Clinical implications of the types of cryoglobulins determined by two-dimensional polyacrylamide gel electrophoresis

BACKGROUND AND OBJECTIVE: Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) is a new method which can be used to study cryoprecipitates from the sera of cryoglobulinemic patients. It led to the identification of a new type of cryoprecipitate, tentatively named II-III, characterized by polyclonal IgG associated with a mixture of polyclonal and monoclonal IgM. Some discrepancies with the conventional classification of cryoglobulins were revealed. The association of particular clinical features with the classification of cryoglobulins by 2-D PAGE is examined. DESIGN AND METHODS: Sixty consecutive patients affected by cryoglobulinemic syndrome with mixed cryoglobulins were included in the study. All patients were evaluated for cutaneous, articular, hepatic, renal and nervous involvement. The washed cryoprecipitates were typed using both techniques: immunofixation electrophoresis (IFE) and 2-D PAGE. RESULTS: Sixteen (6 cases of type II and 10 of type III by IFE) of 60 cryoprecipitates (26.6%) appeared as type II-III by 2-D PAGE analysis. Nine cases were classified differently by IFE and 2-D PAGE. Mixed cryoglobulins of type II-III were not associated with a particular clinical pattern. Examining the clinical findings in the mono group (those with monoclonal IgM alone) and the poly group (those with polyclonal IgM alone or polyclonal and monoclonal IgM) we found clearly significant differences: more severe liver involvement in the poly group, and higher cryocrit and creatinine values, lower C4 level and more severe purpura in the mono group. INTERPRETATION AND CONCLUSIONS: Our results confirm the reliability of 2-D PAGE in characterizing cryoprecipitates. This sensitive method can demonstrate a higher number of monoclonal components, undetectable by IFE. Type II-III cryoglobulins are not associated with a particular clinical pattern. The presence or absence of polyclonal IgM in mixed cryoglobulins seems to be correlated with some clinical findings.     

When a Tritrophic Interaction Goes Wrong to the Third Level: Xanthoxylin From Trees Causes the Honeybee Larval Mortality in Colonies Affected by the River Disease

The “River Disease” (RD), a disorder impacting honeybee colonies located close to waterways with abundant riparian vegetation (including Sebastiania schottiana, Euphorbiaceae), kills newly hatched larvae. Forager bees from RD-affected colonies collect honeydew excretions from Epormenis cestri (Hemiptera: Flatidae), a planthopper feeding on trees of S. schottiana. First-instar honeybee larvae fed with this honeydew died. Thus, we postulated that the nectars of RD-affected colonies had a natural toxin coming from either E. cestri or S. schottiana. An untargeted metabolomics characterization of fresh nectars extracts from colonies with and without RD allowed to pinpoint xanthoxylin as one of the chemicals present in higher amounts in nectar from RD-affected colonies than in nectars from healthy colonies. Besides, xanthoxylin was also found in the aerial parts of S. schottiana and the honeydew excreted by E. cestri feeding on this tree. A larva feeding assay where xanthoxylin-enriched diets were offered to 1^st instar larvae showed that larvae died in the same proportion as larvae did when offered enriched diets with nectars from RD-colonies. These findings demonstrate that a xenobiotic can mimic the RD syndrome in honeybee larvae and provide evidence of an interspecific flow of xanthoxylin among three trophic levels. Further, our results give information that can be considered when implementing measures to control this honeybee disease.

