Molecular diversity in engineered protein libraries

Engineered protein libraries, defined here as a collection of different mutant variants of a single specific protein, are intentionally designed to be rich in molecular diversity and can span ranges from as little as 400 different variants to greater than 10(12) members per library. The goal of engineering libraries is to generate new protein variants, identified upon screening, that possess desired novel properties. Exploitation of the natural organization of the genetic code has led to 'focused' libraries that are lower in overall complexity yet biased towards variants with preferred biophysical properties. An emerging trend, in which computational algorithms are blended with in vivo screens, is also leading towards greater and more rapid success in the field of protein design.     

Morphological and molecular information of a new species of Geleia (Ciliophora, Karyorelictea), with redescriptions of two Kentrophoros species from China

The morphology and infraciliature of three karyorelictean ciliates, Geleia sinica spec. nov. and two poorly known Kentrophoros species, K. flavus and K. gracilis, isolated from the intertidal zone of a beach at Qingdao, China, were investigated. Geleia sinica spec. nov. is distinguished from its congeners by the following combination of characters: body medium-sized and slender-cylindrical; with a conspicuous prebuccal fossa; 28–34 somatic kineties; about 40 short adoral polykineties; intrabuccal kinety composed of 25–34 dikinetids; paroral kineties composed of closely spaced dikinetids. The comparison with similar congeners clearly supports the validity of this new species based on morphological and small subunit (SSU) rRNA gene sequence data. In light of these new data the “well-known” morphotype, Geleia simplex (Fauré-Fremiet, 1951), is redefined. Two Kentrophoros species are redescribed and improved diagnoses are supplied. Kentrophoros flavus Raikov and Kovaleva, 1968 is mainly characterized by having about 33 macronuclei and 12 micronuclei forming a row that extends along the cell meridian, and 12–19 ciliary rows on the right side of the cell. Kentrophoros gracilis Raikov, 1963 is characterized by having about 14 macronuclei, 13 micronuclei and 10–13 kineties on the right side of the cell.     

Pancreatic lipase inhibition activity of trilactone terpenes of Ginkgo biloba

The prevalence of obesity is increasing at an alarming rate, but, unfortunately, only a few drugs are currently available on the market. In the present study, the methanolic extract of Ginkgo biloba L. (Ginkgoaceae) was investigated as an inhibitor of pancreatic lipase (PL) in an attempt to explain its hypolipidaemic activity. In vitro assay of G. biloba leaves extract revealed a substantial PL inhibition activity (IC(50)?=?16.5 μg/mL). Further investigation was performed by employing theoretical docking simulations and experimental testing to uncover the active constituents responsible for G. biloba anti-lipase activity. Virtually, terpene trilactones, including ginkgolides and bilobalide, were found to fit within the binding pocket of PL via several attractive interactions with key amino acids. Experimentally, ginkgolides A, B, and bilobalide were found to inhibit PL significantly (IC(50)?=?22.9, 90.0, and 60.1 μg/mL, respectively). Our findings demonstrated that the hypolipidaemic effects of G. biloba extract can be attributed to the inhibition of PL by, at least in part, terpene trilactones. In conclusion, this work can be considered a new step towards the discovery of new natural safe hypolipidaemic PL inhibitors.     

Oxidative damage induced by chromium (VI) in rat erythrocytes: protective effect of selenium

Excess chromium (Cr) exposure is associated with various pathological conditions including hematological dysfunction. The generation of oxidative stress is one of the plausible mechanisms behind Cr-induced cellular deteriorations. The efficacy of selenium (Se) to combat Cr-induced oxidative damage in the erythrocytes of adult rats was investigated in the current study. Female Wistar rats were randomly divided into four groups of six each: group I served as controls which received standard diet, group II received in drinking water K₂Cr₂O₇ alone (700 ppm), group III received both K₂Cr₂O₇ and Se (0.5 Na₂SeO₃ mg/kg of diet), and group IV received Se (0.5 mg/kg of diet) for 3 weeks. Rats exposed to K₂Cr₂O₇ showed an increase of malondialdehyde and protein carbonyl levels and a decrease of sulfhydryl content, glutathione, non-protein thiol, and vitamin C levels. A decrease of enzyme activities like catalase, glutathione peroxidase, and superoxide dismutase activities was also noted. Co-administration of Se with K₂Cr₂O₇ restored the parameters cited above to near-normal values. Therefore, our investigation revealed that Se was a useful element preventing K₂Cr₂O₇-induced erythrocyte damages.     

