Production of Recombinant Antimicrobial Polymeric Protein Beta Casein-E 50-52 and Its Antimicrobial Synergistic Effects Assessment with Thymol.

Accelerating emergence of antimicrobial resistance among food pathogens and consumers' increasing demands for preservative-free foods are two contemporary challenging aspects within the food industry. Antimicrobial packaging and the use of natural preservatives are promising solutions. In the present study, we used beta-casein-one of the primary self-assembly proteins in milk with a high polymeric film production capability-as a fusion partner for the recombinant expression of E 50-52 antimicrobial peptide in Escherichia coli. The pET21a-BCN-E 50-52 construct was transformed to E. coli BL21 (DE3), and protein expression was induced under optimized conditions. Purified protein obtained from nickel affinity chromatography was refolded under optimized dialysis circumstances and concentrated to 1600 µg/mL fusion protein by ultrafiltration. Antimicrobial activities of recombinant BCN-E 50-52 performed against Escherichia coli, Salmonella typhimurium, Listeria monocytogenes, Staphylococcus aureus, Aspergillus flavus, and Candida albicans. Subsequently, the synergistic effects of BCN-E 50-52 and thymol were assayed. Results of checkerboard tests showed strong synergistic activity between two compounds. Time-kill and growth kinetic studies indicated a sharp reduction of cell viability during the first period of exposure, and SEM (scanning electron microscope) results validated the severe destructive effects of BCN E 50-52 and thymol in combination on bacterial cells.

Functional assessment of CTCF sites at cytokine-sensing mammary enhancers using CRISPR/Cas9 gene editing in mice.

The zinc finger protein CTCF has been invoked in establishing boundaries between genes, thereby controlling spatial and temporal enhancer activities. However, there is limited genetic evidence to support the concept that these boundaries restrict the search space of enhancers. We have addressed this question in the casein locus containing five mammary and two non-mammary genes under the control of at least seven putative enhancers. We have identified two CTCF binding sites flanking the locus and two associated with a super-enhancer. Individual deletion of these sites from the mouse genome did not alter expression of any of the genes. However, deletion of the border CTCF site separating the Csn1s1 mammary enhancer from neighboring genes resulted in the activation of Sult1d1 at a distance of more than 95 kb but not the more proximal and silent Sult1e1 gene. Loss of this CTCF site led to de novo interactions between the Sult1d1 promoter and several enhancers in the casein locus. Our study demonstrates that only one out of the four CTCF sites in the casein locus had a measurable in vivo activity. Studies on additional loci are needed to determine the biological role of CTCF sites associated with enhancers.

Genetic identification of endangered North African ungulates using noninvasive sampling.

North African ungulates include several threatened and emblematic species, yet are poorly studied mainly due to their remoteness and elusiveness. Noninvasive sampling provides a useful approach to obtain ecological and genetic information essential to guide conservation actions. The very first and most important step in conservation planning is to accurately identify species, and molecular genetics has been proved to be a useful tool. Several molecular genetics protocols are available for species identification, even for samples with poor quality DNA, such as faeces, hairs or bones. Most of these protocols use mitochondrial DNA for barcoding despite this marker being especially prone to problems, including mtDNA introgression, nuclear insert copies, high intraspecific diversity or heteroplasmy. In this work, we developed a molecular method based on polymorphisms in small fragments of the mitochondrial cytochrome b (cytb, mtDNA) and the nuclear kappa casein genes (KCAS, nDNA) for identifying endangered North African ungulates. These fragments revealed polymorphisms, including species-specific variation, which allowed species identification of nine ungulate species that co-occur in North Africa. The method was validated across more than 400 samples, including different types of noninvasive samples collected in the field. The simplicity, high reliability and relative low cost of the described method make it a promising tool to improve ecological studies of the North African ungulates and consequently, the implementation of more efficient management and conservation plans for these endangered ungulates.

Valuation of milk composition and genotype in cheddar cheese production using an optimization model of cheese and whey production.

