Piezoelectric displacement mapping of compliant surfaces by constant-excitation frequency-modulation piezoresponse force microscopy.

A simple experimental method for piezoresponse force microscopy (PFM) measurements for reliable evaluation of piezoelectric surface displacements even on compliant surfaces is proposed based on atomic force microscopy (AFM) operated in frequency-modulation (FM) dynamic mode with constant excitation (CE), by using non-contact mode cantilevers. Surface displacement by piezoelectric effect after application of an electric potential to the conductive AFM probe translates into a likewise variation of the probe oscillation amplitude, while the related electrostatic forces mainly affect the oscillator resonant frequency, and cantilever bending is limited due to their high stiffness. Our non-contact CE-FM-PFM method is shown to reduce electrostatic force contributions as compared to contact-PFM modes. Converse piezoelectric effect mapping is demonstrated on poly(vinylidenefluoride) nanofibers obtained by electrospinning.

Synthesis of esters of androgens with unsaturated fatty acids for androgen requiring therapy.

Androgens' metabolism and activity are gaining a more and more important role in human physiology particularly referring to aging and to neurodegenerative diseases. Androgen treatment is often required for long-lasting disorders. In order to improve their duration and effects, androgens can be administered as esters of carboxylic acids. The novelty of our research is the use of esters of androgens with specific unsaturated fatty acids, in order to reduce possible side effects particularly related to chronic pathologies with altered lipid homeostasis such as X-linked adrenoleukodystrophy and cardiovascular disorders. Thus the esters of the main androgenic substances testosterone, dihydrotestosterone (DHT) and their metabolite 5α-androstan-3α,17ß-diol were chemically obtained by coupling with different unsaturated fatty acids. To this aim, fatty acids with various degree of unsaturation and belonging to different series were selected. Specifically, oleic acid (18:1, n-9), linoleic acid (18:2, n-6), and the n-3 fatty acids, α-linolenic acid (18:3), eicosapentaenoic acid (EPA, 20:5), and docosahexaenoic acid (DHA, 22:6) were used obtaining corresponding esters with acceptable yields and good degree of purity. All the synthesized compounds were tested for their cytotoxic activities in mouse NIH3T3 and human astrocyte cell lines. The esters demonstrated good tolerability and no in vitro cytotoxic effect in both cell cultures. After these promising preliminary results, the esters will be suitable for in vivo studies in order to ascertain their pharmacokinetic characteristics and their biological effects.

New perspectives in cell communication: Bioelectromagnetic interactions.

This paper explores physical signalling in biological communications, the so-called biophysical pathways, and especially the role of electromagnetic signalling in cell-cell interactions. The experiments were designed to evaluate whether different cell populations physically interfere when incubated in separate Petri dishes placed in close proximity. Two different cell populations, immortalized mouse fibroblasts (NIH3T3) and adult human microvascular endothelial cells (HMVECad) were selected and seeded in separate polystyrene Petri dishes. Dishes seeded with NIH3T3 were then placed on top of those seeded with HMVECad at distances of 4mm and 11mm. A black filter was placed between dishes containing the two cell populations in another experiment, to prevent transmission of electromagnetic radiation between the two. Cell number and morphology of NIH3T3 and endothelial cells were found to be modified in dishes without the black filter, suggesting that specific signals emitted by the cells were transmitted through the polystyrene wall, affecting cell proliferation rate and morphology, even though the cells were growing in separate dishes.

Long-term safety and efficacy of lentiviral hematopoietic stem/progenitor cell gene therapy for Wiskott-Aldrich syndrome.

