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Monoolein-based cubic and hexagonal mesophases were investigated as matrices for insulin loading, at low pH, as a function of temperature and in the presence of increasing amounts of oleic acid, as a structural stabilizer for the hexagonal phase. Synchrotron small angle X-ray diffraction, rheological measurements, and attenuated total reflection-Fourier transform infrared spectroscopy were used to study the effects of insulin loading on the lipid mesophases and of the effect of protein confinement in the 2D- and 3D-lipid matrix water channels on its stability and unfolding behavior. We found that insulin encapsulation has only little effects both on the mesophase structures and on the viscoelastic properties of lipid systems, whereas protein confinement affects the response of the secondary structure of insulin to thermal changes in a different manner according to the specific mesophase: in the cubic structure, the unfolding toward an unordered structure is favored, while the prevalence of parallel β-sheets, and nuclei for fibril formation, is observed in hexagonal structures.

A method is designed to quickly form protein hydrogels, based on the self-assembly of highly concentrated lysozyme solutions in acidic conditions. Their properties can be easily modulated by selecting the curing temperature. Molecular insights on the gelation pathway, derived by in situ FTIR spectroscopy, are related to calorimetric and rheological results, providing a consistent picture on structure-property correlations. In these self-crowded samples, the thermal unfolding induces the rapid formation of amyloid aggregates, leading to temperature-dependent quasi-stationary levels of antiparallel cross β-sheet links, attributed to kinetically trapped oligomers. Upon subsequent cooling, thermoreversible hydrogels develop by the formation of interoligomer contacts. Through heating/cooling cycles, the starting solutions can be largely recovered back, due to oligomer-to-monomer dissociation and refolding. Overall, transparent protein hydrogels can be easily formed in self-crowding conditions and their properties explained, considering the formation of interconnected amyloid oligomers. This type of biomaterial might be relevant in different fields, along with analogous systems of a fibrillar nature more commonly considered.

The dispute over the paternity of western paper is a longstanding problem. In Italy, a country that has a long tradition of papermaking, many cities compete for the leadership in the production of this material; on the other hand, several studies attribute to Fabriano (13th century), a little center in Marche region, the birth of modern paper and its diffusion throughout Europe. During the 14thcentury, the nearby town of Camerino imposed itself on the paper market, triggering a historic rivalry with the manufacture of Fabriano. As scientific studies on these different productions are missing, a comparative, multidisciplinary investigation has been carried out to characterize a set of thirteenth-fifteenth century handmade papers from Camerino-Fabriano area. Several analytical techniques–some non-invasive such as Fourier-transform infrared spectroscopy (FTIR) and portable magnetic resonance (unilateral NMR), joined to micro-destructive ones such as scanning electron microscopy (SEM), 13C CPMAS NMR spectroscopy, thermo-gravimetric analysis (TGA), sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and reverse-phase liquid chromatography (RP-HPLC)–have been employed in order to study the morphology of the paper documents, to identify the substances used in production process and to define their preservation state. Moreover principal component analysis (PCA) has been applied to the spectroscopic data, allowing the clustering of Fabriano documents with respect to Camerino ones and dividing the samples in two sets. This work improves and confirms knowledges on paper production in Marche region, and the experimental data collected highlight similarities and differences between Camerino and Fabriano manufactures.

The roles of mesophilic lactobacilli in cheese manufacturing and ripening have been widely studied, but their impacts, especially as additives in preserving liquids, on the high-moisture mozzarella cheese quality parameters remained underexplored. The current study studied and compared the effects of four preserving liquid formulations - brine solution (sodium chloride, as control), a salt mixture solution, and two lactobacilli solutions (Lactiplantibacillus plantarum IMC 509 in brine solution) or SYNBIO® (a 1:1 ratio of Lacticaseibacillus rhamnosus IMC 501 and Lacticaseibacillus paracasei IMC 502) - on the microbial, chemical (volatile fatty acids), physicochemical (moisture, weight, pH, colour), texture (adhesiveness, hardness, cohesiveness, gumminess, springiness, chewiness) and sensorial qualities of mozzarella stored for 30 days at 4 °C. The quality of the governing solution, including microbial content, lactobacilli viability, pH, turbidity, and smell, was also monitored. For the first 10 days of storage, all samples demonstrated similar physicochemical variations: the firmness and chewiness of the mozzarella lessened, the pH values of the liquid decreased, but cheese microbial growth increased, as did the levels of free fatty acids, the mozzarella pH values and skin whiteness. At day 20, mozzarella stored in mesophilic lactobacilli liquid, especially SYNBIO®, had a spongy structure, tasted more bitter and sour, and had lower pH values than mozzarella in the simple brine solution. Moreover, the cheese sample and liquid of SYNBIO® also presented higher coliform and Pseudomonas spp. counts than that detected in the control, the SYNBIO® liquid, especially in the later period of storage, exhibited a yogurt smell and increased turbidity. By contrast, Mozzarella packed into salt mixture liquid exhibited somewhat more adhesiveness and gumminess, a saltier taste and slightly higher microbial counts than the control sample. The mozzarella samples differed slightly in colour and texture as well. The overall quality changes suggested that the use of the studied mesophilic lactobacilli as governing liquid additives may not be appropriate for high-moisture mozzarella cheese preservation.

