Biofilm Formation on Stainless Steel by Streptococcus thermophilus UC8547 in Milk Environments Is Mediated by the Proteinase PrtS.

In Streptococcus thermophilus, gene transfer events and loss of ancestral traits over the years contribute to its high level of adaptation to milk environments. Biofilm formation capacity, a phenotype that is lost in the majority of strains, plays a role in persistence in dairy environments, such as milk pasteurization and cheese manufacturing plants. To investigate this property, we have studied S. thermophilus UC8547, a fast-acidifying dairy starter culture selected for its high capacity to form biofilm on stainless steel under environmental conditions resembling the dairy environment. Using a dynamic flow cell apparatus, it was shown that S. thermophilus UC8547 biofilm formation on stainless steel depends on the presence of milk proteins. From this strain, which harbors the prtS gene for the cell wall protease and shows an aggregative phenotype, spontaneous mutants with impaired biofilm capacity can be isolated at high frequency. These mutants lack the PrtS expendable island, as confirmed by comparison of the genome sequence of UC8547Δ3 with that of the parent strain. The prtS island excision occurs between two 26-bp direct repeats located in the two copies of the ISSth1 flanking this genomic island. The central role of PrtS was confirmed by analyzing the derivative strain UC8547Δ16, whose prtS gene was interrupted by an insertional mutation, thereby making it incapable of biofilm formation. PrtS, acting as a binding substance between the milk proteins adhered to stainless steel and S. thermophilus cell envelopes, mediates biofilm formation in dairy environments. This feature provides S. thermophilus with an ecological benefit for its survival and persistence in this environment.IMPORTANCE The increased persistence of S. thermophilus biofilm has consequences in the dairy environment: if, on the one hand, the release of this microorganism from biofilm can promote the fermentation of artisanal cheeses, under industrial conditions it may lead to undesirable contamination of dairy products. The study of the molecular mechanism driving S. thermophilus biofilm formation provides increased knowledge on how an ancestral trait affects relevant phenotypes, such as persistence in the environment and efficiency of growth in milk. This study provides insight into the genetic factors affecting biofilm formation at dairy plants.

Transcriptome characterisation and simple sequence repeat marker discovery in the seagrass Posidonia oceanica.

Posidonia oceanica is an endemic seagrass in the Mediterranean Sea, where it provides important ecosystem services and sustains a rich and diverse ecosystem. P. oceanica meadows extend from the surface to 40 meters depth. With the aim of boosting research in this iconic species, we generated a comprehensive RNA-Seq data set for P. oceanica by sequencing specimens collected at two depths and two times during the day. With this approach we attempted to capture the transcriptional diversity associated with change in light and other depth-related environmental factors. Using this extensive data set we generated gene predictions and identified an extensive catalogue of potential Simple Sequence Repeats (SSR) markers. The data generated here will open new avenues for the analysis of population genetic features and functional variation in P. oceanica. In total, 79,235 contigs were obtained by the assembly of 70,453,120 paired end reads. 43,711 contigs were successfully annotated. A total of 17,436 SSR were identified within 13,912 contigs.

Leptin gene haplotypes are associated with change in immunological and hematological variables in dairy cow during the peripartum period.

In this study, the effect of polymorphisms in the leptin gene on the hematological variables in periparturient dairy cows was investigated. The hematological profile of 67 Holstein cows was assessed for 6 wk around calving. The DNA of the cows was genotyped at 6 polymorphic loci within the leptin gene, and 7 haplotypes were reconstructed. Significant haplotype substitution effects were found, for haplotype 1, on total white blood cell count for 2 wk around calving (+0.70 10(3)/µL, P = 0.05; +1.38 10(3)/µL, P = 0.0001); on neutrophil cell count in the first week after calving (+0.94 10(3)/µL, P = 0.001); on lymphocyte count during the 3 wk before and the first week after calving (+0.32 10(3)/µL, P = 0.05; +0.27 10(3)/µL, P = 0.03; +0.26 10(3)/µL, P = 0.04; +0.34 10(3)/µL, P = 0.01); on red blood cell count during the last week before calving and wk 1 and 2 after calving (+0.21 10(6)/µL, P = 0.02; +0.23 10(6)/µL, P = 0.01; +0.20 10(6)/µL, P = 0.03); on mean corpuscular volume (-1.35 fL, P = 0.01; -1.29 fL, P = 0.002; -1.18 fL, P = 0.004; -1.09 fL, P = 0.008; -1.23 fL, P = 0.003; -1.31 fL, P = 0.003); and on mean corpuscular hemoglobin (-0.37 pg, P = 0.05; -0.38 pg, P = 0.02; -0.39 pg, P = 0.01; -0.34 pg, P = 0.03; -0.40 pg, P = 0.01; -0.40 pg, P = 0.01) during all 6 wk analyzed. Significant haplotype substitution effects, but opposite those of haplotype-1, were found for haplotype-2 on white blood cell count (-1.10 10(3)/µL, P = 0.01; -1.30 10(3)/µL, P = 0.002; -1.09 10(3)/µL, P = 0.01) and neutrophil count (-0.82 10(3)/µL, P = 0.02; -0.95 10(3)/µL, P = 0.005; -0.92 10(3)/µL, P = 0.01). Haplotype-3 influenced red blood cell count (-0.23 10(6)/µL, P = 0.03; -0.28 10(6)/µL, P = 0.01; -0.34 10(6)/µL, P = 0.002) during the last 2 wk before and the first week after calving, and also, with effects evident only in wk 3 and 2 before calving, mean corpuscular volume (+1.38 fL, P = 0.03; +0.97 fL, P = 0.05; +1.08 fL, P = 0.05), mean corpuscular hemoglobin (+0.58 pg, P = 0.02; +0.38 pg, P = 0.04; 0.51 pg, P = 0.01), and red blood cell distribution width (-0.56% P = 0.02; -0.47%, P = 0.05). The current study provided evidence that several polymorphisms in the leptin gene play a role in the variability of hematological variables during the peripartum period, and might be used as genetic markers for improving the immunological conditions of dairy cows in critical productive periods.