Fungal populations and mycotoxins in silage in Assiut and Sohag governorates in Egypt,with a special reference to characteristic Aspergilli toxins

Forty silage samples were collected from Assiut and Sohag governorates in Egypt to measure the presence of fungal population in silage. Forty-three species and 2 species varieties belonging to 17 genera were isolated using glucose Czapeks and Sabourauds dextrose agar media at 28 ^°C. The most prevalent genera were Aspergillus (57.5 and 100 of the samples), Penicillium (100 and 55%) on the two mentioned media, respectively. Also, Fusarium oxysporum and Gibberella fujikurori were recovered in moderate incidences. Mycotoxin profiles were also determined in these samples: Aflatoxins showed the highest incidence rates of occurrence, it occurred in 22.5% of all samples analyzed. Other mycotoxins were detected from all samples (T₂ toxins and sterigmatocystin at incidence of 7.5 and 5%, respectively). The screening of the characteristics mycotoxins of different isolates of Aspergillus isolated from silage samples was tested. The results clarified that some mycotoxins (aflatoxins – aspergillic acid – beta nitro propionic acid – cyclopiazonic acid– kojic acid and sterigmatocystin) were produced by some isolates of A. flavus. Some isolates of A.fumigatus could produce gliotoxin and verrucologen. All of A. niger isolates tested were able to produce kojic acid. One isolate of A. ochraceous formed ochratoxin A and other isolate produced penicillic acid. Concerning A. terreus isolates, the results showed that 5 isolates were able to produce citrinin and 4 isolates had ability to produce patulin. A. versicolor isolates showed the ability to produce ochratoxin A.     

Signalling by PI3K isoforms: insights from gene-targeted mice

Phosphoinositide 3-kinases (PI3Ks) generate lipids that control a wide variety of intracellular signalling pathways. Part of this diversity in PI3K actions stems from the broad range of protein effectors of the PI3K lipids. A further layer of complexity is added by the existence of multiple isoforms of PI3K. Gene-targeting studies in the mouse have recently uncovered key roles for specific PI3K isoforms in immunity, metabolism and cardiac function. Remarkably, some of these actions do not require PI3K catalytic activity. In addition, loss-of-expression of certain PI3K genes leads to increased PI3K signalling following insulin stimulation. PI3K gene targeting has, in many cases, led to altered expression of the non-targeted PI3K subunits, making it difficult to exclude that some of the reported phenotypes result from 'knock-on' effects of PI3K gene deletion. Targeting strategies that take into account the complex interplay between members of the PI3K family will be crucial to gain a full understanding of the physiological roles of the isoforms of PI3K.     

Effects of ocean acidification on the growth and biochemical composition of a green alga (Ulva fasciata) and its associated microbiota

In marine ecosystems, fluctuations in surface-seawater carbon dioxide (CO₂), significantly influence the whole metabolism of marine algae, especially during the early stages of macroalgal development. In this study, the response of the green alga Ulva fasciata for elevating ocean acidification was investigated using four levels of pCO₂ ~ 280, 550, 750 and 1050 µatm. Maximum growth rate (6.6% day⁻¹), protein (32.43 %DW) and pigment (2.9 mg/g) accumulation were observed at pCO₂-550 with an increase of ~2-fold compared to control. On the other hand, lipid and carbohydrate contents recorded their maximum production (4.23 and 46.96 %DW, respectively) at pCO₂-750 while control showed 3.70 and 42.37 %DW, respectively. SDS-PAGE showed the presence of unique bands in response to pCO_2, especially at 550 µatm. Dominant associated bacteria was shifted from Halomonas hydrothermalis of control to Vibrio toranzoniae at pCO₂-1050. These findings suggest that ocean acidification at 550 µatm might impose noticeable effects on growth, protein, pigments, and protein profile of U. fasciata, which could be a good source for fish farming. While, pCO₂-750 was recommended for energetic purpose, due to its high lipid and carbohydrate contents.     

A primer-based approach to genome walking

A genome walking strategy based on annealing and ligation of single-stranded DNA primers to 3′ overhangs following restriction endonuclease digestion was developed. A set of primers contains 4 nucleotides at the 3′ end that are complementary to overhangs formed by restriction endonucleasesApaI;PstI;SacI andSphI. Following ligation, 5′ end overhangs are formed on the DNA, which serves as sites for the adaptor primers and nested primers for PCR amplification in combination with the gene-specific primers. This strategy was verified by the amplification of up to 4 kb of a potato leafroll virus full-length infectious clone. The procedure could be adopted to target any upstream and downstream regions flanking known sequences within the plant genome.