Development of a bacteriophage-based system for the selection of structured peptides

Short structured peptides can provide scaffolds for protease-resistant peptide therapeutics, serve as useful building blocks in biomedical and biotechnological applications, and shed light on the role of secondary structure elements in protein folding. It is well known that directed evolution is a powerful method for creating proteins and peptides with novel properties, and a system for the selection of short peptides based on structure from a randomized library would be an important advancement. In this study, phage particles monovalently displaying a short peptide and an N-terminal 6×His tag on their P3 coat protein were bound to nickel agarose resin and were subsequently challenged with a protease that specifically cleaves at a site within the peptide. The extent to which phage is proteolytically released from the resin was found to be dependent on the structural properties of the inserted peptide sequences. As proofs-of-concept, a structured peptide has been isolated from a pool of flexible peptides using a trypsin selection, and a flexible peptide has been isolated from a pool of structured peptides using a chymotrypsin selection. This selection system will be a strong technological platform for the creation of short peptides with interesting structural properties using directed evolution.     

Meal frequency differentially alters postprandial triacylglycerol and insulin concentrations in obese women

Objective:

The aim of this study was to compare postprandial lipemia, oxidative stress, antioxidant activity, and insulinemia between a three and six isocaloric high‐carbohydrate meal frequency pattern in obese women.

Design and Methods:

In a counterbalanced order, eight obese women completed two, 12‐h conditions in which they consumed 1,500 calories (14% protein, 21% fat, and 65% carbohydrate) either as three 500 calorie liquid meals every 4‐h or six 250 calorie liquid meals every 2‐h. Blood samples were taken every 30 min and analyzed for triacylglycerol (TAG), total cholesterol, high‐density lipoprotein cholesterol, low‐density lipoprotein cholesterol, oxidized low‐density lipoprotein cholesterol, myeloperoxidase, paraoxonase‐1 activity, and insulin.

Results:

The TAG incremental area under the curve (iAUC) during the three meal condition (321 ± 129 mg/dl·12 h) was significantly lower (P = 0.04) compared with the six meal condition (481 ± 155 mg/dl·12 h). The insulin iAUC during the three meal condition (5,549 ± 1,007 pmol/l^.12 h) was significantly higher (P = 0.05) compared with the six meal condition (4,230 ± 757 pmol/l^.12 h). Meal frequency had no influence on the other biochemical variables.

Conclusions:

Collectively, a three and six isocaloric high‐carbohydrate meal frequency pattern differentially alters postprandial TAG and insulin concentrations but has no effect on postprandial cholesterol, oxidative stress, or antioxidant activity in obese women.     

Dystrophin-glycoprotein complex and Ras and Rho GTPase signaling are altered in muscle atrophy

The dystrophin-glycoprotein complex (DGC)is a sarcolemmal complex whose defects cause muscular dystrophies. Thenormal function of this complex is not clear. We have proposed thatthis is a signal transduction complex, signaling normal interactionswith matrix laminin, and that the response is normal growth andhomeostasis. If so, the complex and its signaling should be altered inother physiological states such as atrophy. The amount of some of the DGC proteins, including dystrophin, -dystroglycan, and-sarcoglycan, is reduced significantly in rat skeletal muscleatrophy induced by tenotomy. Furthermore, H-Ras, RhoA, and Cdc42decrease in expression levels and activities in muscle atrophy. Whenthe small GTPases were assayed after laminin or -dystroglycandepletion, H-Ras, Rac1, and Cdc42 activities were reduced, suggesting aphysical linkage between the DGC and the GTPases. Dominant-negativeCdc42, introduced with a retroviral vector, resulted in fibers thatappeared atrophic. These data support a putative role for the DGC intransduction of mechanical signals in muscle.

