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The crested porcupine is a mainly nocturnal mammal that shows both moonlight avoidance and some diurnal motor activity, the latter as an extension of its night-time foraging behaviour. Furthermore, a peculiar daytime behaviour, described as “sunbathing”, was reported as episodic in H. africaeaustralis. Between 2016 and 2019 a camera-trapping monitoring was performed within 10 porcupine settlements in order to detect the diurnal motor activity and to verify and describe the sunbathing behaviour in crested porcupine. Out of 1,003 trap days, a total of 148 events of diurnal motor activity were recorded. The diurnal motor activity occurred throughout the year mainly between December and June from 15:00 to 16:00, with no statistical difference between cubs, youngsters and adults. The sunbathing behaviour was detected for a total of 36 episodes recorded. Sunbathing was performed mainly by cubs. The sunbathing behaviour occurred only between April and June during the hottest hours of the day (11:00 to 12:00). Diurnal motor activity and sunbathing behaviour of porcupine are discussed in relation to food availability and porcupine physiology.

The continuous increasing of engineered nanomaterials (ENMs) in our environment, their combinatorial diversity, and the associated genotoxic risks, highlight the urgent need to better define the possible toxicological effects of ENMs. In this context, we present a new high-throughput screening (HTS) platform based on the cytokinesis-block micronucleus (CBMN) assay, lab-on-chip cell sorting, and automated image analysis. This HTS platform has been successfully applied to the evaluation of the cytotoxic and genotoxic effects of silver nanoparticles (AgNPs) and silica nanoparticles (SiO 2 NPs). In particular, our results demonstrate the high cyto- and genotoxicity induced by AgNPs and the biocompatibility of SiO 2 NPs, in primary human lymphocytes. Moreover, our data reveal that the toxic effects are also dependent on size, surface coating, and surface charge. Most importantly, our HTS platform shows that AgNP-induced genotoxicity is lymphocyte sub-type dependent and is particularly pronounced in CD2+ and CD4+ cells. Refereed/Peer-reviewed

In this study, we show that 15 nm citrate-capped AuNPs exert a remarkable toxicity in living systems. The assessment was performed by using well-characterized AuNPs, the combination of in vitro and in vivo models (namely two different cell lines and Drosophila melanogaster), exposure to low dosages of nanoparticles (in the sub-nanomolar concentration range), along with the application of several biological assays to monitor different aspects of the toxic effects, such as viability, genotoxicity, and molecular biomarkers.

To obtain conformational data on the monomeric form of insulin, which is believed to be the physiologically active form of the hormone, insulin in sodium dodecyl sulfate solution was studied by Fourier-transform infrared (FTIR) spectroscopy and circular dichroism, and results were compared with those obtained with des(B23-30) octapeptide insulin (DOI) and dimeric insulin in buffer. The FTIR amide I band (1600-1700 cm-1) was examined, and a quantitative evaluation of the secondary structure fractions of the various conformations showed less of a beta-sheet component for both insulin in SDS and DOI in buffer than for insulin in buffer, corresponding to a lack of monomers binding to form dimers. At the concentrations used for FTIR (> or = 2 mg/mL), the CD spectra of insulin in SDS and DOI in buffer were qualitatively identical but different from that of insulin in buffer, which is associated at these concentrations. The CD spectrum pattern of insulin in very dilute solution (80 nM), where it is prevalently monomeric, is very similar to that of monomeric insulin in SDS, which suggests that the conformation of the hormone in the two cases is very similar.

