Methods: Herein, we report a comprehensive study of the dynamics of H5N1 mutations by analysis of the aligned overlapping nonamer positions (1-9, 2-10, etc.) of more than 13,000 protein sequences of avian and human influenza A (H5N1) viruses, reported over at least 50 years. Entropy calculations were performed on 9,408 overlapping nonamer position of the proteome to study the diversity in the context of immune system. The nonamers represent the predominant length of the binding cores for peptides recognized by the cellular immune system. To further dissect the sequence diversity, each overlapping nonamer position was quantitatively analyzed for four patterns of sequence diversity motifs: index, major, minor and unique.

Results: Almost all of the aligned overlapping nonamer positions of each viral proteome exhibited variants (major, minor, and unique) to the predominant index sequence. Each variant motif displayed a characteristic pattern of incidence change in relation to increased total variants. The major variant exhibited a restrictive pyramidal incidence pattern, with peak incidence at 50% total variants. Post this peak incidence, the minor variants became the predominant motif for majority of the positions. Unique variants, each sequence observed only once, were present at nearly all of the nonamer positions. The diversity motifs (index and variants) demonstrated complex inter-relationships, with motif switching being a common phenomenon. Additionally, 25 highly conserved sequences were identified to be shared across viruses of both hosts, with half conserved to several other influenza A subtypes.

Materials and Methods: Forty-five semen samples, 15 each were extended with either BX, TEY, or CEY extender which contained different concentrations (0.0 - control, 0.5, 1.0, 1.5, 2.0, and 3.0 mM/mL) of BHT. The extended semen samples were frozen at a concentration of 20×106/mL in 0.25 mL straws and stored in liquid nitrogen for 2weeks. The frozen samples were thereafter thawed, proteins extracted and analyzed for quantities of protein P25b through direct sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel densitometry. Peptides were confirmed by Western blotting (WB).

Results: Results showed that supplementation of BHT improved (p<0.05) quantity of protein P25b at concentrations of 0.5mM/mL for BX and at 1.0 mM/mL for TEY and CE when compared with the controls and other treatments.

METHODS: TEM, SEM and ATP efflux assay were used to evaluate the effect of hybrid peptides on the integrity of the pneumococcal cell wall/membrane. DNA retardation assay was assessed to measure the impact of hybrid peptides on the migration of genomic DNA through the agarose gel. In vitro synergistic effect was checked using the chequerboard assay. ICR male mice were used to evaluate the in vivo toxicity and antibacterial activity of the hybrid peptides in a standalone form and in combination with ceftriaxone.

RESULTS: The results obtained from TEM and SEM indicated that the hybrid peptides caused significant morphological alterations in Streptococcus pneumoniae and disrupting the integrity of the cell wall/membrane. The rapid release of ATP from pneumococcal cells after one hour of incubation proposing that the antibacterial action for the hybrid peptides is based on membrane permeabilization and damage. The DNA retardation assay revealed that at 62.5 µg/ml all the hybrid peptides were capable of binding and preventing the pneumococcal genomic DNA from migrating through the agarose gel. In vitro synergy was observed when pneumococcal cells treated with combinations of hybrid peptides with each other and with conventional drugs erythromycin and ceftriaxone. The in vivo therapeutic efficacy results revealed that the hybrid peptide RN7-IN8 at 20 mg/kg could improve the survival rate of pneumococcal bacteremia infected mice, as 50% of the infected mice survived up to seven days post-infection. In vivo antibacterial efficacy of the hybrid peptide RN7-IN8 was signficantly improved when combined with the standard antibiotic ceftriaxone at (20 mg/kg + 20 mg/kg) as 100% of the infected mice survived up to seven days post-infection.

MATERIALS AND METHODS: Five groups of rats were intravitreally administered with vehicle or Aβ(1-40) in doses of 1.0, 2.5, 5 and 10 nmol. Animals were sacrificed and eyes were enucleated at weeks 1, 2 and 4 post-injection. The retinae were subjected to morphometric analysis and TUNEL staining. Optic nerve sections were stained with toluidine blue and were graded for neurodegenerative effects. The estimation of BDNF and markers of oxidative stress in retina were done using ELISA technique.

METHODS: Human respiratory epithelial cells were serially passaged using a co-culture system and a conventional dispase-dissociation technique. The growth kinetics and gene expression levels of the cultured respiratory epithelial cells were compared. Four genes were investigated, namely cytokeratin-18, a marker for ciliated and secretory epithelial cells; cytokeratin-14, a marker for basal epithelial cells; MKI67, a proliferation marker; and MUC5B, a marker for mucin secretion. Immunocytochemical analysis was performed using monoclonal antibodies against the high molecular-weight cytokeratin 34 beta E12, cytokeratin 18, and MUC5A to investigate the protein expression from cultured respiratory epithelial cells.

RESULTS: Respiratory epithelial cells cultured using both methods maintained polygonal morphology throughout the passages. At passage 1, co-cultured respiratory epithelial showed a 2.6-times higher growth rate compared to conventional dispase dissociation technique, and 7.8 times higher at passage 2. Better basal gene expression was observed by co-cultured respiratory epithelial cells compared to dispase dissociated cells. Immunocytochemical analyses were positive for the respiratory epithelial cells cultured using both techniques.