Morphology and properties of neutralized chitosan-cellulose nanocrystals biocomposite films.

Chitosan/cellulose nanocrystals (CH-CN) films were obtained by casting of dispersions, and treated with NaOH for neutralization proposes. The composition of films was varied from 1 to 10wt.% of CN. Changes in the morphology of the systems were correlated with the different properties studied. FTIR revealed the presence of a weak interaction between the polymer matrix and nanofiller, confirmed by a slightly increase in thermal stability. SEM images suggested that incorporating CN amounts higher than 3wt.% generates phase-segregated systems, and SAXS showed that CH avoid the typical organization of CN at concentrations below 5wt.%. Improved performance against water was obtained in composite materials comparing to the pure polymer matrix, as well as CH and CH-CN films did not show antibacterial activity demonstrating that remnant acetic acid, when no neutralization step is done, plays an important role in this property.

Treatment of in vitro enterohemorrhagic Escherichia coli infection using phage and probiotics.

AIMS: To assay the combination of phage and probiotics against EHEC in vitro on infected Hep-2 cells. METHODS AND RESULTS: Phage and probiotics treatments on EHEC O157:H7-infected Hep-2 cells were assayed individually or combined. The effect of freeze-drying on phage and probiotic antimicrobial activity was also studied. While treatment with phage alone increased cell detachment caused by EHEC infection, the treatments with MM alone or in combination with phage proved to effectively diminish cell damage caused by EHEC infection. Combined treatment showed a decrease in apoptotic cell count of 57·3% and a reduction in EHEC adhesion to cell monolayer of 1·2 log CFU. The simultaneous use of phage and probiotics showed no antagonistic effect, and freeze-drying did not affect their antipathogenic activity. CONCLUSIONS: The combination of phage and probiotics has great potential for reducing the number of pathogens adhered to epithelial cells during EHEC O157:H7 infection and attenuating the cytotoxic effect derived from it. Further in vivo assays are needed for assessing the actual effectiveness of the treatment. SIGNIFICANCE AND IMPACT OF THE STUDY: This study presents a freeze-dried formulation of phage and probiotics capable of controlling EHEC infections and reducing epithelial cell damage in vitro.

Kefir-isolated bacteria and yeasts inhibit Shigella flexneri invasion and modulate pro-inflammatory response on intestinal epithelial cells.

The aim of this work was to evaluate the ability of a kefir-isolated microbial mixture containing three bacterial and two yeast strains (MM) to protect intestinal epithelial cells against Shigella flexneri invasion, as well as to analyse the effect on pro-inflammatory response elicited by this pathogen. A significant decrease in S. flexneri strain 72 invasion was observed on both HT-29 and Caco-2 cells pre-incubated with MM. Pre-incubation with the individual strains Saccharomyces cerevisiae CIDCA 8112 or Lactococcus lactis subsp. lactis CIDCA 8221 also reduced the internalisation of S. flexneri into HT-29 cells although in a lesser extent than MM. Interestingly, Lactobacillus plantarum CIDCA 83114 exerted a protective effect on the invasion of Caco-2 and HT-29 cells by S. flexneri. Regarding the pro-inflammatory response on HT-29 cells, S. flexneri infection induced a significant activation of the expression of interleukin 8 (IL-8), chemokine (C-C motif) ligand 20 (CCL20) and tumour necrosis factor alpha (TNF-α) encoding genes (P<0.05), whereas incubation of cells with MM did not induce the expression of any of the mediators assessed. Interestingly, pre-incubation of HT-29 monolayer with MM produced an inhibition of S. flexneri-induced IL-8, CCL20 and TNF-α mRNA expression. In order to gain insight on the effect of MM (or the individual strains) on this pro-inflammatory response, a series of experiments using a HT-29-NF-κB-hrGFP reporter system were performed. Pre-incubation of HT-29-NF-κB-hrGFP cells with MM significantly dampened Shigella-induced activation. Our results showed that the contribution of yeast strain Kluyveromyces marxianus CIDCA 8154 seems to be crucial in the observed effect. In conclusion, results presented in this study demonstrate that pre-treatment with a microbial mixture containing bacteria and yeasts isolated from kefir, resulted in inhibition of S. flexneri internalisation into human intestinal epithelial cells, along with the inhibition of the signalling via NF-κB that in turn led to the attenuation of the inflammatory response.

