The dynamic changes and structural patterns of DNA methylation of genes without CpG islands are poorly characterized. The relevance of CpG to the non-CpG methylation equilibrium in transcriptional repression is unknown. In this work, we analyzed the DNA methylation pattern of the 5'-flanking of the myogenin gene, a positive regulator of muscle differentiation with no CpG island and low CpG density, in both C2C12 muscle satellite cells and embryonic muscle. Embryonic brain was studied as a non-expressing tissue. High levels of both CpG and non-CpG methylation were observed in non-expressing experimental conditions. Both CpG and non-CpG methylation rapidly dropped during muscle differentiation and myogenin transcriptional activation, with an active demethylation dynamics. Non-CpG demethylation occurred more rapidly than CpG demethylation. Demethylation spread from initially highly methylated short CpC-rich elements to a virtually unmethylated status. These short elements have a high CpC content and density, share some motifs and largely coincide with putative recognition sequences of some differentiation-related transcription factors. Our findings point to a dynamically controlled equilibrium between CpG and non-CpG active demethylation in the transcriptional control of tissue-specific genes. The short CpC-rich elements are new structural features of the methylation machinery, whose functions may include priming the complete demethylation of a transcriptionally crucial DNA region.

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The lengths of the dinucleotide (TG)m and mononucleotide Tn repeats, both located at the intron 8/exon 9 splice acceptor site of the cystic fibrosis transmembrane conductance regulator (CFTR) gene whose mutations cause cysticfibrosis (CF), have been shown to influence the skipping of exon 9 in CFTR mRNA. This exon 9-skipped mRNA encodes a nonfunctional protein and is associated with various clinical manifestations in CF As a result of growing interest in these repeats, several assessment methods have been developed, most of which are, however, cumbersome, multi-step, and time consuming. Here, we describe a rapid methodfor the simultaneous assessment of the lengths of both (TG)m and Tn repeats, based on a nonradioactive cycle sequencing procedure that can be performed even without DNA extraction. This method determines the lengths of the (TG)m and Tn tracts of both alleles, which in our samples ranged from TG8 to TG12 in the presence of T5, T7, and T9 alleles, and also fully assesses the aplotypes. In addition, the repeats in the majority of these samples can be assessed by single-strand sequencing, with no need to sequence the other strand, thereby saving a considerable amount of time and effort.

Ruminal bacterial counts and in vivo digestibility were determined on four Mediterranean buffalo bulls and four Friesian bulls, all fistulated at the rumen, and given at maintenance level (50 g/kg M0·75 per day of dry matter) four different diets with the same crude protein content (N ✕ 6·25 = 140 g/kg dry matter) and with forage: concentrate ratios as follows: diet D12·5 = 0·875: 0·125; diet D25·0 = 0·75: 0·25; diet D37·5 = 0·625: 0·375; diet D50·0 = 0·5: 0·5. All the animals received the diets during four consecutive periods in a Latin-square design. Buffaloes had higher total microbial counts (10·78 v. 10·08 log10 cells per g dry rumen content, P < 0·01) as compared with cattle; differences in total ruminal bacterial counts among the diets were only observed within the buffalo species (diet D12·5 v. diets D25·0, D37·5, D50·0: 10·04 v. 10·92, 10·98, 11·17 log10 cells per g dry rumen content, P 0·01) and when comparing the two species for each diet, significantly higher values for bacterial counts in buffaloes were found for diets D25·0: 10·92 v. 10·28 (P 0·05), D37·5: 10·98 v. 10·08 (P 0·01) and D50·0: 11·17 v. 9·76 (P 0·01) log10 cells per g dry rumen content. Cattle showed significantly higher digestibility values for: organic matter (0·696 v. 0·676, P 0·05), neutral-detergent fibre (NDF; 0·548 v. 0·511, P 0·05) and cellulose (0·621 v. 0 · 509, P 0·01), while the crude protein digestibility (CPD) values were similar (0·667 and 0·671). Comparing the two species for each diet, cattle showed significantly higher digestibility values for organic matter in diet D50·0 only (0·714 v. 0·688, P 0·01), for NDF in diet D12·5 only (0·578 v. 0·531, P 0·05) and for cellulose in all diets (0·660 v. 0·546, 0·630 v. 0·525, 0·605 v. 0·505, 0·588 v. 0·460, P 0·01); in contrast buffaloes showed higher values of the CPD for diet D12·5 (0·662 v. 0·632, P 0·05).

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Recent studies have explored the seropositivity of Bovine alphaherpesvirus 1 (BoHV-1) in water buffaloes, suggesting the urgency for developing strategies to eradicate the virus involving both cattle and water buffaloes. However, in Europe, the glycoprotein E (gE) deleted marker vaccines against BoHV-1 are commercially available only for the cattle industry. This study, for the first time, evaluated the safety and efficacy of a commercial inactivated gE-deleted marker vaccine in water buffalo. Five animals devoid of BoHV-1-neutralizing antibodies were vaccinated via intramuscular route. Five additional animals served as an unvaccinated control group. Sixty days after the first immunization, all animals were experimentally infected with a virulent BoHV-1via intranasal route. A detectable BoHV-1-humoral immune response was observed in the vaccinated group on post-vaccination day 30, whereas the antibodies appeared on post-challenge day 10 in the control group. Moreover, the vaccinated animals neither show viral shedding nor clinical signs compared to the control upon challenge. However, post-challenge, the BoHV-1-specific humoral and cell-mediated immune responses were significantly more increased in vaccinated animals than the control animals. Overall, the present study provides evidence of both the safety and efficacy of an inactivated gE-deleted marker vaccine against BoHV-1 in water buffaloes.

The results of two different protocols of neonatal cystic fibrosis (CF) screening in the Lazio region of Italy are reported. The first study, conducted from 1992 to 2000 on about 200,000 newborns, consisted of an immunoreactive trypsin (IRT) protocol without mutation analysis of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, referred to as the IRT/IRT protocol. Approximately 5% of the newborns with a positive first IRT test were also positive at the second test; approximately 57% of the newborns with a high IRT level at the second test were subsequently found to be affected by CF. In September 1998, a second protocol that included mutation analysis (IRT/DNA/IRT protocol) was started. Comparison of the two different screening protocols in terms of sensitivity in detecting CF patients demonstrated that the IRT/DNA/IRT protocol is more effective because it is able to detect a higher number of CF patients than the IRT/IRT protocol. In the same period, in addition to the overall diagnosis performed on a screening basis, 64 other subjects were identified as being affected by CF on the basis of symptomatic findings. The overall incidence of CF (screening + symptoms) was 1 : 2982, while that for carriers was 1 : 27. The sensitivity of the screening program increased over the period from 1992 to 2000, with the enhanced sensitivity in the past 2 years being due to the introduction of the IRT/DNA/IRT protocol.

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