Background In the scientific literature, there are only a few manuscripts available on small animal faecal proteomics. Methods In the present pilot study, this evaluation was performed using pooled faecal samples from 10 clinically healthy dogs and, for the first time, in 10 clinically healthy cats by mean of two‐dimensional electrophoresis followed by liquid chromatography‐tandem mass spectrometry. Results Our results showed the presence of nine (albumin, alkaline phosphatase, chymotrypsin‐C‐like, cytosol aminopeptidase, elastase‐3B/proteinase E, immunoglobulins and nuclear pore membrane glycoprotein 210) and 14 (albumin, caspase recruitment domain‐containing protein, chymotrypsin‐like, deleted in malignant brain tumours 1 protein‐like, hypothetical protein LOC107375, immunoglobulin, kallikrein‐1, superoxide dismutase, transthyretin precursor, interstitial collagenase‐like) different proteins in canine and feline faeces, respectively. Conclusion These preliminary findings document the presence of a range of proteins in the faeces of apparently healthy dogs and cats and may serve as a basis for larger, prospective studies to establish reference proteomic data against which diseased populations can be compared.

Amyloids are proteinaceous deposits considered an underlying pathological hallmark of several degenerative diseases. The mechanism of amyloid formation and its inhibition still represent challenging issues, especially when protein structure cannot be investigated by classical biophysical techniques as for the intrinsically disordered proteins (IDPs). In this view, the need to find an alternative way for providing molecular and structural information regarding IDPs prompted us to set a novel, to our knowledge, approach focused on UV Resonance Raman (UVRR) spectroscopy. To test its applicability, we study the fibrillation of hen-egg white lysozyme (HEWL) and insulin as well as their interaction with resveratrol, employing also intrinsic fluorescence spectroscopy, Fourier transform infrared (FTIR) spectroscopy, and atomic force microscopy (AFM). The increasing of the β-sheet structure content at the end of protein fibrillation probed by FTIR occurs simultaneously with a major solvent exposure of tryptophan (Trp) and tyrosine (Tyr) residues of HEWL and insulin, respectively, as revealed by UVRR and intrinsic fluorescence spectroscopy. However, because the latter technique is successfully used when proteins naturally contain Trp residues, it shows poor performances in the case of insulin, and the information regarding its tertiary structure is exclusively provided by UVRR spectroscopy. The presence of an increased concentration of resveratrol induces mild changes in the secondary structure of both protein fibrils while remodeling HEWL fibril length and promoting the formation of amorphous aggregates in the case of insulin. Although the intrinsic fluorescence spectra of proteins are hidden by resveratrol signal, UVRR Trp and Tyr bands are resonantly enhanced, showing a good sensitivity to the presence of resveratrol and marking a modification in the noncovalent interactions in which they are involved. Our findings demonstrate that UVRR is successfully employed in the study of aggregation-prone proteins and of their interaction with ligands, especially in the case of Trp-lacking proteins.

Breast cancer (BC) is the most common cancer in women and, among different BC subtypes, triple negative (TN) and human epidermal growth factor receptor 2 (HER2)-positive BCs have the worst prognosis. In this study, we investigated the anticancer activity of the root ethanolic and hexane extracts from Lithospermum erythrorhizon, a traditional Chinese herbal medicine known also as tzu ts’ao or tzu-ken, against in vitro and in vivo models of TNBC and HER2-positive BC. Treatment with L. erythrorhizon root extracts resulted in a dose-dependent inhibition of BC cell viability and in a significant reduction of the growth of TNBC cells transplanted in syngeneic mice. Acetylshikonin, a naphthoquinone, was identified as the main bioactive component in extracts and was responsible for the observed antitumor activity, being able to decrease BC cell viability and to interfere with autochthonous mammary carcinogenesis in Δ16HER2 transgenic mice. Acetylshikonin anticancer effect depends on its ability to act as a potent inhibitor of dihydrofolate reductase (DHFR), to down-regulate key mediators governing cancer growth and progression, such as HER2, Src and STAT3, and to induce apoptosis by caspase-3 activation. The accumulation of acetylshikonin in blood samples as well as in brain, kidney, liver and tumor tissues was also investigated by liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) highlighting that L. erythrorhizon treatment is effective in delivering the active compound into the target tissues. These results provide evidence that L. erythrorhizon extract and in particular its main component acetylshikonin are effective against aggressive BC subtypes and reveal new acetylshikonin mechanisms of action.

Background: Vitamin D and calcium are important dietary compounds that affect bone mass, even if other minerals (potassium, zinc, etc.) and vitamins (A, C and K) are also involved. Vitamin D and certain minerals, in fact, play an important role in calcium homeostasis and calcium absorption. Hip fracture incidence is higher in Europe and the United States, where calcium is frequently included in the human diet; while the occurrence of these fractures is lower in developing countries, where diets are often poor in calcium. This condition is named the “calcium paradox”, and may be partially explained by phosphate toxicity, which can negatively affect mineral metabolism. It is important to maintain correct dietary calcium-phosphate balance in order to have a healthy life, reducing the risk of osteoporotic fractures in older people. Vitamin D can also act as a hormone; vitamin D2 (ergocalciferol) is derived from the UV-B radiation of ergosterol, the natural vitamin D precursor detected in plants, fungi, and invertebrates. Vitamin D3 (cholecalciferol) is synthesized by sunlight exposure from 7-dehydrocholesterol, a precursor of cholesterol that can also act as provitamin D3. Dietary intake of vitamin D3 is essential when the skin is exposed for short periods to ultraviolet B light (UV-B), a category of invisible light rays such as UV-A and UV-C. This can be considered the usual situation in northern latitudes during the winter season, or the typical lifestyle for older people and/or for people with very white delicate skin. The actual recommended daily intake of dietary vitamin D is strictly correlated with age, ranging from 5 μg for infants, children, teenagers, and adults—including pregnant and lactating women—to 15 μg for people over 65 years.