Effect of kappa-casein polymorphism on milk composition in the Orobica goat.

The aim of this work was to study the effects of isoelectrofocusing (IEF) milk protein variants on milk composition in the Italian Orobica goat breed, which is characterized by a rather high frequency of the kappa-casein (CSN3) B(IEF) allele. Significant associations were found between the IEF phenotype and protein and casein percentages. A favorable effect of the CSN3 B(IEF) variant was found for both protein and casein percentages, with a codominance trend for the 3 phenotypes: BB > AB > AA. Depending on the selection purpose, emphasis could be given to different kappa-casein variants in breeding. The high frequency of B(IEF) could be exploited in breeding strategies to improve the protein and casein percentages when cheese making is a selection objective.

Focusing on the goat casein complex.

The analysis of casein polymorphisms in goat species is rather difficult, because of a large number of mutations at each locus, and the tight linkage involving the 4 casein genes. Three goat breeds from Northern Italy, Orobica, Verzasca, and Frisa, were analyzed at the casein complex by milk isoelectrofocusing and analyses at the DNA level to identify the majority of all known polymorphisms. The casein gene structure of the 3 local breeds at alpha(S1)-casein (CSN1S1), beta-casein (CSN2), alpha(S2)-casein (CSN1S2), and kappa-casein (CSN3) was compared with that of Camosciata, a more widely distributed breed. A new allele was identified and characterized at CSN2 gene, which seemed to be specific to the Frisa breed. It was named CSN2*E, and was characterized by a transversion TCT --> TAT responsible for the amino acid exchange Ser(166) --> Tyr(166) in the mature protein. The casein haplotype structure is highly different among breeds. A total of 26 haplotypes showed a frequency higher than 0.01 in at least 1 of the 4 breeds considered, with 12, 3, 5, and 19 haplotypes in Frisa, Orobica, Verzasca, and Camosciata breeds, respectively. Only 13 haplotypes occurred at a frequency higher than 0.05 in at least 1 breed. With the molecular knowledge of each locus, the ancestral haplotype coding for CSN1S1*B, CSN2*A, CSN1S2*A, and CSN3*B protein variants can be postulated. A protein evolutionary model considering the whole casein haplotype is proposed.

Short communication: predominance of beta-casein (CSN2) C allele in goat breeds reared in Italy.

A protocol for the rapid and simultaneous genotyping of A, C, and 0 'CSN2 alleles in goat was developed by single strand conformational polymorphism polymerase chain reaction (SSCP-PCR) technique. Screening the CSN2 variability in 7 goat breeds reared in Italy validated the genotyping test. The SSCP-PCR technique was also suitable for monitoring CSN2 polymorphism. In particular, the discrimination between CSN2*A and CSN2*C is important because the 2 corresponding protein variants cannot be separated by standard typing techniques. The monitoring of CSN2 variability in the goat breeds indicates the predominance of the C allele. In most breeds, CSN2*C occurred with the highest frequency, except in Saanen where CSN2*A and CSN2*C showed similar frequencies. Variant CSN2*C occurred with a frequency of 0.68 (Camosciata), 0.70 (Jonica), 0.71 (Garganica), 0.82 (Maltese), 0.87 (Cilentana), and 0.97 (Orobica). The alignment among the mature CSN2 sequences of different species suggests that CSN2*A is the ancestral allele compared with CSN2*C. Interestingly, the CSN2*A goat variant showed higher frequencies in selected breeds (Saanen and Camosciata).

Casein haplotype structure in five Italian goat breeds.

