Engineering a high-affinity anti-IL-15 antibody: crystal structure reveals an α-helix in VH CDR3 as key component of paratope

Interleukin (IL) 15 is an inflammatory cytokine that plays an essential role in the activation, proliferation, and maintenance of specific natural killer cell and T-cell populations, and has been implicated as a mediator of inflammatory diseases. An anti-IL-15 antibody that blocked IL-15-dependent cellular responses was isolated by phage display and optimised via mutagenesis of the third complementarity-determining regions (CDRs) of variable heavy (VH) and variable light chains. Entire repertoires of improved variants were recombined with each other to explore the maximum potential sequence space. DISC0280, the most potent antibody isolated using this comprehensive strategy, exhibits a 228-fold increase in affinity and a striking 40,000-fold increase in cellular potency compared to its parent. Such a wholesale recombination strategy therefore represents a useful method for exploiting synergistic potency gains as part of future antibody engineering efforts. The crystal structure of DISC0280 Fab (fragment antigen binding), in complex with human IL-15, was determined in order to map the structural epitope and paratope. The most remarkable feature revealed lies within the paratope and is a novel six-amino-acid α-helix that sits within the VH CDR3 loop at the center of the antigen binding site. This is the first report to describe an α-helix as a principal component of a naturally derived VH CDR3 following affinity maturation.     

Two conformational states of Glu242 and pKas in bovine cytochrome c oxidase.

Cytochrome c oxidase (CcO) is the terminal enzyme in the respiratory electron transport chain of aerobic organisms. It catalyses the reduction of atmospheric oxygen to water, and couples this reaction to proton pumping across the membrane; this process generates the electrochemical gradient that subsequently drives the synthesis of ATP. The molecular details of the mechanism by which electron transfer is coupled to proton pumping in CcO is poorly understood. Recent calculations from our group indicate that His291, a ligand of the Cu(B) center of the enzyme, may play the role of the pumping element. In this paper we describe calculations in which a DFT/continuum electrostatic method is used to explore the coupling of the conformational changes of Glu242 residue, the main proton donor of both chemical and pump protons, to its pKa, and the pKa of His291, a putative proton loading site of our pumping model. The computations are done for several redox states of metal centers, different protonation states of Glu242 and His291, and two well-defined conformations of the Glu242 side chain. Thus, in addition to equilibrium redox/protonation states of the catalytic cycle, we also examine the transient and intermediate states. Different dielectric models are employed to investigate the robustness of the results, and their viability in the light of the proposed proton pumping mechanism of CcO. The main results are in agreement with the experimental measurements and support the proposed pumping mechanism. Additionally, the present calculations indicate a possibility of gating through conformational changes of Glu242; namely, in the pumping step, we find that Glu242 needs to be reprotonated before His291 can eject a proton to the P-site of membrane. As a result, the reprotonation of Glu can control proton release from the proton loading site.     

Dental caries in disabled children

The aim of the study was to evaluate oral health conditions and dental caries status in disabled and healthy children. Two groups of randomly selected children 3-17 years old were examined. The first group comprised 80 children with disabilities (cerebral palsy, mental retardation, Down syndrome, autism and hearing-speaking disorders) and the second (control) group included 80 healthy children. Examined children were selected from several institutions which take care of disabled persons, kindergardens and four elementary schools. Clinical examination was performed by using a mirror and a probe and revealed the presence of dental caries, missing (extracted) and filled teeth. All clinically detected cavitations were registered as dental caries. The degree of oral hygiene was evaluated according to the OHI-S index values, which was determined by marking the plaque with 1% eozine solution. The values of OHI-S index ranged from 3.8-4.53 in disabled children and 2.73-2.84 in healthy children. In disabled children, the average dft values were 3.42 in deciduous teeth and 5.24 in mixed dentition. In healthy children, the average dft values were 1.43 in deciduous teeth and 5.1 in mixed dentition. The average DMFT index in disabled children was 1.41 for mixed and 6.39 for permanent dentitions. In healthy children, the average DMFT values were 1.23 in mixed and 4.76 in permanent dentitions. In general, the results revealed significantly poor level of oral hygiene and quite high level of caries prevalence in both disabled and healthy children, accentuating the need to reorganize preventive care measurements and improve dental care, particularly in disabled children in Croatia.     