A mass balance optimization model was developed to determine the value of the kappa-casein genotype and milk composition in Cheddar cheese and whey production. Inputs were milk, nonfat dry milk, cream, condensed skim milk, and starter and salt. The products produced were Cheddar cheese, fat-reduced whey, cream, whey cream, casein fines, demineralized whey, 34% dried whey protein, 80% dried whey protein, lactose powder, and cow feed. The costs and prices used were based on market data from March 2004 and affected the results. Inputs were separated into components consisting of whey protein, ash, casein, fat, water, and lactose and were then distributed to products through specific constraints and retention equations. A unique 2-step optimization procedure was developed to ensure that the final composition of fat-reduced whey was correct. The model was evaluated for milk compositions ranging from 1.62 to 3.59% casein, 0.41 to 1.14% whey protein, 1.89 to 5.97% fat, and 4.06 to 5.64% lactose. The kappa casein genotype was represented by different retentions of milk components in Cheddar cheese and ranged from 0.715 to 0.7411 kg of casein in cheese/kg of casein in milk and from 0.7795 to 0.9210 kg of fat in cheese/kg of fat in milk. Milk composition had a greater effect on Cheddar cheese production and profit than did genotype. Cheese production was significantly different and ranged from 9,846 kg with a high-casein milk composition to 6,834 kg with a high-fat milk composition per 100,000 kg of milk. Profit (per 100,000 kg of milk) was significantly different, ranging from $70,586 for a high-fat milk composition to $16,490 for a low-fat milk composition. However, cheese production was not significantly different, and profit was significant only for the lowest profit ($40,602) with the kappa-casein genotype. Results from this model analysis showed that the optimization model is useful for determining costs and prices for cheese plant inputs and products, and that it can be used to evaluate the economic value of milk components to optimize cheese plant profits.

Cumulative discounted expressions of sire genotypes for the complex vertebral malformation and beta-casein loci in commercial dairy herds.

Based on discounted gene-flow principles, a set of recursive equations was developed to quantify the value of using sires with a specific genotype for an identified gene in a commercial dairy herd. Two examples were used to demonstrate the usefulness of the method. The first example deals with the implications of using sires that are known carriers of the lethal recessive genetic defect, complex vertebral malformation (CVM). The second example examines the value of using sires homozygous for the A2 allele of beta-casein. Results are presented in terms of cumulative discounted expressions. These are then multiplied by the economic values of specific genotypes to determine the cost or benefit of using these sires. In general, the degree of mortality and the required price reduction for carrier sires increased as the proportion of carrier sires used, the duration of sire use, and the initial frequency in the cow herd increased. A semen discount of 3.10 pound sterling per CVM straw used would be required to offset the expected mortality when 20% of CVM carrier sires are used for 3 yr when 5% of cows are carriers. The cumulative discounted expressions' of using sires homozygous for the A2 allele of beta-casein also increased when the proportion and duration of carrier sire use and the initial frequency of the A2 allele increased. Assuming an A2A2 cow is worth 160 pound sterling more than a non-A2A2 cow, the expected benefit of using A2A2 sires in a 100-cow herd for 5 yr would be 57 pound sterling,120 for a 20-yr planning horizon. The results of this study demonstrate how the starting gene frequency in the herd, and the proportion and duration of use of sires of particular genotypes are critical to the economic implications of using single genes in commercial dairy farms.

Technical note: Detection of bovine kappa-casein variants A, B, C, and E by means of polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP).

We used polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis to screen the most frequent variants (A, B, C, and E) found in the bovine kappa-casein gene. The PCR products (453 bp) were heat-denatured, loaded onto nondenaturing polyacrylamide gels, and silver-stained. Each variant yielded patterns clearly distinguishable from the others. Optimal conditions for the simultaneous detection of the four variants were 12% polyacrylamide gels (100:1 acrylamide:bis-acrylamide ratio) with 5% glycerol and a constant running temperature of 10 degrees C. Eight reference samples initially used for this purpose and 40 anonymous samples of different cattle breeds diagnosed by PCR-RFLP and PCR-SSCP showed no discrepancies between the two methods and confirmed previous results. Because it is cost-effective, sensitive, and fast, PCR-SSCP is strongly recommended to routinely screen kappa-casein variants for industrial purposes or in cattle selection schemes.

[The polymorphism of the kappa-casein gene and its connection to economically valuable traits in cattle]. / Polimorfizm gena kappa-kazeina, ego sviaz' s khoziaistvenno tsennymi priznakami u krupnogo rogatogo skota.

The literature data on milk protein (particularly kappa-casein) polymorphism study in different cattle breeds are represented. Allele variants of kappa-kasein are associated with lactation and have an influence on the composition of milk and its properties, including cheese-making properties. The importance of ways of kappa-casein genetic variants use in concrete selection programs are discussed.

[The determination of beta-casein gene expression and the assessment of hormonal homeostasis in gynecomastia patients and in breast cancer in men]. / Opredelenie ékspressii genov beta-kazeina i otsenka gormonal'nogo gomeostaza u bol'nykh s ginekomastiei i rakom molochnoi zhelezy u muzhchin.

beta-Casein genes expression in breast epithelium was studied in male patients with various forms of gynecomastia and cancer. Blood serum levels of pituitary, sex and glucocorticoid hormones were assayed in 29 patients with gynecomastia and 22 cases of breast cancer, and in 25 of them beta-casein genes expression was evaluated additionally. Activation of the above genes was established in the tissues studied. Their level proved to be in a correlation with that of prolactin.