Glycovaccine Design: Optimization of Model and Antitubercular Carrier Glycosylation via Disuccinimidyl Homobifunctional Linker

Conjugation via disuccinimidyl homobifunctional linkers is reported in the literature as a convenient approach for the synthesis of glycoconjugate vaccines. However, the high tendency for hydrolysis of disuccinimidyl linkers hampers their extensive purification, which unavoidably results in side-reactions and non-pure glycoconjugates. In this paper, conjugation of 3-aminopropyl saccharides via disuccinimidyl glutarate (DSG) was exploited for the synthesis of glycoconjugates. A model protein, ribonuclease A (RNase A), was first considered to set up the conjugation strategy with mono- to tri- mannose saccharides. Through a detailed characterization of synthetized glycoconjugates, purification protocols and conjugation conditions have been revised and optimized with a dual aim: ensure high sugar-loading and avoid the presence of side reaction products. An alternative purification approach based on hydrophilic interaction liquid chromatography (HILIC) allowed the formation of glutaric acid conjugates to be avoided, and a design of experiment (DoE) approach led to optimal glycan loading. Once its suitability was proven, the developed conjugation strategy was applied to the chemical glycosylation of two recombinant antigens, native Ag85B and its variant Ag85B-dm, that are candidate carriers for the development of a novel antitubercular vaccine. Pure glycoconjugates (≥99.5%) were obtained. Altogether, the results suggest that, with an adequate protocol, conjugation via disuccinimidyl linkers can be a valuable approach to produce high sugar-loaded and well-defined glycovaccines

Chromosomal unbalancements in sperm and oocytes of two Italian cattle breeds as determined by dual color fluorescent in situ hybridization (FISH)

Aneuploidy is one of the most important causes of embryonic and foetal mortality in mammals. In order to assess the possible risk of chromosomal abnormalities in germ cells of domestic animals we investigated the aneuploidy rates on partially decondensed sperm and in vitro matured oocytes in two cattle breeds, Italian Friesian (I.F.) and Italian Brown (I.B.), by using FISH with chromosome-specific painting probes (chromosomes X-Y for sperm and chromosomes X-5 for oocytes). For each bull, more than 5,000 sperm were analyzed, for a total of 52,586 and 51,342 sperm cells for the two breeds, respectively. Aneuploid and diploid sperm had, respectively, a frequency of 0.110% and 0.050% in the I.F. and 0.078% and 0.062% in the I.B. breeds. Out of 100 in vitro matured oocytes for each breed, on the average, diploidy affected 11.2% and 18.4% in the I.F. and I.B., respectively, whereas disomy for chromosome X-had a frequency of 2% in the I.F. and 2.5% in the I.B. breeds. Further studies are needed to expand our knowledge on frequency of aneuploidy in sperm and oocytes of domestic animals, in order to assess their impact on productive and reproductive efficiency, also in relation to climatic changes and environmental hazards

Chromosomal unbalancements in sperm and oocytes of two Italian cattle breeds as determined by dual color fluorescent in situ hybridization (FISH)

Aneuploidy is one of the most important causes of embryonic and foetal mortality in mammals. In order to assess the possible risk of chromosomal abnormalities in germ cells of domestic animals we investigated the aneuploidy rates on partially decondensed sperm and in vitro matured oocytes in two cattle breeds, Italian Friesian (I.F.) and Italian Brown (I.B.), by using FISH with chromosome-specific painting probes (chromosomes X-Y for sperm and chromosomes X-5 for oocytes). For each bull, more than 5,000 sperm were analyzed, for a total of 52,586 and 51,342 sperm cells for the two breeds, respectively. Aneuploid and diploid sperm had, respectively, a frequency of 0.110% and 0.050% in the I.F. and 0.078% and 0.062% in the I.B. breeds. Out of 100 in vitro matured oocytes for each breed, on the average, diploidy affected 11.2% and 18.4% in the I.F. and I.B., respectively, whereas disomy for chromosome X-5 had a frequency of 2% in the I.F. and 2.5% in the I.B. breeds. Further studies are needed to expand our knowledge on frequency of aneuploidy in sperm and oocytes of domestic animals, in order to assess their impact on productive and reproductive efficiency, also in relation to climatic changes and environmental hazards

Cytogenetic characterization of the yak (Bos Grunniens) and comparison with cattle (Bos taurus)