Non linear anthropometric predictors in swimming

In this paper we have tried to identify the significance and character of the linear and non-linear relations between simple anthropometric predictors: body height (BH), body weight (BW), and body mass index, and swimming performance: freestyle swimming 50 (FS50) and 400 meters (FS400), in a sample of young (15 years old on average) male (N = 40) and female (N = 28) swimmers. Linear (general model: y = a+bx) and nonlinear regression (general model: y = a+bx+cx2) were calculated simultaneously. Morphological variables are a significantly better predictor of the FS50 in males (BH mostly), and FS400 in females (BW mostly). This study emphasized some of the main advantages in the nonlinear regression calculation (including an interpretation of the relationships at a more superior level), and consequently allowed a precise anthropometric modeling in swimming using simple and easily measurable variables. For example, the best results in FS400 can be expected for the subjects that are average in BW (which guarantees solid muscle mass--the generator of force), but above average in BH (because of the physical law of lever). In conclusion, nonlinear regressions allow one to define the real nature of the relationships between variables, but only if compared with the linear ones. Additionally, this study emphasized one of the most important factors in defining possible specification-equation (e.g. structure of the influence of the different dimensions on the sport achievement) in different sports. In short, it underlines the importance of sampling the appropriate sample of the subject--highly skilled athletes exclusively.     

Haloarchaeal communities in the crystallizers of two adriatic solar salterns

Solar salterns operate only for short dry periods of the year in the north shore of the Adriatic Sea because of its relatively humid and cold Mediterranean climate. In a previous paper, we showed that the NaCl precipitation ponds (crystallizers) of Northern Adriatic Secovlje salterns have different haloarchaeal populations from those typically found in dry and hot climates such as Southern Spain. To check whether there is a common pattern of haloarchaeal diversity in these less extreme conditions, diversity in crystallizers of other Adriatic solar salterns in Ston, Croatia was ascertained by molecular and culture methods. In addition, the cultivation approach was used to further describe haloarchaeal diversity in both salterns. Over the period of two solar salt collection seasons, isolates related to species of the genera Haloferax, Haloarcula, and Haloterrigena were recovered from both salterns. Within the same sampling effort, relatives of the genus Halorubrum and a Natrinema-like isolate were cultivated from Slovenian Secovlje salterns while Halobacterium related isolates were obtained from the Croatian Ston salterns. Concurrent with our previous findings, a library of Croatian saltern crystallizer PCR-amplified 16S rRNA genes was dominated by sequences related to the genus Halorubrum. The microbial community structure was similar in both salterns but diversity indices showed greater values in Slovenian salterns when compared with Croatian salterns.     

High-Frequency Microdomain Ca2+ Transients and Waves during Early Myelin Internode Remodeling

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Application of the Bradford Assay for Cell Lysis Quantification: Residual Protein Content in Cell Culture Supernatants

Frequently measured mammalian cell culture process indicators include viability and total cell concentration (TCC). Cell lysis, an additional important process characteristic that substantially contributes to the overall product purity profiles, is often not addressed in detail. In the present study, an inexpensive and simple application of the Bradford assay is developed to determine the residual protein content (RPC) in cell culture supernatants. The reliability and reproducibility of the method are tested in a long‐term study and compared with lysis quantification via the DNA measurement. The results show that its performance is more robust and accurate over time and the respective concentration range. Additionally, both methods are used for cell lysis process monitoring in a recombinant Chinese hamster ovary fed‐batch process. In the presented process, by applying the established assay, the lysis rate k _DL is determined to be constant over time at 4.6 × 10 ^?4 lysed cell concentration (LCC) per TCC and time (LCC/TCC/h). In contrast, DNA data did not confirm the constant lysis rate due to variations of the content per cell during cultivation. Thus, information on the RPC can facilitate the determination of the optimal harvest time point with respect to purity and in improving process characterization.     

Exopolysaccharide Production and Ropy Phenotype Are Determined by Two Gene Clusters in Putative Probiotic Strain Lactobacillus paraplantarum BGCG11

Lactobacillus paraplantarum BGCG11, a putative probiotic strain isolated from a soft, white, artisanal cheese, produces a high-molecular-weight heteropolysaccharide, exopolysaccharide (EPS)-CG11, responsible for the ropy phenotype and immunomodulatory activity of the strain. In this study, a 26.4-kb region originating from the pCG1 plasmid, previously shown to be responsible for the production of EPS-CG11 and a ropy phenotype, was cloned, sequenced, and functionally characterized. In this region 16 putative open reading frames (ORFs), encoding enzymes for the production of EPS-CG11, were organized in specific loci involved in the biosynthesis of the repeat unit, polymerization, export, regulation, and chain length determination. Interestingly, downstream of the eps gene cluster, a putative transposase gene was identified, followed by an additional rfb gene cluster containing the rfbACBD genes, the ones most probably responsible for dTDP-l-rhamnose biosynthesis. The functional analysis showed that the production of the high-molecular-weight fraction of EPS-CG11 was absent in two knockout mutants, one in the eps and the other in the rfb gene cluster, as confirmed by size exclusion chromatography analysis. Therefore, both eps and rfb genes clusters are prerequisites for the production of high-molecular-weight EPS-CG11 and for the ropy phenotype of strain L. paraplantarum BGCG11.