     

3D Bioprinting of Gelatin–Xanthan Gum Composite Hydrogels for Growth of Human Skin Cells

In recent years, bioprinting has attracted much attention as a potential tool for generating complex 3D biological constructs capable of mimicking the native tissue microenvironment and promoting physiologically relevant cell–cell and cell–matrix interactions. The aim of the present study was to develop a crosslinked 3D printable hydrogel based on biocompatible natural polymers, gelatin and xanthan gum at different percentages to be used both as a scaffold for cell growth and as a wound dressing. The CellInk Inkredible 3D printer was used for the 3D printing of hydrogels, and a glutaraldehyde solution was tested for the crosslinking process. We were able to obtain two kinds of printable hydrogels with different porosity, swelling and degradation time. Subsequently, the printed hydrogels were characterized from the point of view of biocompatibility. Our results showed that gelatin/xanthan-gum bioprinted hydrogels were biocompatible materials, as they allowed both human keratinocyte and fibroblast in vitro growth for 14 days. These two bioprintable hydrogels could be also used as a helpful dressing material.     

Synthesis of novel DC-SIGN ligands with an alpha-fucosylamide anchor

The dendritic cell-specific intercellular adhesion molecule (ICAM) 3-grabbing nonintegrin (DC-SIGN) is a C-type lectin that appears to perform several different functions. Besides mediating adhesion between dendritic cells and T lymphocytes, DC-SIGN recognizes several pathogens some of which, including HIV, appear to exploit it to invade host organisms. The intriguing diversity of the roles attributed to DC-SIGN and their therapeutic implications have stimulated the search for new ligands that could be used as biological probes and possibly as lead compounds for drug development. The natural ligands of DC-SIGN consist of mannose oligosaccharides or fucose-containing Lewis-type determinants. Using the known 3D structure of the Lewis-x trisaccharide, we have identified some monovalent alpha-fucosylamides that bind to DC-SIGN with inhibitory constants 0.4-0.5 mM, as determined by SPR, and have characterized their interaction with the protein by STD NMR spectroscopy. This work establishes for the first time alpha-fucosylamides as functional mimics of chemically and enzymatically unstable alpha-fucosides and describes interesting candidates for the preparation of multivalent systems able to block the receptor DC-SIGN with high affinity and with potential biomedical applications.     

Robust modeling of RC structures with an “event-by-event” strategy

The sequentially linear analysis is a robust alternative to nonlinear finite element analysis of structures when bifurcation, snap-back or divergence problems arise. The load–displacement response is captured by a series of linear analyses as a sequence of ‘events’. Every ‘event’ is a scaled critical state corresponding to the reaching of some peak of some saw-tooth for some softening element. Now, the approach is extended with a rippled saw-tooth curve which applies to any stress–strain diagram, including compression nonlinearity and yielding of reinforcement. Several RC structural examples demonstrate that both sharp snap-backs as well as ductile failures can be handled correctly.     

General method for the evaluation of complex heat pump cycles

Whenever the fractional temperature lift ΔT/T_c of a heat pump is 0.15, simple cycles with one-stage throttling exhibit unsatisfactory energy performance. The adoption of multi-stage throttling, both in non-regenerative and regenerative cycles, is the most direct way of improving the cycle coefficient of performance (COP). The performance of these complex cycles is found to be a function of the molecular complexity of the working fluid, the reduced evaporation temperature, the fractional temperature lift and the number of stages of throttling. Furthermore, complex cycles are shown to be equivalent to a combination of simple cycles and their performance may be directly inferred by this route. Such calculations show that for a given fractional temperature lift an optimum molecular complexity (between that of R12 and n-butane) exists. Fluids with simpler molecules exhibit excessive vapour superheating during compression, and those with more complex molecules have excessive throttling losses. Also, with complex cycles, regeneration should be applied only to the cycle at the lowest temperature in order to improve the cycle COP and to prevent condensation during compression. As a general trend, however, complex cycles suffer a significant loss in performance compared to optimized simple cycles due to the adverse area of the two-phase diagram in which they work.     