In this work, the profile of the volatile compounds has been determined for the first time in fresh donkey milk and in donkey milk subjected to accelerated shelf-life test, freeze-drying and spray-drying processes. The effects of freeze-drying and spray-drying processes in donkey milk have been analyzed also on three representative whey proteins, β-lactoglobulin, α-lactalbumin and lysozyme, by determining the total amount, the enzymatic activity and the thermal stability. Considering the volatile compounds in fresh donkey milk, 2-heptanone, 1,3-bis (1,1-dimethylethyl)-benzene, nonanal, d -limonene, octanoic acid, and 1-octanol were detected. The accelerated shelf-life test revealed a constant increase of octanoic acid, 2-heptanone, and nonanal compared to fresh donkey milk (P

Background: Vitamin D and calcium are the most important dietary compounds that affect bone mass, even if other minerals (potassium, zinc, etc.) and other vitamins (A, C and K) are also involved. Vitamin D, in fact, plays an important role in calcium homeostasis and calcium absorption. Talking about calcium, it is well known the “calcium paradox”: hip fractures incidence are higher in western countries, where calcium is frequently included in human diet, while the occurrence of these fractures is lower in developing countries, where diets are normally poor in calcium. This paradox may be partially understood considering vitamin D content in serum of local population; a report produced by WHO/FAO experts team investigating on diet, nutrition and the prevention of specific diseases stated that there is enough clinical data demonstrating that an intake of vitamin D and calcium together sufficient to cover dietary requirements can greatly reduce the risk of osteoporotic fracture in older people. Vitamin D can also act as a hormone; vitamin D2 (ergocalciferol) is derived from the UV-B radiation of ergosterol, the vitamin D precursor naturally found in plants, fungi, and invertebrates. Vitamin D3 (cholecalciferol) is originated by sunlight exposure from 7-dehydrocholesterol, a precursor of cholesterol that can also act as a provitamin D3. Dietary intake of vitamin D is very important when skin is exposed for short times to ultraviolet B light (UV-B) one of the three kind of invisible light rays together with UV-A and UV-C. This can be considered the usual situation in some latitude and in winter season, or the typical condition for older people and/or for people with vey white delicate skin. Actually, the recommended daily intake of dietary vitamin D is strictly correlated with age, ranging from 5 μg for infants, children, teen-agers and adults, including women during pregnancy and lactation, to 15 μg for people over 65 years.

Platelet-rich plasma (PRP) is a nontransfusional hemocomponent, considered as a powerful concentrate of growth factors (GFs) therapeutically used to stimulate tissue regeneration. The use of autologous PRP, as the patient&rsquo, s own biological material, for therapeutic purposes represents a safe and effective alternative to conventional treatments in both human and veterinary medicine. The aim of this study was the characterization of canine PRP from rheological and biological points of view. Thus, a characterization of the viscoelastic properties of the PRP systems was performed in order to clarify the influence of different calcium concentrations, in the presence of autologous thrombin-rich solution, on the PRP gels&rsquo, mechanical properties, from which the applicability of these systems in biomedical treatments is strongly dependent. Then, an evaluation of the content of GFs in PRP, activated or not with thrombin, and stored at different temperatures (37 &deg, C and &minus, 20 &deg, C) was performed over time, outlining, for the first time, the importance of the effect of physiological temperature (37 &deg, C) on the production of GFs. A clinical case study conducted in a dog with a complete rupture of the common calcaneal tendon (Achilles tendon) confirmed the relevance of this hemocomponent in the daily veterinary clinical activity and the potential translational value for human health.

The aim of this study was to compare the carcass characteristics and the chemical and physical parameters of the meat produced by 10 male crossbred donkeys and 10 male mules slaughtered at 16 &plusmn, 1 years of age. The carcass weight and dressing percentage were significantly (p &lt, 0.05) higher in mules. Samples of the muscle Longissimus thoracis (LT) were analyzed. Donkey meat showed a higher fat content, while the glycogen content was higher in the mule LT. The total collagen content was higher in the mule LT, in this muscle, the shear force values were higher compared to the donkey LT. The lightness parameter (L*) was lower in the mule LT, while the redness index (a*) was higher in the mule LT, this muscle showed a higher content of iron, while zinc was higher in the donkey muscle LT. The donkey LT muscle showed a higher content of essential amino acids (52.2%) compared to the mule LT (50.1%). The results obtained demonstrated that the chemical and physical traits of mule and donkey meat were similar to those of other kinds of equid meat.