Native glycosylated and enzymically deglycosylated conglutin γ (a lupin seed oligomeric protein) both showed an unusual resistance to tryptic degradation. The result of this treatment was that a single 40–residue peptide was cleaved from the N-terminus of conglutin γ light subunit. Acid treatment of the two protein forms led to their substantial unfolding, as indicated by CD spectra. After this treatment, both polypeptides were completely degraded by trypsin after a few minutes of incubation. Conversely, trypsin pulse experiments run under renaturing conditions demonstrated a different refolding behaviour of the two proteins: the glycosylated form became resistant to trypsin after a 7-h renaturation, while the deglycosylated form required 42 h renaturation. These results were confirmed by CD spectra and reverse-phase HPLC analyses of the glycosylated and deglycosylated conglutin γ forms. Therefore, it was concluded that the saccharide chain of conglutin γ increased the rate of formation of a trypsin-resistant conformation upon refolding of the acid-treated protein, without playing any direct role in the protection of the native protein from proteolysis.

The effects of high pressure on the solubility and on some structural features of ovalbumin were investigated on protein solutions at neutral pH, containing different protein concentrations and treated at 400−800 MPa for different times. Ovalbumin solutions were prepared in the presence and in the absence of common food ingredients, such as sucrose and NaCl, that could act as “protectants” against treatment-induced modification. Protein insolubilization occurred in the absence of protectants as a function of the protein concentration and the treatment intensity; it appeared to be a consequence of structural modifications involving in particular the tertiary structure of the protein, and it was completely prevented by blocking the free −SH groups in the protein. Several of the structural features of the protein were modified also when it was treated in the presence of protectants. Modifications depended on the treatment pressure and time and on the protectant used. All of the treated proteins showed an inc...

Bacterial adhesion onto abiotic surfaces is an important issue in biology and medicine since understanding the bases of such interaction represents a crucial aspect in the design of safe implant devices with intrinsic antibacterial characteristics. In this framework, we investigated the effects of nanostructured metal substrates on Escherichia coli adhesion and adaptation in order to understand the bio-molecular dynamics ruling the interactions at the interface. In particular, we show how highly controlled nanostructured gold substrates impact the bacterial behavior in terms of morphological changes and lead to modifications in the expression profile of several genes, which are crucially involved in the stress response and fimbrial synthesis. These results mainly demonstrate that E. coli cells are able to sense even slight changes in surface nanotopography and to actively respond by activating stress-related pathways. At the same time, our findings highlight the possibility of designing nanoengineered substrates able to trigger specific bio-molecular effects, thus opening the perspective of smartly tuning bacterial behavior by biomaterial design.

The expected potential benefits promised by nanotechnology in various fields have led to a rapid increase of the presence of engineered nanomaterials in a high number of commercial goods. This is generating increasing questions about possible risks for human health and environment, due to the lack of an in-depth assessment of the physical/chemical factors responsible for their toxic effects. In this work, we evaluated the toxicity of monodisperse citrate-capped gold nanoparticles (AuNPs) of different sizes (5, 15, 40, and 80 nm) in the model organism Drosophila melanogaster, upon ingestion. To properly evaluate and distinguish the possible dose- and/or size-dependent toxicity of the AuNPs, we performed a thorough assessment of their biological effects, using two different dose-metrics. In the first approach, we kept constant the total surface area of the differently sized AuNPs (Total Exposed Surface area approach, TES), while, in the second approach, we used the same number concentration of the four different sizes of AuNPs (Total Number of Nanoparticles approach, TNN). We observed a significant AuNPs-induced toxicity in vivo, namely a strong reduction of Drosophila lifespan and fertility performance, presence of DNA fragmentation, as well as a significant modification in the expression levels of genes involved in stress responses, DNA damage recognition and apoptosis pathway. Interestingly, we found that, within the investigated experimental conditions, the toxic effects in the exposed organisms were directly related to the concentration of the AuNPs administered, irrespective of their size.