Patients with Wiskott-Aldrich syndrome (WAS) lacking a human leukocyte antigen-matched donor may benefit from gene therapy through the provision of gene-corrected, autologous hematopoietic stem/progenitor cells. Here, we present comprehensive, long-term follow-up results (median follow-up, 7.6 years) (phase I/II trial no. NCT02333760 ) for eight patients with WAS having undergone phase I/II lentiviral vector-based gene therapy trials (nos. NCT01347346 and NCT01347242 ), with a focus on thrombocytopenia and autoimmunity. Primary outcomes of the long-term study were to establish clinical and biological safety, efficacy and tolerability by evaluating the incidence and type of serious adverse events and clinical status and biological parameters including lentiviral genomic integration sites in different cell subpopulations from 3 years to 15 years after gene therapy. Secondary outcomes included monitoring the need for additional treatment and T cell repertoire diversity. An interim analysis shows that the study meets the primary outcome criteria tested given that the gene-corrected cells engrafted stably, and no serious treatment-associated adverse events occurred. Overall, severe infections and eczema resolved. Autoimmune disorders and bleeding episodes were significantly less frequent, despite only partial correction of the platelet compartment. The results suggest that lentiviral gene therapy provides sustained clinical benefits for patients with WAS.

Reelin levels are increased in synovial fluid of patients with rheumatoid arthritis.

OBJECTIVES: To evaluate the presence and the glycosylation pattern of reelin in synovial fluid and serum of patients affected by different rheumatic pathologies. METHODS: Reelin levels were evaluated in patients affected by rheumatoid arthritis (RA), psoriatic arthritis (PsA), spondyloarthritis (SpA) and osteoarthritis (OA). Reelin semi-quantitative assays were performed by western blot. The glycosylation pattern was evaluated by immunoblotting performed by sepharose conjugated lectins. RT-PCR was used to detect the presence of mRNA encoding for reelin and its receptors. RESULTS: Reelin is detectable in both sinovial fluids and sera and its levels are more elevated in patients affected by RA with respect to those affected by other inflammatory and non inflammatory joint diseases. The glycosylation pattern of the protein differs in synovial fluid and serum. Fibroblast-like synoviocytes (FLS) express the mRNAs encoding for reelin and its receptors. CONCLUSIONS: Since its levels are higher in RA then in the other analysed pathologies, reelin can represent a candidate suitable for the differential diagnosis of this pathology. Moreover, the observation that this protein is encoded by FLS and differentially glycosylated in blood and synovial fluid supports the hypothesis that it is locally produced in the joints, where it could play an important role in RA development and maintenance.

Impact of involvement of individual joint groups on subdimensions of functional ability scales in juvenile idiopathic arthritis.

OBJECTIVE: To investigate the influence of arthritis in individual joint groups on subdimensions of functional ability questionnaires in children with juvenile idiopathic arthritis (JIA). METHODS: 206 patients were included who had the Childhood Health Assessment Questionnaire (C-HAQ) and the Juvenile Arthritis Functionality Scale (JAFS) completed simultaneously by a parent and received a detailed joint assessment. In each patient, joint involvement (defined as presence of swelling, pain on motion/tenderness and/or restricted motion) was classified in 3 topographic patterns: Pattern 1 (hip, knee, ankle, subtalar and foot joints); Pattern 2 (wrist and hand joints); Pattern 3 (elbow, shoulder, cervical spine and temporomandibular joints). Frequency of reported disability in each instrument subdimension was evaluated for each joint pattern, present either isolatedly or in mixed form. RESULTS: Among patients with Pattern 1, the JAFS revealed the greatest ability to capture and discriminate functional limitation, whereas impairment in the C-HAQ was more diluted across several subdimensions. Both C-HAQ and JAFS appeared to be less reliable in detecting functional impairment in the hand and wrist (Pattern 2) than in other body areas. Overall, the JAFS revealed a superior ability to discriminate the relative functional impact of impairment in individual joint groups among patients with mixed joint patterns. CONCLUSION: In children with JIA, a functional measure focused to assess the function of individual joint groups (the JAFS) may detect with greater precision the functional impact of arthritis in specific body areas than does a standard questionnaire based on the assessment of activities of daily living (the C-HAQ).

Rapid Maxillary Expander and Eruption Guidance Appliance therapy in skeletal Class II: cephalometric considerations.