A class of gold(I) phosphane complexes have been identified as inhibitors of dihydrofolate reductase (DHFR) from E. coli, an enzyme that catalyzes the reduction of dihydrofolate (DHF) to tetrahydrofolate (THF), using NADPH as a coenzyme. In this work, to comprehend the nature of the interaction at the basis of these inhibitory effects, the binding properties of bis- and tris-phosphane gold(I) chloride compounds in regards to DHFR have been studied by emission spectroscopy and spectrophotometric assays. The lack of cysteine and seleno-cysteine residues in the enzyme active site, the most favorable sites of attack of Au(I) moieties, makes this work noteworthy. The interaction with the gold compounds results into the quenching of the DHFR tryptophan&rsquo, s emissions and in an enhancement of their intrinsic emission intensities. Moreover, a modulating action of NADPH is highlighted by means of an increase of the gold compound affinity toward the enzyme, in fact, the dissociation constants calculated for the interactions between DHFR and each gold compound in the presence of saturating NADPH were lower than the ones observed for the apo-enzyme. The fluorimetric data afforded to Kd values ranged from 2.22 &plusmn, 0.25 &micro, M for (PPh3)2AuCl in the presence of NADPH to 21.4 &plusmn, 3.85 &micro, M for 4L3AuTf in the absence of NADPH. By elucidating the energetic aspects of the binding events, we have attempted to dissect the role played by the gold phosphane/protein interactions in the inhibitory activity, resulting in an exothermic enthalpy change and a positive entropic contribution (&Delta, H&deg, = &minus, 5.04 &plusmn, 0.08 kcal/mol and &Delta, S&deg, = 7.34 &plusmn, 0.005 cal/mol&middot, K).

Background: Whole milk is a good source of all the nutrients, and it also contains a sufficient number of vitamins to permit regular the growth of the neonate. Dairy cow milk can create allergy in infants less than 12 months old because of the high caseins and β-lactoglobulin content. In these circumstances, donkey milk can represent a good replacement for dairy cows’ milk in children affected by Cow Milk Protein Allergy (CMPA) because of its close chemical composition with human milk, mainly due to its low protein and low mineral content. Milk vitamin content is highly variable among mammalian species and it is strictly correlated with the vitamin status and the diet administered to the mother. Fat-soluble vitamins content in donkey milk is, on average, lower compared to ruminants’ milk, while vitamin C content determined in donkey milk is higher compared to dairy cows’ milk, showing a great similarity with human milk. In donkey milk, the content of vitamins of the B-complex such as thiamine, riboflavin, niacin, pyridoxine, and folic acid is higher compared to human milk. The use of donkey milk as a new functional food must be further evaluated in interdisciplinary clinical trials in which pediatricians, dietitians, and food scientists must be involved to deepen the knowledge about the positive health impact of donkey milk in different sensitive people, especially children and the elderly.

Kefir is a type of fermented milk obtained thanks to the introduction of "kefir grains" in mammalian milk. Kefir grains consist of lactic and acetic acid bacteria and yeasts in alternative proportions that are held together by a matrix of complex sugars known as "kefiran." Thanks to the fermentative process, the kefir milk is rich in nutraceutical substances such as amino acids, vitamins, and mineral salts. The most valuable compounds of kefir fermentation are mainly lactic acid, exopolysaccharides, and bioactive peptides, the resulting products of proteolytic release from milk proteins (caseins and whey proteins). Among the nutraceutical properties of kefir are antimicrobial and antitumor activity, immunomodulating effect, and cholesterol-lowering effect. Therefore, in light of these intriguing properties of kefir milk, in this work, a proteomic analysis, by two-dimensional electrophoresis followed by mass spectrometry, has been performed. As a result, milk-derived polypeptides were identified in commercial kefir milk from organic farming. In particular, polypeptides deriving from κ-, αs1 -, and αs2 -caseins that may have potentially beneficial effects on human health have been detected.

Different laboratory markers are routinely used in the diagnosis and management of gastrointestinal (GI) disease in dogs. In the present study, starting from feces from both healthy dogs and dogs suffering from food responsive diarrhea (FRD), we tried to find proteins differently expressed in the two groups of dogs, by using a proteomic approach. Interestingly, we found that the immunoglobulin J-chain isoform 1 (species:Canis lupus familiaris) was identified only in diseased dogs (not in healthy). J-chain combines especially IgA monomers to IgA dimers and plays a crucial role for their secretions into mucosal interface. Being the first study of that kind in the dog, it is only possible to hypothesize that their presence could be likely due to an increased activation of the immune system or to a mucosal damage or both in FRD patients. Similarly, it is still impossible to assess whether this protein could be used as diagnostic/prognostic marker of GI disease; however, this study represents a promising first step toward fecal proteomics in canine GI disorders.

Vitellogenin (Vtg) has proven to be a sensitive and simple biomarker in determining sex, sexual maturity, and xenoestrogenic effects in fish. Thus, our investigation has been focused on identification, partial characterization, and quantification of grey mullet (Mugil cephalus) Vtg through the use of a variety of biochemical and immunological analytical techniques. Mullet is considered both a promising aquaculture candidate and an important species for improving sediment quality in polyculture systems. In the first part of this work, grey mullet Vtg was purified from plasma of 17β-estradiol (E2)-induced male fish by a one-step chromatographic protocol, and partially characterized. Specific polyclonal antibodies were then raised against the mullet Vtg, and both an indirect ELISA and an optical immunosensor were set up and validated to quantify plasma Vtg. The indirect ELISA and the optical immunosensor assay developed showed linear measuring in the range 56.8–1047.1 ng mL−1 and 70–739 ng mL−1 Vtg concentrations in standard solutions, respectively. The results obtained suggest that the indirect ELISA allows Vtg detection over a wide dynamic range, thus resulting more suitable for rapid and sensitive sample screening. Therefore, we suggest that the direct immunosensor is a promising tool which needs more investigation to improve the sensitivity.