The domestic yak (Bos grunniens) belongs to the family Bovidae and is one of the most important species for the economy of the Asian high lands. Recently, the interest for this species increased also in European countries, mainly because of its meat and milk characteristics. Furthermore, it is a species well adapted to marginal conditions and it could be used to preserve mountain territories economy. We carried out a detailed investigation on this species with the following main aims: 1) to perform a cytogenetic screening of a yak herd recently introduced in Italy; 36 out of 60 animals were analyzed by conventional and GTG-banding karyotype. All animals were 2n=and no chromosomal abnormalities were found, confirming that the introduced herd is a good nucleus to start up with yak farming in Italy; 2) to determine the genetic stability of the animals reared under Italian conditions; two different tests were used: “Chromatid/chromosome breaks” and “Sister chromatid exchange (SCE)”. Out of 400 analyzed cells, 3.75% showed one or more anomalies; the most represented were chromosome and chromatid breaks. The mean number of SCE/cells was 5.2%. Comparison with cattle revealed that the two species have the same percentage of chromosome anomalies, while SCE frequency is lower in yak compared to cattle; 3) to increase the cytogenetic knowledge about this species and compare Bos taurus and Bos grunniens in relation to male sterility of F1 hybrids; C-, R-, and G-banded karyotypes of yak were prepared and compared with the standard karyotypes of cattle. To identify Nucleolar Organizer Regions, sequential NOR’s/R banded karyotypes were performed. Despite the two species share a very high degree of banding homology, some differences can be pointed out; the most remarkable is on the centromeric region of chromosome 15, where a strong positive band is present in Bos grunniens while absent in Bos taurus. Molecular data indicate that in yak, unlike in cattle, the X chromosome has retained a homologous sequence (182 bp) to the Y chromosome. To verify if larger blocks of chromatin are involved, FISH analysis of yak metaphases using X and Y cattle painting probe was performed. The lack of unspecific signal indicated that no large blocks of chromatin are interested. Finally, we mapped two genes (SRY: sex-determining region Y gene and ZFY: zinc finger protein, Y-linked) on the yak Y chromosome to verify if this chromosome, like in other species, such as Bos indicus, underwent rearrangement during evolution. The two genes mapped at the same position compared to cattle, indicating that the sterility of F1 hybrids seems not to be due to Y chromosomal rearrangements relatively to the these two genes

A Cytogenetic comparison between yak (Bos grunniens) and cattle (Bos taurus)

A sample of 28 yaks (Bos grunniens) (9 males and 19 females), kept in the province of Teramo (Italy), was cytogenetically analyzed in order to investigate similarities or differences with cattle (Bos taurus). The results were as follows: (a) the chromosomal makeup of the yak was 2n060,XY, as for cattle; (b) no numerical as well as structural chromosomal abnormalities were found in the sample investigated; (c) the incidence of chromosome + chromatid breaks was 3.7 vs 3.0 % as for cattle; (d) the GTG- RBG- and RBAbanded karyotypes were all similar to the cattle standard karyotypes; (e) the CBA-banding pattern was similar to that of cattle; (f) the mean rate of SCE/cell at 10 μg/ml (f.c.) of BrdU was 5.2±2,23 (range 1–13), similar to that of cattle; (g) silver staining revealed the presence of telomeric NORs on five pairs of autosomes n. 2,3,4,11 and 25, as for cattle; (h) Zoo-FISH with bovine painting probes derived from microdissected chromosomes 5-X-Xcen and Y- upon yak metaphase chromosomes showed complete hybridization; (i) FISH-mapping of bovine BAC-clones containing ZFY- and SRY- genes revealed the same location on the yak Y-chromosome. All these data demonstrate the close evolutionary relationships between yak and cattle. However, the fact that Bos taurus x Bos grunniens F1 male hybrids are sterile, while the females are normally fertile, would suggest that the genomes of the two species are not completely homologous and that minute structural differences might exist in the chromosomes of the two species which are worth to be further investigated

Frequency of aneuploidy in in vitro matured MII oocytes and corresponding first polar bodies in two dairy cattle (Bos taurus) breeds as determined by dual color fluorescent in situ hybridization (FISH)

The current study was undertaken to investigate the aneuploidy rates in in vitro-matured meiosis II (MII) oocytes and corresponding first polar bodies in two dairy cattle (Bos taurus) breeds by using dual-color fluorescent in situ hybridization (FISH). A total of 159 and 144 in vitro-matured MII oocytes of the Italian Friesian and Italian Brown breeds, respectively, were obtained according to the standard methods and analyzed by FISH using "Xcen" and "5" chromosome-specific painting probes, produced by chromosome microdissection and Degenerate Oligonucleotide Primer- Polymerase Chain Reaction (DOP-PCR). Oocytes with unreduced chromosome number were 10.1% and 16.7% in the two breeds, respectively. To avoid bias due to possible artifacts, the aneuploidy rates were determined by analyzing only oocytes with the corresponding polar bodies. In the Italian Friesian, 100 of 143 (69.9%) secondary MII oocytes showed clear MII plates with corresponding first polar bodies and were scored for aneuploidy detection; one oocyte was "nullisomic" for chromosome X (1.0%) and one "disomic" for chromosome 5 (1.0%). In the Italian Brown, 100 of 120 (83.3%) MII oocytes with corresponding first polar bodies were analyzed; one oocyte was nullisomic (1.0%) and one was disomic (1.0%), both for chromosome 5. Totally, 303 oocytes were analyzed, 40 of which showed an unreduced chromosome complement (13.2%); of 200 MII oocytes with the corresponding first polar bodies, the aneuploidy rate (nullisomy+disomy) for the two chromosomes scored was 2%. Assuming that each chromosome is equally involved in aneuploidy, it results that in cattle oocytes matured in vitro, at least 30% of the oocytes (1x30 haploid chromosomes) should be aneuploid. Premature separation of sister chromatids (PSSC) was also observed in 2% of the oocytes in the Italian Friesian breed involving chromosome 5 and in 1% of the Italian Brown breed involving the X chromosome. Estimation of the "baseline" level of aneuploidy in the in vitro-matured oocytes of the various domestic animal species and breeds is, to our opinion, a useful reference for improving the in vitro production of embryos as well as for monitoring future trends of the reproductive health of the species/breeds engaged in zootechnical productions, especially in relation to management errors and environmental hazard