AIM: The use of rapid maxillary expander (RME) combined with eruption guidance appliances (EGA) represents a valid method in the treatment of skeletal Class II deep bite patients. In this paper a retrospective cephalometric study of the effects of RME and EGA therapy in Class II deep bite patients is described and compared with the treatment effects of patients with the same malocclusion treated only by RME. MATERIALS AND METHODS: Pre and post treatment cephalometric radiographs of 47 Class II division 1 deep bite growing patients treated with RME followed by EGA (mean age 9.65 years) (Group 1) were compared with those obtained from a control group (Group 2) of 44 patients (mean age 9.34 years) treated with RME only. The following cephalometric values were analized: SNA; SNB; ANB; SN-PNS.ANS; SN-Go.GN; N-Me; S-Go. RESULTS: Improvement in maxillo-mandibular relationship was observed in both groups with significantly higher decrease of SNA and ANB in group 1. The cranio-maxillary relation shows a tendency to grow downward and backward in both groups. Both anterior and posterior total facial heights showed a significant increase in group 1. CONCLUSIONS: RME followed by EGA therapy in Class II skeletal deep bite patients has shown better results compared with RME therapy without functional appliance.

The magnitude of early response to methotrexate therapy predicts long-term outcome of patients with juvenile idiopathic arthritis.

OBJECTIVE: To investigate the relationship between the magnitude of clinical response in the first 6 months of methotrexate (MTX) therapy and long-term outcome in children with juvenile idiopathic arthritis (JIA). METHODS: The clinical charts of 125 JIA patients who were started with MTX and then followed for at least 5 years were reviewed. Based on the level of American College of Rheumatology (ACR) Pediatric response at 6 months, patients were divided in four mutually exclusive groups: (1) non-responders, (2) responders at 30%, (3) responders at 50%, and (4) responders at 70%. The long-term outcome in each response group was evaluated by calculating the percentage change in active and restricted joint counts from baseline to 1, 2 and 5 years and the frequency of inactive disease at 5 years. RESULTS: At 6 months, 42 patients were classified as non-responders, 24 as 30% responders, 26 as 50% responders, and 33 as 70% responders. Patients who had achieved a 70% response showed a significantly greater percentage improvement in active joint count between baseline to 5 years compared with non-responders and 30% responders, and a significantly greater percentage improvement in restricted joint count between baseline to 5 years compared with 30% responders. The 70% responders also had a greater frequency of inactive disease at 5 years compared with 30% responders, CONCLUSIONS: Our results show that the achievement of an ACR Pediatric 70 response at 6 months after start of MTX therapy predicts a more favorable long-term outcome of patients with JIA.

Assessment of climate change effects on mountain ecosystems through a cross-site analysis in the Alps and Apennines.

Mountain ecosystems are sensitive and reliable indicators of climate change. Long-term studies may be extremely useful in assessing the responses of high-elevation ecosystems to climate change and other anthropogenic drivers from a broad ecological perspective. Mountain research sites within the LTER (Long-Term Ecological Research) network are representative of various types of ecosystems and span a wide bioclimatic and elevational range. Here, we present a synthesis and a review of the main results from ecological studies in mountain ecosystems at 20 LTER sites in Italy, Switzerland and Austria covering in most cases more than two decades of observations. We analyzed a set of key climate parameters, such as temperature and snow cover duration, in relation to vascular plant species composition, plant traits, abundance patterns, pedoclimate, nutrient dynamics in soils and water, phenology and composition of freshwater biota. The overall results highlight the rapid response of mountain ecosystems to climate change, with site-specific characteristics and rates. As temperatures increased, vegetation cover in alpine and subalpine summits increased as well. Years with limited snow cover duration caused an increase in soil temperature and microbial biomass during the growing season. Effects on freshwater ecosystems were also observed, in terms of increases in solutes, decreases in nitrates and changes in plankton phenology and benthos communities. This work highlights the importance of comparing and integrating long-term ecological data collected in different ecosystems for a more comprehensive overview of the ecological effects of climate change. Nevertheless, there is a need for (i) adopting co-located monitoring site networks to improve our ability to obtain sound results from cross-site analysis, (ii) carrying out further studies, in particular short-term analyses with fine spatial and temporal resolutions to improve our understanding of responses to extreme events, and (iii) increasing comparability and standardizing protocols across networks to distinguish local patterns from global patterns.