Neuropathic pain is caused by a lesion or disease of the somatosensory nervous system and has a considerable impact on the quality of life. Neuropathic pain has a dynamic and complex aetiology and gives heterogeneous symptoms across patients; therefore, it represents an important clinical challenge. Current pharmacological treatment includes tricyclic antidepressant serotonin-noradrenaline uptake inhibitors such as duloxetine, pregabalin, and gabapentin. However, these drugs do not show efficacy in all patients suffering from neuropathic pain. In this work we used a nerve chronic constriction injury mice model based on the ligation of sciatic nerve to analyse, by two-dimensional electrophoresis and mass spectrometry, blood proteins significantly altered by neuropathic pain one-week after surgery. A sham-ligated group of mice acting as control and a group of ligated mice treated with gabapentin were also analysed. The results indicated that four haptoglobin isoforms were significantly more expressed, while transthyretin and alpha-2-macroglobulin expression decreased in the serum of the murine neuropathic pain model with respect to the control mice. Interestingly, the treatment with the gabapentin reversed these conditions. The outcomes of this study can provide a further understanding of the pathophysiological meaning of the biomarkers involved in neuropathic pain.

Caseins are a family of proteins constituted by α-caseins (αs-1 and αs-2 caseins), β-caseins and κ-caseins. β-caseins, in particular, show a temperature and concentration-dependent self-assembling behaviour. Recently, β-casein micelles have been proposed as natural nanocarriers for the delivery of hydrophobic compounds, promoting their bioavailability. Until now, all studies regarding both chemical-physical characterization and applications of β-caseins have employed the protein of bovine origin. However, it could be interesting to exploit the use of β-caseins from other milk sources for their potential encapsulation ability and immunogenicity but, at present, no information on the self-assembling behaviour is available for β-caseins from the milk of species different from bovine. In this work, for the first time, β-caseins from human milk and from donkey, goat, and sheep milk were purified and their self-assembling behaviour was compared to that of a commercial bovine β-casein, the only one for which the concentration and temperature aggregation behaviour is known. Furthermore, a preliminary evaluation of the immunogenicity potential of β-casein from other milk sources has been performed by cross-reaction experiments using anti-β-casein antibodies from bovine origin. The results indicated a similar self-assembling profile among all β-caseins examined compared to the bovine β-casein, suggesting the possible use of β-casein from other milk sources as nanocarriers. Since donkey and human β-casein do not cross-react with bovine anti-β-casein antibodies, they could be particularly interesting for the development of self-assembling systems with lower hypoallergenic potential.

Basal like breast cancer (BLBC) is a very aggressive subtype of breast cancer giving few chances of survival, against which cisplatin based therapy is a compromise among the anticancer activity, the resistance development and the severe side effects. With the aim of finding new anticancer agents alternative to cisplatin, seven gold(I) azolate/phosphane compounds were evaluated in vitro by MTT tests in human MDA-MB-231, human mammary epithelial HMLE cells overexpressing FoxQ1, and murine A17 cells as models of BLBC. Two compounds, (4,5-dichloro-1H-imidazolate-1-yl)-(triphenylphosphane)-gold(I) 1 and (4,5-dicyano-1H-imidazolate-1-yl)-(triphenylphosphane)-gold(I) 2 were found very active and chosen for an in vivo study in A17 tumors transplanted in syngeneic mice. The compounds resulted to be more active than cisplatin, less nephrotoxic and generally more tolerated by the mice. This study also provides evidence that both gold(I) complexes inhibited the 19 S proteasome-associated deubiquitinase USP14 and induced apoptosis, while compound 1's mechanism of action depends also on its ability to down-regulate key molecules governing cancer growth and progression, such as STAT3 and Cox-2.

Nicotinamide, nicotinic acid, nicotinamide mononucleotide, nicotinamide riboside and nicotinamide adenine dinucleotide represent key metabolites through which a variety of cellular processes is regulated. Here, we report a quantitative, simultaneous determination of nicotinamide mononucleotide, nicotinamide adenine dinucleotide and their pyridine precursors in several murine tissues using a high-performance liquid chromatography with tandem mass spectrometry method based on reversedphase chromatographic separation. All the analytical steps, including sample preparation and compounds extraction, reversed-phase liquid chromatographic separation and electrospray ionization mass spectrometry monitoring and identification, were optimized and validated in order to achieve low limit of quantification, high sensitivity and good robustness for metabolomic studies. In the analyzed tissues, we have quantified the compounds of interest, which were in the concentration range of picomoles/mg of wet tissue.

The effects of slaughter age (8 vs 12 months) were investigated on meat and carcass quality obtained from Martina Franca donkey foals. Sixteen male foals were used, eight were slaughtered at 8 months of age with a mean (±s.e.) final body weight of 101±18kg and the remaining 8 foals slaughtered at 12 months of age with a mean final body weight of 122±13kg. Carcass weight and dressing percentage were higher (P

An unprecedented study on the inhibitory activities of a class of phosphane gold(I) complexes on E. coli dihydrofolate reductase (DHFR) is reported. The gold(I) complexes considered in this work consist of azolate or chloride ligands and phosphane as co-ligands. The ligands have been functionalized with polar groups (–COOH, –COO−, NO2, Cl, CN) to obtain better solubility in polar media. Neutral, anionic and cationic gold(I) complexes have been tested as DHFR inhibitors by means of a continuous direct spectrophotometric method. X-ray structural characterizations were performed on ((triphenylphosphine)-gold(I)-(4,5-dicyanoimidazolyl-1H-1yl) and on the analog (triphenylphosphine)-gold(I)-(4,5-dichloroimidazolyl-1H-1yl). The inhibition constants obtained from the enzyme tests range from 20 μM to 63 nM (auranofin) and are conducive to promoting these compounds as potential DHFR inhibitors.