The aim of this work was to investigate the genetic structure of the casein gene cluster in 5 Italian goat breeds and to evaluate the haplotype variability within and among populations. A total of 430 goats from Vallesana, Roccaverano, Jonica, Garganica, and Maltese breeds were genotyped at alphas1-casein (CSN1S1), alphas2-casein, (CSN1S2), beta-casein (CSN2), and kappa-casein (CSN3) loci using several genomic techniques and milk protein analysis. Casein haplotype frequencies were estimated for each breed. Principal component analysis was carried out to highlight the relationship among breeds. Allele and haplotype distributions indicated considerable differences among breeds. The haplotype CSN1S1*F- CSN1S2*F-CSN3*D occurred in all breeds with frequencies >0.100 and was the most common haplotype in the Southern breeds. A high frequency of CSN1S1*0-CSN1S2*C-CSN3*A haplotype was found in Vallesana population (0.162). Principal component analysis clearly separated the Northern and Southern breeds by the first component. The variability of the caprine casein loci and variety of resulting haplotypes should be exploited in the future using specific breeding programs aiming to preserve biodiversity and to select goat genetic lines for specific protein production.

Single nucleotide polymorphisms in the ovine casein genes detected by polymerase chain reaction-single strand conformation polymorphism.

Casein genetic polymorphisms are important and well known due to their effects on quantitative traits and technological properties of milk. At the DNA level, polymerase chain reaction (PCR)-single-strand conformation polymorphism (SSCP) allows for the simultaneous typing of several alleles at casein loci, as well as the detection of unknown polymorphisms. Here we describe the usefulness of the PCR-SSCP technique for casein typing in sheep. In particular, three single-nucleotide polymorphisms (SNP) are described at CSN1S1, CSN2, and CSN3, all resulting in amino acid exchanges. At CSN1S1, a transition T-->C was found, resulting in the deduced amino acid exchange Ile186-->Thr186. A transition A-->G resulting in the deduced amino acid exchange Met183-->Val183 was identified at CSN2. The 2 SNP showed a rather high frequency (ranging from 0.12 to 0.26) in 3 Italian breeds (Sarda, Comisana, Sopravissana). Another transition C-->T (Ser104-->Leu104) was found at CSN3 in one heterozygous animal.

Genetic polymorphism of goat kappa-casein (CSN3) in different breeds and characterization at DNA level.

Data on genetic differences at CSN3 in goat breeds including a DNA based typing method and the mutations responsible for variation on protein and DNA level are presented here. Isoelectric focusing (IEF) in ultrathin polyacrylamide gels with carrier ampholytes was used to demonstrate CSN3 polymorphism in milk samples of Italian (Orobica n=88; Ionica n=68) and German goat breeds (Bunte Deutsche Edelziege n=244; Weisse Deutsche Edelziege n=134; Toggenburger n=25; Thüringer Waldziege n=70). A further CSN3 band was found presenting a more cathodic migration than CSN A. After chymosin action, the genetic polymorphism was also observed in the para-kappa-casein fraction. The new allele CSN3(B) was spread mainly in Orobica (37%), Bunte Deutsche Edelziege (11%) and Ionica (10%). CSN3(B) occurred in low frequency (<3%) in Thüringer Waldziege and in Weisse Deutsche Edelziege, and could not be demonstrated in milk samples of Toggenburger. The populations were in Hardy-Weinberg equilibrium at CSN3. The codominant genetic control of CSN3(B) was confirmed by genetic studies. Discrimination of CSN3 alleles A and B was also obtained by DNA SSCP analysis. Sequencing of CSN3(B) revealed four transitions at position 247, 309, 471, and 591 compared with CSN3(A). From these transitions, the following amino acid substitutions are deduced: 44 Gln --> Arg, 65 Val --> Ile, 119 Val --> Ile, and 159 Ser --> Pro. Among the four mutations, only the 44 Gln --> Arg can be revealed by milk protein IEF analysis while at DNA level three further genetic variants should exist in addition to CSN3(A) and CSN3(B).

Single nucleotide polymorphisms (SNPs) within Bov-A2 SINE in the second intron of bovine and buffalo k-casein (CSN3) gene.