Continuous multilayered composite hydrogel as osteochondral substitute.

Cartilage is a highly organized avascular soft tissue that assembles from nano-to macro-scale to produce a complex structural network. To mimic cartilage tissue, we developed a stable multilayered composite material, characterized by a tailored gradient of mechanical properties. The optimized procedure implies chemical crosslinking of each layer directly onto the previous one and ensures a drastic reduction of the material discontinuities and brittleness. The multilayered composite was characterized by infrared spectroscopy, differential scanning calorimetry, thermogravimetry, and scanning electron microscopy in order to compare its physico-chemical characteristics with those of cartilage tissue. The rheological behavior of the multilayered composite was similar to that of human cartilage. Finally its cytocompatibility toward chondrocytes and osteoblasts was evaluated.

Iron release, membrane protein oxidation and erythrocyte ageing.

The aerobic incubation of erythrocytes in phosphate buffer for 24-60 h (a model of rapid in vitro ageing) induced progressive iron release and methemoglobin formation. Membrane proteins showed electrophoretic alterations and increase in carbonyl groups (as documented by IR spectroscopy). None of these phenomena were seen when the erythrocytes were incubated under anaerobic conditions. The membranes from aerobically incubated cells bound a much higher amount of autologous IgG than those from anaerobically incubated ones, suggesting that the aerobic incubation gives rise to the senescent antigen. The addition of ferrozine during the aerobic incubation prevented both the IgG binding and the protein alterations seen in the IR spectra, suggesting an intracellular chelation of the released iron by ferrozine.

Synthesis and hypoglycemic activity of 4-substituted 3-mercaptopicolinic acids.

3-Mercapto-4-methylpicolinic acid one of very few compounds derived from 3-mercaptopicolinic acid (3-MPA) to have hypoglycemic activity. In an effort to find compounds with greater potency than 3-MPA, several 4-substituted 3-mercaptopicolinic acids (4-OMe, OC6H5, SMe, SH, Cl, NH2, Et; 1-7) were prepared and tested in 48-h fasted rats. None was hypoglycemic in this test system after oral dosing of 150 mg/kg.

Reduced natural killer cell activity in major depression: neuroendocrine implications.

Natural killer cell activity (NKCA) was significantly reduced in a group of depressed patients, melancholic subtype, compared to sex- and age-matched controls. Corticotropin and cortisol values were significantly higher in the depressed subjects than in the controls, but no correlation between high hormone levels and low immunological activity was found in the patients.

Conformation of human plasma proteins at polymer surfaces: the effectiveness of surface heparinization.

Studies were made on the adsorption of two human plasma proteins, albumin (HSA) and fibrinogen (HFg), onto three different polymeric surfaces: commercial pellethane 2363-80AE (PU); pellethane crosslinked with a poly(amido-amine) (PUPA); and heparinized PUPA, using in situ ATR/FTIR spectroscopy (attenuated total reflection Fourier transform infrared spectroscopy). Conformational changes were found to occur on the two proteins upon adsorption onto bare PU and PUPA, and the protein unfolding on bare PU was also found to be time dependent. On the contrary, the two proteins do not change conformation when they are adsorbed onto the heparinized surface, emphasising the effectiveness of surface heparinization.

Physiochemical characterization and coating of polyurethane with a new heparin-adsorbing material.

The coating of polyurethane devices was obtained by using a solution of a new heparin-adsorbing material, PUPA. The structure of the coated material was investigated at different depths by FTIR techniques. The bond with heparin is electrostatic, as revealed by subtracting the spectrum of the native PUPA from that of the heparinized sample.

The influence of molecular weight on the biological activity of heparin like sulphated hyaluronic acids.

Sulphated hyaluronic acids having a sulphation degree of 3.5 per disaccharide unit, HyalS3.5, were prepared with different molecular weights corresponding to 21 x 10(3), 320 x 10(3) and 3500 x 10(3). The thrombin inhibition in plasma and in the presence of purified molecules, i.e. fibrinogen, antithrombin III (AT III) and heparin cofactor II (HC II), were studied for the three different MW compounds at different concentrations. The thrombin time in plasma depended on the length of the chain, and the two lower MW HyalS3.5 inhibited thrombin both by direct aspecific interaction and via HC II, whereas the activity of the highest MW compound was mainly related to the electrostatic interaction with HC II. The inactivation of FXa serine protease was only attributed to HyalS3.5-AT III complex.

