Rational design of glycoengineered interferon-ß analogs with improved aggregation state: experimental validation.

Recombinant human interferon-ß (rhIFN-ß) used clinically has lower efficacy than expected due to protein instabilities such as aggregation. Increasing molecular stability, glycoengineering has been used to improve clinical efficacy for a number of therapeutics; however, often labor-intensive trail-and-error approaches are used to identify additional glycosylation sites. In this study two rhIFN-ß analogs with one additional glycosylation site, L6T and S75N, identified by a rational in silico approach, were characterized. These rhIFN-ß analogs were synthesized in parallel with a Chinese hamster ovary (CHO) codon-optimized natural human IFN-ß (Opt-IFN-ß) and expressed in CHO cells using the same expression system. The molecular weights for both analogs were observed to be higher than Opt-IFN-ß, consistent with hyper-glycosylation. The in vitro biological assay showed the hyper-glycosylated analogs and the Opt-IFN-ß had similar activity. The aggregation studies demonstrated that both analogs had lower tendencies to aggregate compared to the Opt-IFN-ß. These experimental studies validate the in silico strategy to predict suitable glycosylation sites that would be glycosylated, while maintaining biological function. Moreover, this work describes hyper-glycosylated rhIFN-ß analogs with improved solubility (i.e. lower aggregation). These findings, together with the rational in silico design, will allow us to increase protein glycosylation with the goal to enhance therapeutic efficacy.

Efficacy of combined or single use of Lactobacillus crispatus LT116 and L. johnsonii LT171 on broiler performance.

1. The objective of this research was to investigate the efficacy of combined or single use of Lactobacillus crispatus LT116 and Lactobacillus johnsonii LT171 on broiler performance. 2. A total of 320 one-d-old male Ross broiler chicks were allocated in 4 experimental treatments for 6 weeks. The experimental treatments received a maize-soybean meal basal diet that was supplemented as follows: 'control', with no other additions; 'LJ', 1 × 10(6) CFU of L. johnsonii LT171; 'LC', 1 × 10(6) CFU of L. crispatus LT116; and 'LCJ', 0·5 × 10(6) CFU of L. johnsonii LT171 + 0·5 × 10(6) CFU of L. crispatus LT116/g of the diet. A suspension of sheep red blood cells (SRBC) was injected into the breast of 8 birds from each treatment on d 14 and 30, and the antibody titre was measured on d 20, 26, 36 and 42. 3. Body weight was improved when compared with control for broilers fed diets supplemented with LCJ. The feed conversion ratio (FCR) decreased in LC and LCJ groups compared with control. The number of coliforms in the ileum of LJ, LC and LCJ birds was lower than that from the control birds. However, only the LCJ treatment significantly decreased the number of coliforms in the caecum. The LCJ group had greater villus height in the duodenum than the LC group, and both LCJ and LC groups showed increased villus height in the duodenum and jejunum relative to the control. Antibody titre against SRBC was higher for the LCJ group than for the LJ and control groups in terms of secondary immune response (mean of 36 and 42 d). 4. This study showed, compared with the control, that the combination of Lactobacillus spp. could positively affect body weight, coliform numbers in the caecum and immune response.

Screening of lactic acid bacteria toward their selection as a source of chicken probiotic.

In this study, 332 lactic acid bacteria of broiler gut origin were investigated as putative probiotic candidates. Sixty-two bacteria showed the highest aggregation while showing antibacterial effects against Salmonella Enteritidis, Salmonella Typhimurium, and Escherichia coli O78:K80. Enzymatic activities of the selected bacteria were examined. No lipase activity was detected, but all of them showed almost similar proteolytic activities. However, they had different amylase and phytase activities. Four of the 62 bacterial strains were selected as a result of aggregation and amylase tests. They were chosen for the following attributes: high cell surface hydrophobicity, co-aggregation, and resistance to bile salts and also acidic conditions. Finally, Lactobacillus crispatus was selected as a source of potential probiotic because of its predominant characteristics in comparison to the other isolated strains from the gastrointestinal tract of chickens. This study has provided a good arrangement of rapid methods for screening a high number of bacteria: a series of beneficial tests that are used together for the first time on chickens, and not any other animals.