Gene expression involved in the sexual development of Best Beluga hybrid sturgeons.

The scope of this study is to investigate the expression of dmrt1, foxl2, ar, star and sox9 genes in the context of the gonad development stage of 21 month-old Best Beluga individuals. No significant difference was observed between males and females but the cyp17a1 gene showed higher expression in male than in female gonads. The results suggest that during sampling the females were in perinucleolar stage and the males in early spermatogenesis stage which led to specific patterns of expression for the investigated genes.

Large-Scale Biophysical Evaluation of Protein PEGylation Effects: In Vitro Properties of 61 Protein Entities.

PEGylation is the most widely used method to chemically modify protein biopharmaceuticals, but surprisingly limited public data is available on the biophysical effects of protein PEGylation. Here we report the first large-scale study, with site-specific mono-PEGylation of 15 different proteins and characterization of 61 entities in total using a common set of analytical methods. Predictions of molecular size were typically accurate in comparison with actual size determined by size-exclusion chromatography (SEC) or dynamic light scattering (DLS). In contrast, there was no universal trend regarding the effect of PEGylation on the thermal stability of a protein based on data generated by circular dichroism (CD), differential scanning calorimetry (DSC), or differential scanning fluorimetry (DSF). In addition, DSF was validated as a fast and inexpensive screening method for thermal unfolding studies of PEGylated proteins. Multivariate data analysis revealed clear trends in biophysical properties upon PEGylation for a subset of proteins, although no universal trends were found. Taken together, these findings are important in the consideration of biophysical methods and evaluation of second-generation biopharmaceutical drug candidates.

Assessment of genetic diversity in main local sheep breeds from Romania using microsatellite markers.

The state of the local breeds of farm animals is increasingly precarious worldwide because of the aggressive introduction of breeds with improved economical traits. The preference of the breeders for local breeds is due to their higher adaptability to the particular climate and relief conditions of the mountain areas, to the high rate of assimilation of the feeds from these regions and to their increased resistance to diseases. This study analyzes the genetic variation of the main four local Romanian sheep breeds (Tsurcana, Tsigai, Ratska and Teleorman Blackhead) in terms of stock and economic importance, using 18 microsatellite markers. The mean number of alleles per locus was of 9.764. The values of genetic diversity parameters exhibited a high degree of polymorphism for the analyzed breeds, although inbreeding was highlighted particularly in Tsurcana and Tsigai. These breeds also showed an intense gene flow among them and were less differentiated in comparison with Ratska and Teleorman Blackhead. The results of this study may be useful for breeding programs and conservation plans since the genetic resources of the local breeds must be preserved so as to maintain an adequate level of biodiversity in animal husbandry.

Engineering hemoglobin to enable homogenous PEGylation without modifying protein functionality.

In order to infuse hemoglobin into the vasculature as an oxygen therapeutic or blood substitute, it is necessary to increase the size of the molecule to enhance vascular retention. This aim can be achieved by PEGylation. However, using non-specific conjugation methods creates heterogenous mixtures and alters protein function. Site-specific PEGylation at the naturally reactive thiol on human hemoglobin (ßCys93) alters hemoglobin oxygen binding affinity and increases its autooxidation rate. In order to avoid this issue, new reactive thiol residues were therefore engineered at sites distant to the heme group and the α/ß dimer/dimer interface. The two mutants were ßCys93Ala/αAla19Cys and ßCys93Ala/ßAla13Cys. Gel electrophoresis, size exclusion chromatography and mass spectrometry revealed efficient PEGylation at both αAla19Cys and ßAla13Cys, with over 80% of the thiols PEGylated in the case of αAla19Cys. For both mutants there was no significant effect on the oxygen affinity or the cooperativity of oxygen binding. PEGylation at αAla19Cys had the additional benefit of decreasing the rates of autoxidation and heme release, properties that have been considered contributory factors to the adverse clinical side effects exhibited by previous hemoglobin based oxygen carriers. PEGylation at αAla19Cys may therefore be a useful component of future clinical products.

Effects of starvation and refeeding on growth performance and stress defense mechanisms of stellate sturgeon Acipenser stellatus juveniles from aquaculture.

Acipenser stellatus represents a species of great economical interest due to its roe used for caviar production. Therefore, it has been intensively captured for decades and nowadays, this species is on the verge of extinction. As a consequence, Acipenser stellatus is intensively raised in fish farms. Aquaculture is focused on optimizing the feeding regime of juveniles. The aim of this study was to investigate if Acipenser stellatus can adapt to a starvation/refeeding regime by assessing the effects of this regime on growth performance, oxidative stress biomarkers and heat shock protein (hsp) gene expression in juveniles raised under aquaculture conditions. The juveniles were subjected to two starvation/refeeding regimes: a 7-day starvation period followed by 21 days of refeeding, and a14-day starvation period followed by 21 days of refeeding. The results had shown that the juveniles subjected to 7/21-day starvation/refeeding regime presented a complete compensatory growth, they were able to counteract the oxidative stress by enhancing activities of the antioxidant enzymes and they presented no significant changes in hsp gene expression. In contrast, 14/21-day starvation/refeeding regime negatively influenced growth performance, it induced a high level of oxidative stress that was impossible to counteract and it determined major changes in the hsp gene expression level in the liver of Acipenser stellatus. Thus, Acipenser stellatus seems to be able to adapt only to the 7/21-day starvation/refeeding regime that does not threaten the growth performance and the welfare of juveniles. Therefore, it could be useful to optimize the feeding practice in aquaculture production.