The use of hyaluronan and its sulphated derivative patterned with micrometric scale on glass substrate in melanocyte cell behaviour.

Surface microfabrication techniques were widely utilised for the spatial control of in vitro cell behaviour. A photo-immobilisation procedure was utilised to create micropatterned surfaces: four different stripe patterns (100, 50, 25 and 10 microm) of hyaluronan (Hyal) and its sulphated derivative (HyalS) on silanised glass substrate were obtained.The morphological analysis showed that the surface topography showed regular stripes of 100, 50, 25 and 10 microm wide and ranging from 300 nm up to 1 microm in thickness. They reproduced the exact photo-mask pattern: glass stripes alternating with polysaccharide ones. On the contrary, Hyal microstructures showed just a topographic pattern as the glass stripes appeared to be covered by a thin layer of the macromolecule by TOF-SIMS. Cell adhesion studies demonstrated that melanocytes adhered and oriented within the first 2h of culture on HyalS microdomains and not on Hyal microstructures where they spread on glass substrate around the patterned area. Double photo-immobilised samples characterised by a 100 microm stripe pattern of Hyal or HyalS on the top of a continuous layer of the two polysaccharides were also created in order to investigate the effect of the topography on cell behaviour. The obtained results demonstrated that melanocytes adhered on HyalS stripes while on the Hyal micropatterned surfaces they spread on silanised glass substrate around the structured area, resulting in the exclusion of the topographic pattern.

Neurotransmitter, opiodergic system, steroid-hormone interaction and involvement in the replacement therapy of sexual disorders.

Dopamine (DA) and serotonin (5-HT) are the neurotransmitters most directly involved in sexual activity. DA plays a stimulatory role while 5-HT has an inhibitory effect. The two monoaminergic systems modulate the secretion of many hormones (GnRH, LH, testosterone, prolactin and endorphins) involved in sexual functional capacity. Furthermore, hormones influence synthesis and storage of brain neurotransmitters. Impotence can often be associated to clinical depression and altered neurotransmitter function. Moreover, stress represents an unbalance between various neurotransmitter systems and can induce impotence especially when disorders of the endorphinic system are present. Replacement therapy is based upon the understanding of these basic concepts. Impotence due to an underlying depressive illness must be treated with dopaminergic antidepressant drugs; while in stressful conditions a good response to the naloxone test is the preliminary criterion to subsequent naltrexone treatment. When a hormonal deficiency has been proved, the hormone replacement therapy is of course highly effective (gonadotropins in hypogonadotropic syndromes, testosterone in aging, etc.). Finally, idiopathic impotence could be treated by DA agonist and/or 5-HT antagonist drugs either alone or better yet in association with psychotherapy.

Blood-interaction performance of differently sulphated hyaluronic acids.

Seven differently sulphated hyaluronic acid derivatives, having a general formula HyalSx where x can be 1, 2, 2.5, 3, 3.5, 3.8, 4, were synthetized. Coagulation tests i.e. whole blood clotting time and thrombin time were performed on these compounds and significant prolongations were observed from HyalS2.5 up to HyalS4. All that means the heparin like activity increases by increasing the sulphation degree of hyaluronic acid. The interaction of each of them with thrombin and FXa was studied in order to understand the mechanism of coagulation inactivation and the role of the sulphate position in the disaccharide unit to favour the protease inhibiting reaction. The bioactivity of HyalSx in terms of FXa and thrombin inactivation increases increasing with sulphation degree but the FXa inactivation seems to be mediated by ATIII, while the aspecific electrostatic interaction seems to play an important role in the inactivation of thrombin. Also the interaction with human serum albumin was studied by ATR/FT-IR technique and no changes of protein conformation was observed, as occurs in the case of heparin.