Nucleotides play a crucial role to cellular functions; they can be obtained from the diet or through the nucleotide salvage pathway, however, in particular situations (occurring mainly in newborns) the metabolic demand of nucleotides exceeds the capacity of their synthesis. These molecules, are receiving attention from a nutraceutical point of view because of their potential direct role in regulating metabolism and infant body condition. Donkey's milk may be considered a good replacer for cow's milk in feeding children with severe Ig-E mediated cow's milk protein allergy, due to its high similarity with human milk. In this study, the presence of cytidine, uridine, CMP, UMP, guanosine, and adenosine, involved in numerous biochemical and physiological activities, were detected for the first time through a RP-HPLC method.

Basal-like breast cancer (BLBC) remains a great challenge because of its clinically aggressive nature and lack of effective targeted therapy. We analyzed the potential anti-neoplastic effects of sanguinarine, a natural benzophenanthridine alkaloid, against BLBC cells. Sanguinarine treatment resulted in a reduction of cell migration, in a dose-dependent inhibition of cell viability and in the induction of cell death by apoptosis in both human (MDA-MB-231 cells) and mouse (A17 cells) in vitro models of BLBC. In vivo experiments demonstrated that oral administration of sanguinarine reduced the development and growth of A17 transplantable tumors in FVB syngeneic mice. Western blotting analysis revealed that suppression of BLBC growth by sanguinarine was correlated with a concurrent upregulation of p27 and downregulation of cyclin D1 and with the inhibition of STAT3 activation. In addition, we identified sanguinarine as a potent inhibitor of dihydrofolate reductase (DHFR), able to impair enzyme activity even in methotrexate resistant MDA-MB-231 cells. These results provide evidence that sanguinarine is a promising anticancer drug for the treatment of BLBC.

Human cytidine deaminase is an enzyme of the pyrimidine salvage pathways that metabolizes several cytosine nucleoside analogs used as prodrugs in chemotherapy. We carried out a characterization of the cytidine deaminase 79A>C and 208G>A Single Nucleotide Polymorphisms, in order to highlight their functional role and provide data that could help fine-tune the chemotherapic use of cytosine nucleosides in patients carrying the above mentioned SNPs. The 79A>C SNP results in a K27Q change in a protein region not involved in the catalytic event. The 208G>A SNP produces an alanine to threonine substitution (A70T) within the conserved catalytic domain. Q27 variant is endowed with a greater catalytic efficiency toward the natural substrates and the antileukemic agent cytarabine (Ara-C), when compared to K27 variant. Molecular modeling, protein stability experiments and site-directed mutagenesis suggest that K27 variant may have an increased stability with respect to Q27 due to an ionic interaction between a lysine residue at position 27 and a glutamate residue at position 24. The T70 variant has a lower catalytic efficiency toward the analyzed substrates when compared to the A70 variant, suggesting that patients carrying the 208G>A SNP may have a greater exposure to cytosine based pro drugs, with possible toxicity consequences.

Hill's plots of oxygen binding isotherms reveal the presence of a transition between two different oxygen affinities at the beginning and end of the isotherm. They correspond to the two conformations anticipated by the MWC model, namely, the T and R conformations at the beginning and end of oxygen binding, when the lower affinity of the T form develops into the higher affinity of the R form. The difference between the binding Gibbs free energy changes of the two affinities (ΔGL) is the free energy of binding cooperativity. Notably, ΔGL is positive in favor of the T form, which moves to a higher energy level upon oxygen release. Osmotic stress reveals a higher volume/surface ratio of deoxyhemoglobin, with a positive ΔGW also in favor of the T form. An increasing protein concentration shifts the isotherms to the right, indicating the formation of intermediate polymeric forms. The enthalpy of the intermediates shows a strong absorption of heat at the third oxygenation step because of polymer formation with quinary, and higher-order, structures. The disassembly of intermediate polymers releases energy with a negative ΔG that compensates and allows the positive values of ΔGL. High-energy polymers are the barrier preventing the relaxation of the T and R conformations into one another. The MWC allosteric model is the best justification of oxygen binding cooperativity. hemoglobin (Hb) with low (T form) and high (R form) oxygen affinity. In the absence of oxygen, the allosteric equilibrium constant Lo = To/Ro is in favor of To, while with an increasing level of Hb saturation, Ro becomes the favored species. Consistent with the model, linear extrapolation of the very first and very last saturation data of Hill's plot isotherms defines the presence of two hyperbolic bindings for the first and last oxygen bound to Hb. 2 They have a higher and lower P50 reflecting a lower and higher oxygen affinity, respectively. It is easy to reconcile the initial low affinity (KT )w ith deoxyhemoglobin in the T form and the final high affinity (KR) with oxyhemoglobin in the R form. The overall binding affinity of the isotherm is defined by the value of PO2 at 50% saturation of the isotherm (P50), or the value of Pm when available.

Donkey milk could be considered a good and safer alternative, compared to other types of milk, for infants affected by cow’s milk protein allergy, when breastfeeding is not possible. Interestingly, donkey milk has low allergenicity, mainly due to the low total casein amount, and the content of some whey proteins that act as bioactive peptides. The amount of lysozyme, an antibacterial agent, is 1.0 g/L, similar to human milk. Lactoferrin content is 0.08 g/L, with this protein being involved in the regulation of iron homoeostasis, anti-microbial and anti-viral functions, and protection against cancer development. Lactoperoxidase, another protein with antibacterial function, is present in donkey milk, but in very low quantities (0.11 mg/L). β-lactoglobulin content in donkey milk is 3.75 g/L—this protein is able to bind and transport several hydrophobic molecules. Donkey milk’s α-lactalbumin concentration is 1.8 g/L, very close to that of human milk. α-lactalbumin shows antiviral, antitumor, and anti-stress properties. Therefore, donkey milk can be considered as a set of nutraceuticals properties and a beverage suitable, not only for the growing infants, but for all ages, especially for convalescents and for the elderly.