Familial combined hyperlipidemia (FCHL) is the most common genetic lipid disorder among young survivors of myocardial infarction. Elevations of plasma total and low-density lipoprotein (LDL) cholesterol and the prevalence of small, dense LDL particles are both involved in the high coronary risk of FCHL patients. We investigated the ability of pravastatin to favorably correct plasma lipid and lipoprotein levels and LDL structure in FCHL patients. Twelve patients with FCHL, documented by studies of first-degree relatives, received pravastatin (40 mg/d) for 12 weeks. Pravastatin significantly lowered plasma total and LDL cholesterol levels by 21% and 32%, respectively. Triglyceride levels did not change, and apolipoprotein B (apoB) concentrations decreased by 9% (P = NS). High-density lipoprotein (HDL) cholesterol increased by 6% because of a significant 73% rise of HDL2 cholesterol. LDL were smaller (diameter, 24.5 +/- 0.5 nm), less buoyant, and apoB-rich (cholesteryl ester-apoB ratio, 1.64 +/- 0.46) in the selected patients compared with patients with familial hypercholesterolemia or healthy control subjects. LDL became even smaller (23.8 +/- 0.6 nm) and richer in apoB (cholesteryl ester-apoB ratio, 1.27 +/- 0.52) after pravastatin treatment. Although pravastatin favorably altered plasma lipid and lipoprotein levels in FCHL patients, the abnormal LDL particle distribution and composition were not affected. Because of the apparent resistance of the small, dense LDL to drug-induced modifications, a maximal lipid-lowering effect is needed to reduce coronary risk in FCHL patients.

Bacterial adhesion onto inorganic/nanoengineered surfaces is a key issue in biotechnology and medicine, because it is one of the first necessary steps to determine a general pathogenic event. Understanding the molecular mechanisms of bacteria-surface interaction represents a milestone for planning a new generation of devices with unanimously certified antibacterial characteristics. Here, we show how highly controlled nanostructured substrates impact the bacterial behavior in terms of morphological, genomic, and proteomic response. We observed by atomic force microscopy (AFM) and scanning electron microscopy (SEM) that type-1 fimbriae typically disappear in Escherichia coli adherent onto nanostructured substrates, as opposed to bacteria onto reference glass or flat gold surfaces. A genetic variation of the fimbrial operon regulation was consistently identified by real time qPCR in bacteria interacting with the nanorough substrates. To gain a deeper insight into the molecular basis of the interaction mechanisms, we explored the entire proteomic profile of E. coli by 2D-DIGE, finding significant changes in the bacteria adherent onto the nanorough substrates, such as regulations of proteins involved in stress processes and defense mechanisms. We thus demonstrated that a pure physical stimulus, that is, a nanoscale variation of surface topography, may play per se a significant role in determining the morphological, genetic, and proteomic profile of bacteria. These data suggest that in depth investigations of the molecular processes of microorganisms adhering to surfaces are of great importance for the design of innovative biomaterials with active biological functionalities.

Structural properties, and especially the differential stability, of complexes between carbonic anhydrase (CA) and three sulfonamide inhibitors, acetazolamide, dorzolamide and methazolamide, were investigated by spectroscopic and electrophoretic techniques. These included denaturant gradient gel electrophoresis either across a urea or a steady-state transverse sodium dodecyl sulfate (SDS) gradient. Acetazolamide, the smallest and most hydrophilic of the sulfonamides, forms the most stable complex in the presence of urea, whereas dorzolamide, with a bulky and hydrophobic structure, is most stable against the effects of SDS. At pH 7.4, complexes with dorzolamide show minimal changes in mobility across the SDS gradient, as if unaffected by the detergent, both in the presence and in the absence of excess ligand in the gel. When bound to both acetazolamide and methazolamide, on the other hand, CA displays an increase in mobility above 0.05% SDS, lower in the presence than in the absence of excess ligand. The finding of a distinct pattern for the unliganded enzyme, however, suggests the complexes can still retain the ligand, although binding of the surfactant changes their charge density. Under saturating conditions and in the presence of SDS, the surface charge of all complexes is much lower than for unliganded, denatured CA. Circular dichroism (CD) spectra clearly indicate that the increase in secondary structure and the decrease in tertiary structure brought about in CA by the presence of low concentrations of SDS are largely prevented by complexing with the inhibitors. These observations point out peculiar properties of each CA inhibitor, of potential value in the definition of their biological activities and also in the potential development of novel antagonist molecules.