Expression Characterization of Six Genes Possibly Involved in Gonad Development for Stellate Sturgeon Individuals (Acipenser stellatus, Pallas 1771).

Nowadays, in sturgeon's aquaculture, there is a necessity for sex identification at early stages in order to increase the efficiency of this commercial activity. The basis for a correct identification is studying the different factors that influence the gonad development. The research has been directed towards molecular methods that have been employed with various degrees of success in identifying genes with different expression patterns between male and female sturgeons during their development stages. For the purpose of understanding the sexual development of 4-year-old stellate sturgeon (Acipenser stellatus) individuals, we have selected six genes (foxl2, cyp17a1, ar, dmrt1, sox9, and star). We analysed the gene expression of the selected genes for gonads, anal fin, liver, body kidney, and white muscle. The cyp17a1, ar, dmrt1, and sox9 genes have a significant higher expression in male gonads than in female gonads, while the data shows no significant differences in the expression of the investigated genes in the other organs. We investigate these genes to shed light on aquaculture sturgeon sexual development.

Colon-available raspberry polyphenols exhibit anti-cancer effects on in vitro models of colon cancer.

BACKGROUND: There is a probable association between consumption of fruit and vegetables and reduced risk of cancer, particularly cancer of the digestive tract. This anti-cancer activity has been attributed in part to anti-oxidants present in these foods. Raspberries in particular are a rich source of the anti-oxidant compounds, such as polyphenols, anthocyanins and ellagitannins. METHODS: A "colon-available" raspberry extract (CARE) was prepared that contained phytochemicals surviving a digestion procedure that mimicked the physiochemical conditions of the upper gastrointestinal tract. The polyphenolic-rich extract was assessed for anti-cancer properties in a series of in vitro systems that model important stages of colon carcinogenesis, initiation, promotion and invasion. RESULTS: The phytochemical composition of CARE was monitored using liquid chromatography mass spectrometry. The colon-available raspberry extract was reduced in anthocyanins and ellagitannins compared to the original raspberry juice but enriched in other polyphenols and polyphenol breakdown products that were more stable to gastrointestinal digestion. Initiation--CARE caused significant protective effects against DNA damage induced by hydrogen peroxide in HT29 colon cancer cells measured using single cell microgelelectrophoresis. Promotion--CARE significantly decreased the population of HT29 cells in the G1 phase of the cell cycle, effectively reducing the number of cells entering the cell cycle. However, CARE had no effect on epithelial integrity (barrier function) assessed by recording the trans-epithelial resistance (TER) of CACO-2 cell monolayers. Invasion--CARE caused significant inhibition of HT115 colon cancer cell invasion using the matrigel invasion assay. CONCLUSION: The results indicate that raspberry phytochemicals likely to reach the colon are capable of inhibiting several important stages in colon carcinogenesis in vitro.

Modulation of drug-linker design to enhance in vivo potency of homogeneous antibody-drug conjugates.

Antibody-drug conjugates (ADCs) are a promising class of anticancer agents which have undergone substantial development over the past decade and are now achieving clinical success. The development of novel site-specific conjugation technologies enables the systematic study of architectural features within the antibody conjugated drug linker that may affect overall therapeutic indices. Here we describe the results of a systematic study investigating the impact of drug-linker design on the in vivo properties of a series of homogeneous ADCs with a conserved site of conjugation, a monodisperse drug loading, a lysosomal release functionality and monomethyl auristatin E as a cytotoxic payload. The ADCs, which differed only in the relative position of certain drug-linker elements within the reagent, were first evaluated in vitro using anti-proliferation assays and in vivo using mouse pharmacokinetics (PK). Regardless of the position of a discrete polymer unit, the ADCs showed comparable in vitro potencies, but the in vivo PK properties varied widely. The best performing drug-linker design was further used to prepare ADCs with different drug loadings of 4, 6 and 8 drugs per antibody and compared to Adcetris® in a Karpas-299 mouse xenograft model. The most efficacious ADC showed complete tumor regression and 10/10 tumor free survivors at a single 0.5mg/kg dose. This study revealed drug-linker design as a critical parameter in ADC development, with the potential to enhance ADC in vivo potency for producing more efficacious ADCs.

Site-selective three-component reaction for dual-functionalization of peptides.

A site-selective dual-functionalization of peptides is presented, involving readily available maleimides as well as N-hydroxylamines. The modification proceeds through a three component 1,3-dipolar cycloaddition, forming a stable product. This was exemplified by the one-pot attachment of two molecular imaging moieties to a tumor binding cyclic peptide, and was extended to the conjugation of a DOTA chelator to a 12 kDa protein.

Synthesis of hybrid transition-metalloproteins via thiol-selective covalent anchoring of Rh-phosphine and Ru-phenanthroline complexes.

The preparation of hybrid transition metalloproteins by thiol-selective incorporation of organometallic rhodium- and ruthenium complexes is described. Phosphine ligands and two rhodium-diphosphine complexes bearing a carboxylic acid group were coupled to the cysteine of PYP R52G, yielding a metalloenzyme active in the rhodium catalyzed hydrogenation of dimethyl itaconate. The successful coupling was shown by (31)P NMR spectroscopy and ESI mass spectroscopy. In addition wild-type PYP (PYP WT), PYP R52G and ALBP were successfully modified with a (eta(6)-arene) ruthenium(II) phenanthroline complex via a maleimide linker.