The effects of two different slaughter age (2 vs 5 months) on carcass traits and on meat quality obtained from muscle longissimus thoracis taken from 28 Fabrianese lambs were studied. Carcass weight and dressing percentage were significantly higher ( P 0.05) in older animals. Slaughter age affected average daily gain, that was reduced in older lambs. Lamb meat from older animals showed a significant higher content of fat and protein, while cholesterol content and the ratio between Ʃ n-6/Ʃ n-3fatty acids were not statistically different. Rumenic acid (CLA) content was significantly (P

The use of surfactants as drug permeability enhancers across epithelial barriers remains a challenge. Although many studies have been performed in this field using synthetic surfactants, the possibility of employing surfactants produced by bacteria (the so called biosurfactants") has not been completely explored. Among them, one of the most well characterized class of biosurfactants are rhamnolipids. The aim of the study was to investigate the effect of rhamnolipids on the epithelial permeability of fluorescein isothiocyanate-labelled dextrans 4kDa and 10kDa (named FD4 and FD10, respectively) as model for macromolecular drugs, across Caco-2 and Calu-3monolayers. These cell lines were selected as an in vitro model for the oral and respiratory administration of drugs. Before performing permeability studies, rhamnolipids mixture was analysed in terms of chemical composition and quantification through mass analysis and HPLC. Cytotoxicity and transepithelial electrical resistance (TEER) studies were also conducted using Caco-2 and Calu-3 cell lines. A dose-dependent effect of rhamnolipids on TEER and FD4 or FD10 permeability across both cell lines was observed at relatively safe concentrations. Overall, results suggest the possibility of using rhamnolipids as absorption enhancers for macromolecular drugs through a reversible tight junction opening (paracellular route), despite more investigations are required to confirm their mechanism of action in term of permeability.

Clams have long been a fisheries and aquaculture sector of great importance in Italy, the main resource of fisheries is the Chamelea gallina of indigenous origin, whereas clams breeding is supported almost entirely by the Tapes philippinarum, a species of Indo-Pacific origin. Bivalve molluscs quality depends mainly on the water quality, and then by a series of factors such as water temperature and salinity, gametogenic cycle, food availability, and environmental conditions, that affect the Condition Index. In this work crude extracts obtained from the edible part of Chamelea gallina and Tapes philippinarum were analyzed by a proteomic approach based on a two-dimensional gel electrophoresis followed by liquid chromatography-tandem mass spectrometry, in order to detect biomarkers useful for identification of the two kinds of clams and to assess their nutritional characteristics. As a result, four differentially expressed spots were found and identified, namely enolase, cyclophilin-A, ribosomal protein L13 and actin-1.

Background: Microemulsions are attractive delivery systems for therapeutic proteins and peptides due to their ability to enhance bioavailability. Although different proteins and peptides have been successfully delivered through such ternary systems, no information can be found about protein loading and the formulation stability when such microemulsions are prepared with pharmaceutically-approved oils and surfactants. The aim of this work was to optimise a ternary system consisting of water/ethyl oleate/SpanO 80-TweenO 80 and to determine its protein loading capacity and stability, using bovine serum albumin (BSA) as a model of biomolecule. Methods: The optimization was carried out using a Central Composite Design and all the prepared formulations were characterised through dynamic light scattering, rheology, optical and polarized microscopy. Subsequently, the maximum loading capacity was determined and the stability of the final microemulsion with the highest content of protein was followed over six months. To investigate the structural features of the protein, BSA was recovered from the microemulsion and analysed through fluorescence spectroscopy. Results: After incorporation of the protein in the microemulsion, a decrease of its aqueous solubility was observed. However, the formulation remained stable over six months and the native-like state of the recovered protein was demonstrated by fluorescence spectroscopy Conclusion: This study demonstrated the feasibility of preparing microemulsions with the highest content of protein and their long-term stability.

The industrial extraction and further applications of isofuranodiene are limited because at room temperature it spontaneously converts to curzerene, a structurally less active isomer. This work definitively identified the structure of isofuranodiene in the solid state, showing the two methyl groups in syn position. In addition, two bioactive metal cations, namely, silver(I) and copper(II) ions, were used in the attempt to obtain the chemical stability of isofuranodiene: in the case of silver(I), a labile adduct was formed, while in the case of copper(II), a more stable 1:1 adduct was achieved. In the former, the presence of silver did not significantly affect the biological activities of isofuranodiene, while in the latter, the copper(II) coordination suppressed them. The biological activities of the isofuranodiene adducts were then evaluated as antiproliferative agents against human tumor cell lines (HCT116, MDA-MB 231, and T98G). In addition, for the first time, isofuranodiene was tested as an inhibitor of DHFR (DiHydroFolateReductase) from Escherichia coli. Anticancer activity was observed in the isofuranodiene with the AgCF3SO3 adduct, in the tested cell lines, with IC50 values ranging from 4.89 μM to 13.06 μM, while inhibition assays highlighted a Ki of 6.22 μM for isofuranodiene and of 0.17 μM for the related silver adduct. Docking studies indicated a binding mode score of − 6.83 Kcal/mol for isofuranodiene, and an energy value of − 11.82 Kcal/mol for methotrexate (a classic DHFR inhibitor).