The genetic polymorphism of an entire Bov-A2 element located in the second intron of the buffalo and bovine k-casein (CSN3) gene was investigated by amplification and sequencing of PCR products. Single nucleotide polymorphisms were detected. A PCR-RFLP method was developed to detect an A or G mutation at position 605 of bovine Bov-A2 element which creates a BfaI polymorphic site. The frequencies of the B allele, with the BfaI site, were for 0.275, 0.775, 0.750, 0.975, respectively, for Italian Holstein Friesian, Grey Alpine, Friuli Red Pied and Reggio bovine breeds. The mutation rate (substitutions and deletions/insertions per nucleotide site per year) was 2.5 x 10(-9) for Bov-A2 sequences in the second intron of CSN3. The comparison with other Bov-A2 elements suggests that this retroelement might be an important source of single nucleotide polymorphism for analysis of Bovidae genomes.

A case of familial nesidioblastosis: prenatal diagnosis of foetal hyperinsulinism.

Persistent neonatal hyperinsulinaemic hypoglycaemia due to nesidioblastosis is a rare condition probably transmitted by an autosomal recessive inheritance. Recurrent hypoglycaemic episodes become evident after birth and cause severe neurological damage without intensive treatment. The intrauterine detection of hypoglycaemia and hyperinsulinism in newborns subsequently diagnosed as affected by nesidioblastosis has not yet been reported. We describe a case of familial nesidioblastosis in which an intrauterine diagnosis could be suggested by high levels of insulin and C-peptide and low values of glucose in the amniotic fluid.

[Screening for obesity in a schoolchildren population of the 20th zone of Milan and a nutritional education intervention]. / Screening dell'obesità nella popolazione scolastica della zona 20 di Milano ed intervento di educazione alimentare.

Our study was performed in 1986-'87 and 1987-'88 school years on 12.354 three to eighteen years old students (the whole scholastic population of zone 20 of Milan) in order to apply dietary education on obese subjects. Mean prevalence of obesity was 13.4% with elevated percentages in 11 to 13 years old students (17.9%), with respect to primary (14.1%), high school (12.4%) and nursery school (4.7%). The 36% of obese subjects (more than 50% of adolescents) had already tempted to reduce body weight. Intervention reduced % weight excess (from 33.6 +/- 0.5% to, 28.8 +/- 0.5% after 12 months, p less than 0.001); 67% of obese subjects lost weight and body weight returned within normal limits in 31% of subjects. An educational dietetic strategy may be successful in childhood obesity.

Variability of reduced glutathione levels in Massese ewes and its effect on daily milk production.

The variability of the erythrocyte concentration of the tripeptide glutathione was studied in four flocks of Massese sheep. A relationship was found between glutathione level and stage of lactation in which the second month showed the lowest content. Genotype at transferrin and haemoglobin loci was associated with glutathione levels. Homozygotes at the transferrin locus yielded higher levels, while the D allele at the same locus was associated with lower concentrations. Animals with haemoglobin AA showed higher contents of glutathione. Also, a negative correlation was found between daily milk production and glutathione level.

[Diagnostic contribution of auditory and somesthetic evoked potentials in multiple sclerosis]. / Contribution diagnostique des potentiels évoqués auditifs et somesthésiques dans la sclérose en plaques.

Results of BERA measured on 52 patients with possible Multiple Sclerosis were compared with the diagnosis pronounced one year after the initial functional exploration. In 21 of these patients, somesthetic evoked potentials were recorded at the same time as the BERA. One year later, diagnosis was considered as likely in 23 cases, as eliminated in 15 cases, and still possible in 14 cases. 38 cases were kept after elimination of the group with "possible diagnosis"; in 68.5% of them, an agreement was found between the results of initially recorded BERA and the diagnosis pronounced one year later. A parallel study of the correlation carried out in 16 patients, for whom somesthetic evoked potentials were recorded, showed a diagnosis agreement in 69% of the cases.