Association analyses of single nucleotide polymorphisms in the LEP and SCD1 genes on the fatty acid profile of muscle fat in Simmental bulls.

The aim was to investigate the effect of the genetic polymorphisms of leptin (LEP) and stearoyl-CoA desaturase (SCD1) genes on the fatty acid (FA) composition of the muscle of 103 Simmental bulls. Ten single nucleotide polymorphisms (SNP) were detected in exons 2 and 3 of the LEP gene, two of them encoding non-synonymous mutations. Allelic substitution effects of all the SNP on 28 single fatty acids, monounsaturated (MUFA) and polyunsaturated (PUFA) and desaturation indexes were estimated. Both the SCD1 SNP, as well as three SNP of the leptin gene, affected, to different extents, the desaturation of FA into MUFA. Because it was previously proposed that leptin's metabolic action involves down-regulation of SCD1, it is possible that, beyond the mere additive effect of SCD1 gene on FA desaturation, the non-synonymous mutations in the leptin gene also contribute to the variability of FA composition in muscle fat.

Fatty acid composition and sensory properties of Italian Simmental beef as affected by gene frequency of Montbéliarde origin.

The aim of this study was to investigate the effect of Montbéliarde (Mb) gene frequency on fatty acid composition and sensory properties of Italian Simmental (IS) steaks (longissimus thoracis m.). Twenty-seven bulls belonging to three strains with different percentages of Mb genes: traditional (ISt), without Mb ascendants (ISt=0% Mb genes), cross-strain (ISmt=37.5-50% Mb genes), Montbéliarde strain (ISm=87.5-100% Mb genes) and balanced for stearoyl Co-A desaturase genotype were considered. ISt has the highest C20:4 n-6 (P<0.01), C22:4 n-6 (P<0.05) and total PUFA n-3 level (P<0.01), while ISt and ISmt have higher C18:3 n-3 (P<0.05) and slightly lower MUFA (P=0.08) than ISm. Sensory tests indicated that the three experimental groups can be differentiated; moreover, ISmt meat is perceived as less hard (P<0.01), less chewable (P<0.01) and less fibrous (P<0.05) than ISt meat.

Short communication: Effect of stearoyl-coenzyme A desaturase polymorphism on fatty acid composition of milk.

The effect of the stearoyl-CoA desaturase (SCD) gene on milk fatty acid composition was tested. Cows of 3 breeds of northern Italy, Piedmontese, Valdostana, and Jersey, were genotyped at exon 5 of the SCD gene. This has been suggested as a primary candidate gene to change the proportion of saturated vs. unsaturated fatty acids in milk, wherein a single nucleotide polymorphism (C/T) gives rise to a different AA codon. It was possible to ascribe a reduced desaturase activity to the T allele only in the case of caproleic and myristoleic fatty acids. In contrast with the findings of SCD effects on carcass fat, it was not possible to confirm the higher desaturation activity of this single nucleotide polymorphism on long-chain fatty acids, due to the different pathways that originate milk fatty acids of different carbon length; long-chain fatty acids are highly influenced by the complex metabolic events that affect the ingested nutrients during their transfer to milk fat.

Meat molecular traceability: How to choose the best set of microsatellites?

To set up routine assays for molecular meat traceability along the food chain, the availability of a simple and low cost test for the identification of cattle carcasses is required. For this purpose, we evaluated 13 microsatellites for their ability in the identification of animals belonging to four Italian cattle breeds. Here we propose a criterion for a microsatellite-based test with the best reliability when reducing the number of loci to be analysed. The method is based on the observation that in the same loci breeds can show differences in frequencies and number of fixed alleles. This non-uniform distribution of alleles between breeds results in differences in the informative content of the same loci in different breeds. Taking into account these differences, it is possible to perform tests for the allocation of samples to specific animals utilizing a small number of microsatellites. The proposed approach allows cost reduction and ease in performing the analyses.

