distal filter or proximal occlusion neuroprotection devices were analysed (N=1523 ; mean age 72 years [237 ≥80 years, 15.5%]). Indications included symptomatic stenoses ≥50% (366, 24.1%) and asymptomatic stenoses ≥80% (1157, 75.9%). CAS success was 99.6% and the 30-day all-stroke/death rate was 1.5% (minor stroke 11 [0.7%], major stroke 8 [0.5%], death 5 [0.3%]). The risk was 1.2% for asymptomatic patients and 2.7% for symptomatic patients (p=0.042). Regarding octogenarians this risk was 2.1% versus 1.5% for patients ≤79 years (p=0.47). Symptomatic octogenarians had a higher risk than other groups (OR 3.9, 95% Cl 1.06 to 14.0): asymptomatic

In the present study we describe the in vitro pharmacological characterization of the nociceptin/orphanin FQ (N/OFQ) receptor (NOP) ligand Ac-RYYRWK-NH2 and the synthesis and biological evaluation of 13 Trp5 substituted Ac-RYYRWK-NH2 analogs. Results indicate that Ac-RYYRWK-NH2 behaves as a highly potent and selective partial agonist at the NOP receptors and that the whole indole moiety of the Trp5 side chain is not required, being a phenyl-ethyl side chain already sufficient for maintaining high potency.

[(pF)Phe 4 Aib 7 Arg 14 Lys 15 ]N/OFQ-NH 2 (UFP-112) has been designed as a novel ligand for the nociceptin/orphanin FQ (N/OFQ) peptide receptor (NOP) by combining into the same peptide different chemical modifications reported to increase N/OFQ potency. In vitro data obtained in the electrically stimulated mouse vas deferens demonstrated that UFP-112 behaved as a high potency (pEC 50 9.43) full agonist at the NOP receptor. UFP-112 effects were sensitive to the NOP antagonist UFP-101 but not to naloxone and no longer evident in tissues taken from NOP −/− mice. In vitro half life of UFP-112 in mouse plasma and brain homogenate was 2.6- and 3.5-fold higher than that of N/OFQ. In vivo, in the mouse tail withdrawal assay, UFP-112 (1–100 pmol, i.c.v.) mimicked the actions of N/OFQ producing pronociceptive effects after i.c.v. administration and antinociceptive effects when given i.t.; in both cases, UFP-112 was approximately 100-fold more potent than the natural peptide and produced longer lasting effects. UFP-112 also mimicked the hyperphagic effect of N/OFQ producing a bell shaped dose response curve with the maximum reached at 10 pmol. The hyperphagic effects of N/OFQ and UFP-112 were absent in NOP −/− mice. Equi-effective high doses of UFP-112 (0.1 nmol) and N/OFQ (10 nmol) were injected i.c.v. in mice and spontaneous locomotor activity recorded for 16 h. N/OFQ produced a clear inhibitory effect which lasted for 60 min while UFP-112 elicited longer lasting effects (>6 h). In conscious rats, UFP-112 (0.1 and 10 nmol/kg, i.v.) produced a marked and sustained decrease in heart rate, blood pressure, and urinary sodium excretion and a profound increase in urine flow. Collectively, these findings demonstrate that UFP-112 behaves in vitro and in vivo as a highly potent and selective ligand able to produce full and long lasting activation of NOP receptors.

The excellent pharmacological profile displayed by the selective nociceptin/orphanin FQ (N/OFQ) peptide (NOP) receptor antagonist SB-612111 [(-)-cis-1-methyl-7-[[4-(2,6-dichlorophenyl)piperidin-1-yl]methyl]-6,7,8,9-tetrahydro-5H-benzocyclohepten-5-ol] in vitro prompted us to investigate the actions of this compound in vivo. In the mouse tail withdrawal assay, SB-612111 given i.p. up to 3 mg/kg did not modify per se tail withdrawal latencies but was able to prevent the pronociceptive and the antinociceptive action of 1 nmol of N/OFQ given i.c.v. and i.t., respectively. In food intake studies performed in sated mice, SB-612111 (1 mg/kg i.p.) had no effect on food consumption but fully prevented the orexigenic effect of 1 nmol of N/OFQ i.c.v. In 17-h food-deprived mice, the opioid receptor antagonist naltrexone (1 mg/kg s.c.), but not SB-612111 (1 and 10 mg/kg i.p.), produced a statistically significant reduction of food intake. In the mouse forced swimming and tail suspension tests, SB-612111 (1-10 mg/kg) reduced immobility time. The antidepressant-like effect elicited by SB-612111 in the forced swimming test was reversed by the i.c.v. injection of 1 nmol of N/OFQ and no longer evident in mice knockout for the NOP receptor gene. In conclusion, the present findings demonstrate that SB-612111 behaves in vivo as a potent and selective NOP antagonist and suggest that the N/OFQ-NOP receptor endogenous system plays an important role in regulating mood-related behaviors. The use of SB-612111 in future pathophysiological studies will certainly contribute to define the therapeutic potential of selective NOP receptor antagonists in different disease areas.