The truncated mutant Met-adrenodoxin-(4-107)-peptide of bovine adrenal ferredoxin was expressed as apoprotein in Escherichia coli BL21 and could be reconstituted to the holoform by chemical or enzymatic methods. The reconstituted protein had spectroscopic, functional and redox properties similar to the Met-adrenodoxin-(4-108)-peptide of adrenal ferredoxin, into which the cluster was inserted upon expression in the same Escherichia coli strain. Rate of in vitro cluster insertion into the Met-adrenodoxin-(4-107) apoprotein was much lower than for the Met-adrenodoxin-(4-108) apoprotein under identical conditions. Comparative thermodynamic studies with the Met-adrenodoxin-(4-108)-peptide indicated that removal of Pro108 resulted in an extensive decrease of the overall stability of the protein in either oxidation state. The Met-adrenodoxin-(4-107)-peptide showed a higher sensitivity to urea denaturation and had a sensibly lower denaturation temperature, 44.8 degrees C, compared with 51.7 degrees C for mutant Met-adrenodoxin-(4-108). The stability of the reduced state of both mutants is slightly lower than that of the oxidized state indicating that this protein region does not undergo major structural changes upon reduction.

Gemfibrozil, a widely used fibric acid derivative, corrects hypercholesterolemia in a non-negligible fraction of patients. To investigate the mechanism of the cholesterol-lowering activity of fibric acids, a study was performed in 12 type IIa hyperlipidemic patients treated with gemfibrozil for 12 weeks. Changes in low density lipoprotein (LDL) structure and composition, agonist capacity of LDL against the LDL-receptor in human skin fibroblasts, LDL-receptor activity in mononuclear cells, lecithin:cholesterol acyltransferase (LCAT) and cholesterol ester transfer protein (CETP) activity, were evaluated. Plasma total and LDL cholesterol levels decreased by 17% and 20% after 12 weeks of treatment, the reduction being directly correlated with the baseline levels (r = 0.75 and 0.78, respectively). The mean LDL diameter increased significantly, from 25.5 to 26.1 nm, while the relative content of small LDL particles (< 25.1 nm) increased from 23.4% to 32.8% of total LDL. Neither the apolipoprotein (apo) B secondary structure nor the affinity of LDL for the LDL-receptor of fibroblasts were affected. The LDL-receptor activity in patients' mononuclear cells increased 3-fold, the rise being unrelated to the plasma cholesterol reduction. LCAT activity did not change, while CETP activity was reduced by 25% (P = 0.13) after treatment. These findings indicate that gemfibrozil causes significant changes in LDL structure that do not, however, affect the LDL interaction with peripheral cells.

Semiconductor nanocrystals, or Quantum Dots (QDs), have gained considerable attention due to their unique size-dependent optical and electronic properties that make them attractive for a wide range of applications, including biology and nanomedicine. Their widespread use, however, poses urgent questions about their potential toxicity, especially because of their heavy metal composition that could cause harmful effects to human health and environment. In this work, we evaluated in vivo the long-term toxicity of CdSe-ZnS QDs with different surface coatings, probing oral administration in the model system Drosophila melanogaster. In particular, we found that all the differently coated QDs significantly affect the lifespan of treated Drosophila populations and induce a marked increase in reactive oxygen species (ROS) levels. Furthermore, we observed that these QDs induce severe genotoxic effects and increased rate of apoptosis in Drosophila haemocytes. These toxic effects were found to be mainly related to the in vivo degradation of QDs with consequent release of Cd(2+) ions, while the coating of QDs can modulate their bioaccumulation in the organism, partly decreasing their overall toxicity.