Single nucleotide polymorphism detection in promoter III of the acetyl-CoA carboxylase-alpha gene in sheep.

Three novel SNPs were identified in the locus OAR292286, encoding the DNA sequence of promoter III of the ovine acetyl-CoA carboxylase-alpha gene, in Italian sheep of four breeds: Gentile di Puglia (25 individuals) and Sopravissana (31) which are triple-purpose local endangered breeds, Comisana (25) which is a local non-selected, non-endangered dairy breed and Sarda (15) which is a popular selected high yielding dairy breed. Variant alleles are: G/T at 1330 bp, C/G at 1338 bp and C/T at 1430 bp. Frequencies of the variant alleles were calculated and chi-squared analysis of the differences in allele frequency between breed pairs was performed. Allele frequencies of the Sarda breed differ significantly from the other considered breeds.

Gene silencing by S-adenosylmethionine in muscle differentiation.

A well-characterised experimental system, the myogenin gene in C2C12 muscle cell culture, was chosen to better understand the methylation mechanism underlying the regulation of gene expression. We already demonstrated that demethylation dynamics of a specific CpG site in the 5'-flanking region of myogenin well correlates with gene expression and terminal differentiation. Here we demonstrate that S-adenosylmethionine-sulphate-p-toluenesulphonate (SAM) inhibits myogenin expression and myoblast differentiation by delaying the demethylation of specific CpG in differentiating myoblasts. These results suggest new perspectives in methylation mechanisms and the use of SAM in the partial silencing of gene expression, as it could be required in disease treatment.

Melanoma cells stimulate osteoclastogenesis, c-Src expression and osteoblast cytokines.

Malignant melanomas metastasise to the bone and enhance osteoclast bone resorption. We demonstrated that a 48-h-B16 melanoma cell conditioned media (B16CM) induced osteoclastogenesis in mouse bone marrow cultures, without the requirement of B16 cell-bone marrow cell co-culture. B16 cells transcriptionally expressed detectable levels of TGFbeta1, IL-6, M-CSF, GM-CSF and TNFalpha mRNAs, albeit to a lower extent compared with levels in osteoblasts, and failed to express PTHrP, OPGL, OPG and IL-1beta. Interestingly, B16CM greatly upregulated IL-1beta, IL-6 and GM-CSF, and modestly enhanced TNFalpha and OPGL mRNA expression in osteoblasts, suggesting a potential indirect stimulation of osteoclastogenesis via the osteogenic lineage. B16CM barely upregulated c-Fos, but strongly and time-dependently enhanced c-Src expression in the total bone marrow cultures during osteoclast differentiation. Moreover, c-Src expression was enhanced in differentiated and purified osteoclast preparations to higher levels than in stromal cells. In conclusion, melanoma induces osteoclast generation with a paracrine mechanism independent of cell-cell contact, specifically upregulating c-Src in osteoclasts and cytokine expression in osteoblasts.

Erythroid differentiation and regulatory gene expression are modulated by adenosine derivatives interfering with S-adenosylmethionine metabolic pathway.

The differentiation of murine erythroleukemia cells and the expression of SCL, Id1 and c-myc regulatory genes were studied. The first gene is a positive regulator of differentiation, while the other two are both negative regulators of differentiation and positive regulators of proliferation. Accordingly, our data show that when differentiation is stimulated SCL is upregulated while Id1 and c-myc are, coordinately, downregulated. The cultures were treated with two adenosine derivatives, 3-deazaadenosine and 3-deazaaristeromycin, known to act on the metabolic pathway of the methyl donor S-adenosylmethionin, in order to assess the possibility of a coordinated modulation, by these drugs, of regulatory gene expression and erythroid cell differentiation. 3-Deazaaristeromycin caused the simultaneous downregulation of Id1 and c-myc, whereas 3-deazaadenosine caused their upregulation; both drugs produced a transient increase in SCL expression. The use of these drugs evidenced a predominant regulatory effect of negative regulators in the control of erythroid differentiation. The distinct effects of the two drugs on regulatory gene expression led to an increased differentiation induced by 3-deazaaristeromycin and to a reduced differentiation induced by 3-deazaadenosine, if compared with controls. Southern analysis of DNA digested with methylation-specific restriction endonucleases showed that the administration of 3-deazaaristeromycin resulted in hypomethylation of SCL and c-myc, thus evidencing, in these cells, a clear correlation between DNA hypomethylation and differentiation but no straightforward correlation between DNA methylation and gene expression.

Dermorphin and deltorphin glycosylated analogues: synthesis and antinociceptive activity after systemic administration.

In the present paper we describe the synthesis of some dermorphin and deltorphin analogues beta-O- and alpha-C-glycosylated on the C-terminal amino acid residue and report their opioid receptor affinity and selectivity as well as their analgesic potency after subcutaneous injection in mice.