Some cellular mechanisms such as migration and invasion, are determined and regulated by cell mechanical properties, especially during embryonic development or tumorigenesis. In these cases, cells attitude to undergo deformation and to displace their whole body is a crucial point for survival and metastasis formation. Extracellular environment three-dimensionality is a key feature influencing cell mechanics, and micro- and nano-fabricated structures represent a promising tool to study cell behavior. Here we engineer 3D- cage-like microstructures realized by two-photon polymerization to analyze cell migration through pores significantly smaller than the cell nucleus. We found that cells spontaneously interact with the cages by applying force at the cell-cage contact points while rearranging their cytoskeleton. The ability to traverse pores of different area depends on the invasiveness of the cell lines, allowing to discriminate between cells with different invasive potential.

Cancer cell motility is one of the major events involved in metastatic process. Tumor cells that disseminate from a primary tumor can migrate into the vascular system and, being carried by the bloodstream, transmigrate across the endothelium, giving rise to a new tumor site. However, during the invasive process, tumor cells must pass through the extracellular matrix, whose structural and mechanical properties define the parameters of the migration process. Here, we propose 3D-complex cage-like microstructures, realized by two-photon (TP) direct laser writing (DLW), to analyze cell migration through pores significantly smaller than the cell nucleus. We found that the ability to traverse differently sized pores depends on the metastatic potential and on the invasiveness of the cell lines, allowing to establish a pore-area threshold value able to discriminate between non-tumorigenic and tumorigenic human breast cells.

It was recently reported that the hexapeptide Ac-RYYRIK-ol binds with high affinity nociceptin/orphanin FQ (N/OFQ) peptide (NOP) receptors and competitively antagonizes N/OFQ actions in the mouse vas deferens assay. Here we further describe the in vitro and in vivo pharmacological features of this NOP receptor ligand. In mouse brain homogenate the degradation half life of Ac-RYYRIK-ol (2.48 min) was significantly higher than that of the parent compound Ac-RYYRIK-NH 2 (1.20 min). In the electrically stimulated mouse vas deferens, Ac-RYYRIK-ol (10–1000 nM) competitively antagonized the inhibitory effect of N/OFQ (pA 2 = 8.46), while in the isolated mouse colon the hexapeptide mimicked N/OFQ contractile effects thus behaving as a NOP receptor agonist (pEC 50 = 9.09). This latter effect was no longer evident in colon tissues taken from mice knock out for the NOP receptor gene (NOP −/− ). In vivo in mice, similarly to N/OFQ, Ac-RYYRIK-ol (dose range 0.001–1 nmol) produced: i) pronociceptive effects after intracerebroventricular (i.c.v.) administration and antinociceptive actions when given intrathecally (i.t.) in the tail withdrawal assay; ii) inhibition of locomotor activity and iii) stimulation of food intake after supraspinal administration. Finally, in the forced swimming test, Ac-RYYRIK-ol was inactive per se, but reversed the antidepressant-like effects elicited by the NOP receptor selective antagonist UFP-101 ([Nphe 1 ,Arg 14 ,Lys 15 ]N/OFQ-NH 2 ). Thus, in all these in vivo assays Ac-RYYRIK-ol mimicked the actions of N/OFQ showing however higher potency. In conclusion, Ac-RYYRIK-ol displayed a complex pharmacological profile which is likely due to the low efficacy agonist nature of this novel ligand of the NOP receptor. The high potency, selectivity of action, and in vivo effectiveness make Ac-RYYRIK-ol a useful pharmacological tool for future studies in the field of N/OFQ and its NOP receptor.

Following the discovery of nociceptin/orphanin FQ (N/OFQ) peptide receptor (NOP) and its endogenous ligand, an extensive search has started to find selective agonists and antagonists targeting this novel receptor-ligand system due to their therapeutic potentials. By the help of the combinatorial chemistry a series of hexapeptides with a general formula of Ac-RYY-R/K-W/I-R/K-NH(2) having high NOP receptor affinity and selectivity were identified. On the basis of this information we developed a number of novel compounds. The detailed structure-activity studies on the partial agonist Ac-RYYRIK-NH(2) are reported in this communication. Besides the modifications on N- and C-terminal, Arg-Cit exchange was performed on the template structure. The novel hexapeptides were analyzed in radioligand binding, functional biochemical [(35)S]GTPgammaS binding assays by using membranes from rat brains and Chinese hamster ovary cells expressing human NOP receptor. The agonist/antagonist properties were also tested on in the mouse vas deferens bioassay. C-terminal modification yielded a high affinity, selective and potent NOP ligand (Ac-RYYRIK-ol) with a partial agonist property. Several analogs of this compound were synthesized. The presence of the positively charged arginine residue at the first position turned out to be crucial for the biological activity of the hexapeptide. The N-terminal modifications with various acyl groups (ClAc, pivaloyl, formyl, benzoyl, mesyl) decreased the affinity of the ligand towards the receptor and the intrinsic activity for stimulating the G-protein activation was also decreased. The structure-activity studies on the hexapeptide derivatives provided some basic information on the structural requirements for receptor binding and activation.