CD38 (EC 3.2.2.6, NAD(+)-glycohydrolase) is a multifunctional enzyme catalyzing the synthesis and hydrolysis of cyclic ADP-ribose from NAD(+) to ADP-ribose. The loss of CD38 function is associated with impaired immune responses, metabolic disturbances, and behavioral modifications. Notably, it has been linked to HIV infection, leukemias, myelomas, solid tumors, Type II Diabetes mellitus, bone metabolism, as well as Autism Spectrum Disorder. Taking into account the crucial role played by CD38 in many diseases and in clinical practice, here we assessed the distribution of CD38 NADase activity in a healthy population (104 sex-matched unrelated individuals, 12-98 years) and determined its main predictors among genetic and physiological factors (age and sex). The mean value of CD38 NADase activity was 0.051±0.023 mU/mg (0.010-0.099 mU/mg), following a normal distribution in the study population (Kolmogorov-Smirnov test P=0.200). The TNF-α -308G>A (rs1800629) resulted the main predictor (β=0.364, P=0.00008), followed by Age (β=0.280, P=0.002) and the CD38 184C>G (rs6449182) (β=0.193, P=0.033). Our study contributes to understanding CD38 enzyme physiological functions, by reporting, for the first time, its activity distribution in healthy individuals and demonstrating a significant positive correlation with age. Moreover, the possible use of TNF-α -308G>A (rs1800629) and the CD38 184C>G (rs6449182) SNPs as predictive genetic markers of CD38 activity, clearly point toward possible pharmacogenomic applications and to a more refined use of CD38 in clinical settings.

Dihydrofolate reductase (DHFR) is a ubiquitous enzyme involved in major biological process, including DNA synthesis and cancer inhibition, and its modulation is the object of extensive structural, kinetic, and pharmacological studies. In particular, earlier studies showed that green tea catechins are powerful inhibitors of bovine liver and chicken liver DHFR. In this article, we report the results of inhibition kinetics for the enzyme from another source (DHFR from E. coli) exerted by (−)-epigallocatechingallate (EGCG). Using different analytical techniques, we reported that EGCG acts as a bisubstrate inhibitor on the bacterial DHFR. Moreover, the combined approach of biosensor, kinetic, and molecular modelling analysis disclosed the ability of EGCG to bind to the enzyme both on substrate (DHF) and cofactor (NADPH) site. Collectively, our data have confirmed the selectivity of antifolate compounds with respect to the different source of enzyme (bacterial or mammalian DHFR) and the possible role of tea catechins as chemopreventive agents. Proteins 2008. © 2008 Wiley-Liss, Inc.

It is well known that disorders of pyrimidine pathways may lead to neurological, hematological, immunological diseases, renal impairments, and association with malignancies. Nucleotide homeostasis depends on the three stages of pyrimidine metabolism: de novo synthesis, catabolism and recycling of these metabolites. Cytidine and uridine, in addition to be used as substrates for pyrimidine nucleotide salvaging, also act as the precursors of cytidine triphosphate used in the biosynthetic pathway of both brain's phosphatidylcholine and phosphatidylethanolamine via the Kennedy cycle. The synthesis in the brain of phosphatidylcholine and other membrane phosphatides can utilize, in addition to glucose, three compounds present in the blood stream: choline, uridine, and a polyunsaturated fatty acids like docosahexaenoic acid. Some authors, using rat models, found that oral administration of two phospholipid precursors such as uridine and omega-3 fatty acids, along with choline from the diet, can increase the amount of synaptic membrane generated by surviving striatal neurons in rats with induced Parkinson's disease. Other authors found that in hypertensive rat fed with uridine and choline, cognitive deficit resulted improved. Uridine has also been recently considered as a neuroactive molecule, because of its involvement in important neurological functions by improving memory, sleep disorders, anti-epileptic effects, as well as neuronal plasticity. Cytidine and uridine are uptaken by the brain via specific receptors and successively salvaged to the corresponding nucleotides. The present review is devoted to the enzymology of pyrimidine pathways whose importance has attracted the attention of several researchers investigating on the mechanisms underlying the physiopathology of brain.

This paper deals with the rheological and thermal characterisation of the self-assembling behaviour of different Poloxamer 407 systems (15-30%, w/v), both alone or after the addition of various amounts of hydroxypropyl beta-cyclodextrin (5-20%, w/v). The knowledge of the interactions existing between the two kinds of molecules could allow the development of systems for parenteral administration containing also proteins or peptides drugs. A rheology (temperature sweep and frequency sweep test) study has been performed to characterise the thermogelation process while thermal analysis (nanoDSC) allowed the determination of both micellization and sol/gelation transition processes. These two techniques were also utilised to outline the variation in the critical micelle temperature (cmT) or in the sol/gel temperature (sgT,) of the systems containing also hydroxypropyl beta-cyclodextrin (HP beta-CD). Both the rheology and thermal analysis showed the presence of interesting interactions between the HP beta-CD and the Poloxamer 407, which cause a shift of both cmT and sgT. The presence of HP beta-CD modified also Poloxamer samples elastic characters and microrheological structure as demonstrated by the G'-G'' mechanical spectra. Rheological and thermal results outlined how these new systems could open the possibility to join the thermogelling behaviour offered by Poloxamer 407 with the well-known stabilization and protection ability of HP beta-CD, which could make possible the formulation of systems for the parenteral delivery of peptides and proteins.