The precursors of the legumin-like storage protein from developing white lupin seeds (35 days after flowering) are trimers composed of protomers of M(r) 72,000 or 67,000. Some subunits of these oligomers contain processed precursor polypeptides, namely alpha polypeptides of either 52,000 or 44,000 linked through disulphide bonds to a beta polypeptide of 21,000, typical of the mature legumin. The prolegumin is glycosylated. Legumin oligomers purified from the same seeds are both trimers and hexamers; some of their subunits are still made of precursor polypeptides. The hexamer contains less precursor polypeptide than the trimer. A low level or absence of precursor appears to be a condition of hexamer assembly. The heterogenous prolegumin and legumin oligomers represent intermediates in the processing of the prolegumin to mature legumin. Hydrophobic-interaction chromatography on TSK-phenyl-5PW and titration with the hydrophobic probe 8-anilino-1-naphthalenesulphonate indicate that the legumin is less hydrophobic than the prolegumin. This is attributed to structural rearrangements at processing of the propolypeptide, made evident by the behaviour in CD and by the second-derivative ultraviolet spectra of the two proteins. The total protein extract of developing cotyledons at 40 days after flowering contains endopeptidases, similar to those existing in the resting seeds, which cause a limited cascade degradation of the prolegumin and legumin.

The reaction of human serum albumin (HSA) with aldoses (C3-C6) and acetaldehyde has been studied. U.v. and fluorescent spectra of the HSA-glyceraldehyde and HSA-GlcN adducts reveal yellow chromophores absorbing at 300-350 nm and emitting at 435 nm. However, even limited reaction of HSA with acetaldehyde induced perturbation in the Trp microenvironment. C.d. spectra of the adducts show an average 20% decrement in mean residual ellipticity [theta], which is independent of the extent of the reaction and the aldose used. It is concluded that most of the reactions with aldoses occur at the surface of the HSA molecule. With the exception of the GlcN adduct, the HSA adducts rearrange to produce pyrrole rings on the protein surface. I.e.f. analysis shows that the pI values of the modified HSA are almost linearly correlated with the chain length of the reacting aldose: from pI 4.2 for HSA-glyceraldehyde up to pI 5.0 for HSA-GlcN.

The isoelectric properties of serum and urinary albumin from normal subjects and patients with nephrotic syndrome have been investigated in various conditions of denaturation, obtained by using urea (0-8 M) as a support in isoelectric focusing. In normal human serum, albumin is rather acidic (pI = 4.7) when focused in glycerol while the denatured form obtained by exposing the protein to 8 M urea has a much higher pI (6.1). Albumin from nephrotic patients is acidic in glycerol but at very low levels of urea (2M) it shifts from pI 4.7 to pI 6.1; the same effect has been induced by treating albumin with activated charcoal at low pH. In order to obtain more information on urea-induced changes, we have recorded the circular dichroic spectra of albumin when exposed to the concentration of urea used in gels, and we found that no conformational transition occurs for urea concentrations less than 5 M. Taken together, these observations reveal that variation of the pI of albumin in nephrotic syndrome occurs mainly due to a dissociating effect of urea on charged substances bound to this protein.

Several molecular variants of human apolipoproteins, particularly of apo AI and apo E1, have been described since 1980, when the apolipoprotein AI-Milano (AIM) mutant was identified in an Italian family2,3. The apo E mutants are often associated with severe hyperlipidemia, primarily dysbetalipoproteinemia, and premature atherosclerosis. By contrast, with the exception of the apo AIM, there is no convincing evidence M associating any of the described apo AI variants with pathological conditions or with significant alterations of lipid and lipoprotein metabolism.

Positively charged insulin is described to induce aggregation of phosphatidylcholine vesicles containing 10 mol% sulfatide at acidic pH. Techniques including light-scattering, Sepharose chromatography, centrifugation, trapped volume determintion, circular dichroism and fluorescence polarization, demonstrate that large amounts of negatively charged insulin remain firmly associated to the vesicles upon raising the pH to 7. This is surprising, since only trace amounts of insulin associate to the sulfatide-containing vesicles upon direct incubation at pH 7. The possible molecular explanation of the phenomenon and the relevance of these findings to the actions of insulin in vivo are discussed.