Miniaturization of analytical procedures has a significant impact on diagnostic testing since it provides several advantages such as: reduced sample and reagent consumption, shorter analysis time and less sample handling. Lab-on-a-chip (LoC), usually silicon, glass, or silicon-glass, or polymer disposable cartridges, which are produced using techniques inherited from the microelectronics industry, could perform and integrate the operations needed to carry out biochemical analysis through the mechanical realization of a dedicated instrument. Analytical devices based on miniaturized platforms like LoC may provide an important contribution to the diagnosis of high prevalence and rare diseases. In this paper we review some of the uses of Lab-on-a-chip in the clinical diagnostics of immune-mediated diseases and we provide an overview of how specific applications of these technologies could improve and simplify several complex diagnostic procedures.

PURPOSE To report a prospective feasibility study of cutting balloon angioplasty (CBA) applied in the predilation phase of carotid artery stenting (CAS) in highly calcified lesions. METHODS From January 2003 to February 2007, 178 consecutive patients (109 men; mean age 73.1+/-7.3 years) with highly calcified carotid lesions underwent CAS with CBA applied as a pre-specified strategy in the predilation phase of the procedure. All steps in the procedure were performed under cerebral filter protection. The cutting balloon ranged in diameter from 3 to 4 mm and was inflated at nominal pressures in the target lesion. Pre-CBA dilation with a low-profile coronary balloon was performed only when the cutting balloon was not able to cross the lesion. Selection of the filters and stents was at the operator's discretion. Primary endpoints were the all stroke and death rates at 30 days and 6 months. Secondary endpoints included cutting balloon success (positioning and full balloon inflation), CAS technical success (residual angiographic stenosis

Two-photon (2P) lithography shows great potential for the fabrication of three-dimensional (3-D) micro- and nanomechanical elements, for applications ranging from microelectromechanical systems to tissue engineering, by virtue of its high resolution (

Knockout and pharmacological studies have shown that delta opioid peptide (DOP) receptor signalling regulates emotional responses. In the present study, the in vitro and in vivo pharmacological profile of the DOP ligand, H-Dmt-Tic-NH-CH(CH2-COOH)-Bid (UFP-512) was investigated. In receptor binding experiments performed on membranes of CHO cells expressing the human recombinant opioid receptors, UFP-512 displayed very high affinity (pKi 10.20) and selectivity (>150-fold) for DOP sites. In functional studies ([35S]GTP gamma S binding in CHOhDOP membranes and electrically stimulated mouse vas deferens) UFP-512 behaved as a DOP selective full agonist showing potency values more than 100-fold higher than DPDPE. In vivo, in the mouse forced swimming test, UFP-512 reduced immobility time both after intracerebroventricular (i.c.v.) and intraperitoneal (i.p.) administration. Similar effects were recorded in rats. Moreover, UFP-512 evoked anxiolytic-like effects in the mouse elevated plus maze and light-dark aversion assays. All these in vivo actions of UFP-512 were fully prevented by the selective DOP antagonist naltrindole (3 mg/kg, s.c.). In conclusion, the present findings demonstrate that UFP-512 behaves as a highly potent and selective agonist at DOP receptors and corroborate the proposal that the selective activation of DOP receptors elicits robust anxiolytic- and antidepressant-like effects in rodents.