     

Biogenesis,characterization, and the effect of vicenin‐gold nanoparticles on glucose utilization in 3T3‐L1 adipocytes: A bioinformatic approach to illuminate its interaction with PTP 1B and AMPK

This study reported the synthesis of Vicenin‐2 gold nanoparticles (VN‐AuNPs) and evaluated their effect on the glucose utilization efficiency of 3T3‐L1 adipocytes. The VN‐AuNPs were characterized by microscopic, DLS and spectral analysis. The bio‐reducing efficiency of Vicenin‐2 (VN) was computed and confirmed by HPLC analysis. The stability of VN‐AuNPs in various physiological media was explored. The cytotoxicity and glucose uptake assays were performed in 3T3‐L1 adipocytes. The docking of VN with PTP1B and AMPK was also performed. The color change and UV absorption at 537 nm preliminarily confirmed the VN reduced gold nanoparticles. The VN‐AuNPs appeared as spherical particles (57 nm) and face centered cubic crystals under TEM and XRD analysis, respectively. Its zeta potential was found to be ?6.53 mV. The FT‐IR spectra of VN and its AuNPs confirmed its stability. The computed reducing potential of VN was similar to the extent of VN utilized during the synthesis of VN‐AuNPs. The VN‐AuNPs showed a remarkable stability in different physiological media. At 100 µM concentration, VN‐AuNPs displayed 78.21% cell viability. A concentration dependent increase in glucose uptake was noted in 3T3‐L1 adipocytes when incubated with VN‐AuNPs. The docking data revealed a strong interaction of VN with the binding pockets of PTP1B and AMPK. This demonstrates that the fabricated VN‐AuNPs might enhance the intracellular VN availability mediated cellular glucose utilization and this would serve as a novel nanodrug for the management of diabetes. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1096–1106, 2015     

3,4-Disubstituted benzofuran P1′ MMP-13 inhibitors: Optimization of selectivity and reduction of protein binding

Potent 3,4-disubstituted benzofuran P1′ MMP-13 inhibitors have been prepared. Selectivity over MMP-2 was achieved through a substituent at the C4 position of the benzofuran P1′ moiety of the molecule. By replacing a backbone benzene with a pyridine and valine with threonine, compounds (e.g., 44) with greatly reduced plasma protein binding were also obtained.     

The Feasibility of Using High Resolution Genome Sequencing of Influenza A Viruses to Detect Mixed Infections and Quasispecies

Background

The rapidly expanding availability of de novo sequencing technologies can greatly facilitate efforts to monitor the relatively high mutation rates of influenza A viruses and the detection of quasispecies. Both the mutation rates and the lineages of influenza A viruses are likely to play an important role in the natural history of these viruses and the emergence of phenotypically and antigenically distinct strains.

Methodology and Principal Findings

We evaluated quasispecies and mixed infections by de novo sequencing the whole genomes of 10 virus isolates, including eight avian influenza viruses grown in embryonated chicken eggs (six waterfowl isolates - five H3N2 and one H4N6; an H7N3 turkey isolate; and a bald eagle isolate with H1N1/H2N1 mixed infection), and two tissue cultured H3N2 swine influenza viruses. Two waterfowl cloacal swabs were included in the analysis. Full-length sequences of all segments were obtained with 20 to 787-X coverage for the ten viruses and one cloacal swab. The second cloacal swab yielded 15 influenza reads of ∼230 bases, sufficient for bioinformatic inference of mixed infections or quasispecies. Genomic subpopulations or quasispecies of viruses were identified in four egg grown avian influenza isolates and one cell cultured swine virus. A bald eagle isolate and the second cloacal swab showed evidence of mixed infections with two (H1 and H2) and three (H1, H3, and H4) HA subtypes, respectively. Multiple sequence differences were identified between cloacal swab and the virus recovered using embryonated chicken eggs.

Conclusions

We describe a new approach to comprehensively identify mixed infections and quasispecies in low passage influenza A isolates and cloacal swabs and add to the understanding of the ecology of influenza A virus populations.