Ectopic expression of pRb2/p130 suppresses the tumorigenicity of the c-erbB-2-overexpressing SKOV3 tumor cell line

We investigated the in vitro and in vivo effects of the ectopic expression of the pRb2/p130 cell cycle regulator on c-erbB-2-associated tumorigenicity. SKOV3 ovarian cancer cells, which display c-erbB-2 gene amplification and oncoprotein (p185HER2) overexpression, were stably transfected with a plasmid containing the coding sequence for human wild-type pRb2/p130 (wtRb2), or with pcDNA3 empty vector. Three wtRb2-transfected clones (cl. 24, ci. 49, cl. 100) and one empty vector-transfected clone (cl. mock) were randomly picked and further analysed. Western blot analysis revealed high levels of pRb2/p130 in the three clones compared to mock cells. Levels of p185HER2 and the extent of its tyrosine phosphorylation were similar in all transfectant clones, as were levels of pRb1 and p107. In anchorage-independent growth assays, the number of colonies from wtRb2 clone-transfectants was about 90% less than that arising from mock cells (P<0.001). Tumor take rates of the three wtRb2-transfected clones xenografted in nu/nu mice were much lower than those of mock cells, and tumor volume was decreased by 80% (P<0.001). A mutant version of pRb2/p130 deleted of the pocket region (mut-Rb2) was also transfected into SKOV3 cells and studied in parallel with the wtRb2-transfected and pcDNA empty vector-transfected bulk populations. mut-Rb2 transfected cells showed no inhibition of in vitro colony formation and were fully tumorigenic. Together, these findings indicate that Rb2 acts as a tumor suppressor gene in vivo and in vitro in SKOV3 cells and that the intact pocket region is required for the suppressor activity.     

A potent integrin antagonist from a small library of cyclic RGD pentapeptide mimics including benzyl-substituted azabicycloalkane amino acids

A small library of cyclic RGD pentapeptide mimics, including benzyl-substituted azabicycloalkane amino acids, was synthesized with the aim of developing active and selective integrin antagonists. In vitro binding assays established one particular compound with affinity for both the alpha(v)beta(3) and the alpha(v)beta(5) integrins. The synthesis in solution and the in vitro screening of these RGD derivatives, as well as the determination of the conformational properties of the integrin ligands by spectroscopic and computational methods are described.     

Stirling engines using working fluids with strong real gas effects

Real gas effects typical of the critical region of working fluids are a powerful tool to increase the energy performances of Stirling cycles, mainly at low top temperatures. To carry out the compression near the critical region the working fluids must have a critical temperature near environmental conditions and the use of organic working substances (pure or in suitable mixtures) as a matter of fact begins compulsory. The moderate thermal stability of the organic working fluids limits the maximum temperatures to 300–400 °C and as a consequence, the achievable cycles efficiencies result rather low. Carbon dioxide, with a critical temperature of 31 °C, is, among the traditionally inorganic gases, an exception and is considered here in comparison with organic substances. But the good thermodynamics of the cycles allows, in the considered cases, conversion efficiencies of about 20%, with good specific powers. The good energy performance of real gas Stirling cycles is obtained at the cost of high maximum cycle pressure, in the range of at least 100–300 bar. These high pressures nevertheless have large positive effects on the heat power transferred per unit of pumping mechanical power, and the low top temperatures have a positive influence on the material problems for the hottest engine parts.     

Historical brick-masonry subjected to double flat-jack test: Acoustic emissions and scale effects on cracking density

The results obtained varying the size of the masonry prism involved in the double flat-jack test are described. In these tests, not only the deformations have been acquired, but also the acoustic emissions (AE) events, in order to get information about local cracking in the specimens. In addition, a meso-scale numerical model of the test is presented, where every brick of the masonry is modeled in details. Discrete cracks can arise both in the mortar joints and in the brick units. A good correlation is found between the amount of cracking simulated numerically and the experimental AE events for different prism sizes. The model is also able to catch the decrease in the compressive strength with increasing size. Although a quantitative relation between the AE events and the amount of cracking is not easy to obtain, we have been able to prove that the two quantities are simply proportional to each other when increasing specimen sizes are considered, whereas both of them are not proportional to the same sizes, but rather obey a power-law.