The compound SB-612111 [(-)-cis-1-methyl-7-[[4-(2,6-dichlorophenyl)piperidin-1-yl]methyl]-6,7,8,9-tetrahydro-5H-benzocyclohepten-5-ol] was recently identified as a selective antagonist for the nociceptin/orphanin FQ (N/OFQ) peptide receptor (NOP). In the present study, the in vitro pharmacological profile of SB-612111 at human recombinant NOP receptors expressed in Chinese hamster ovary (CHO) cells [receptor binding, guanosine 5'-O-(3-[(35)S]thio)triphosphate (GTPgamma[(35)S]) binding, and cAMP level experiments] as well as at native NOP receptors expressed in peripheral (mouse and rat vas deferens, guinea pig ileum) and central (mouse cerebral cortex synaptosomes releasing [(3)H]5-HT) preparations was evaluated and compared with that of the standard nonpeptide antagonist (+/-)J-113397 [(+/-)-trans-1-[1-cyclooctylmethyl-3-hydroxymethyl-4-piperidyl]-3-ethyl-1,3-dihydro-2H-benzimidazol-2-one]. SB-612111 produced a concentration-dependent displacement of [(3)H]N/OFQ binding to CHO(hNOP) cell membranes, showing higher affinity and NOP selectivity over classical opioid receptors than (+/-)J-113397. SB-612111 and (+/-)J-113397 competitively antagonized the effects of N/OFQ on GTPgamma[(35)S] binding in CHO(hNOP) cell membranes (pK(B), 9.70 and 8.71, respectively) and on cAMP accumulation in CHO(hNOP) cells (pK(B), 8.63 and 7.95, respectively), being per se inactive. In isolated peripheral tissues of mice, rats, and guinea pigs and in mouse cerebral cortex synaptosomes preloaded with [(3)H]5-HT, SB-612111 competitively antagonized the inhibitory effects of N/OFQ, with pA(2) values in the range of 8.20 to 8.50. In parallel experiments, (+/-)J-113397 was found to be 2- to 9-fold less potent than SB-612111. In the electrically stimulated tissues, 1 microM SB-612111 did not modify the effects of classical opioid receptor agonists. In conclusion, the results of the present study demonstrated that SB-612111 is among the most potent and NOP-selective nonpeptide antagonists identified to date.

Previous structure-activity and NMR studies on nociceptin/orphanin FQ (N/OFQ) demonstrated that Aib substitution of Ala(7) and/or Ala(11) increases the peptide potency through an alpha helix structure induction mechanism. On these bases we synthesised and evaluated pharmacologically in the mouse vas deferens assay a series of N/OFQ-NH(2) analogues substituted in position 7 and 11 with Calpha,alpha-disubstituted cyclic, linear and branched amino acids. None of the 20 novel N/OFQ analogues produced better results than [Aib(7)]N/OFQ-NH(2). Thus, this substitution was combined with other chemical modifications known to modulate peptide potency and/or efficacy generating compound 21 [Nphe(1)Aib(7)Arg(14)Lys(15)]N/OFQ-NH(2) (coded as UFP-111), compound 22 [(pF)Phe(4)Aib(7)Arg(14)Lys(15)]N/OFQ-NH(2) (UFP-112) and compound 23 [Phe(1)Psi(CH(2)-NH)Gly(2)(pF)Phe(4)Aib(7)Arg(14)Lys(15)]N/OFQ-NH(2) (UFP-113). These novel peptides behaved as highly potent NOP receptor ligands showing full (UFP-112) and partial (UFP-113) agonist and pure antagonist (UFP-111) activities in a series of in vitro functional assays performed on pharmacological preparations expressing native as well as recombinant NOP receptors.

Knockout and pharmacological studies demonstrated that the activation of delta opioid peptide (DOP) receptors produces antidepressant-like effects in rodents. Here we report the results obtained with the novel DOP ligand H-Dmt-Tic-NH-CH 2 -Bid (UFP-502). UFP-502 bound with high affinity (p K i 9.43) to recombinant DOP receptors displaying moderate selectivity over MOP and KOP. In CHO hDOP [ 35 S]GTPγS binding and mouse vas deferens experiments, UFP-502 behaved as a potent (pEC 50 10.09 and 10.70, respectively) full agonist. In these preparations, naloxone, naltrindole and N , N (CH 3 ) 2 Dmt-Tic-OH showed similar p A 2 values against UFP-502 and DPDPE and the same rank order of potency. In vivo in mice, UFP-502 mimicked DPDPE actions, producing a significant reduction of immobility time after intracerebroventricular administration in the forced swimming test and a clear antinociceptive effect after intrathecal injection in the tail withdrawal assay. However, while the effects of DPDPE were fully prevented by naltrindole those evoked by UFP-502 were unaffected (tail withdrawal assay) or only partially reversed (forced swimming test). In conclusion, UFP-502 represents a novel and useful chemical template for the design of selective agonists for the DOP receptor.

To date, J-113397 represents the most potent and selective non peptide NOP receptor antagonist widely used in pharmacological studies. However, the synthesis, purification, and enantiomer separation of this molecule, which contains two chiral centers, is rather difficult and low-yielding. Here, we synthesized and tested a series of simplified J-113397 analogues to investigate the importance of the stereochemistry and the influence of the substituents at position 3 of the piperidine nucleus and on the nitrogen atom of the benzimidazolidinone nucleus. The compound coded as Trap-101, an achiral analogue of J-113397, combines a pharmacological profile similar to that of the parent compound with a practical, high-yielding preparation.