Conjugation of folate via gelonin carbohydrate residues retains ribosomal-inactivating properties of the toxin and permits targeting to folate receptor positive cells

Conjugation of folate to proteins permits receptor-mediated endocytosis via the folate receptor (FR) and delivery of the conjugate into the cytoplasm of cells. Since many cancers up-regulate the FR it has enabled the targeting of toxins to tumor cells resulting in specific cell death. However, current conjugation methods rely on chemistries that can affect certain catalytic subunits, such as the A-chain of the plant toxin gelonin. As a result many folate-targeted toxins are a compromise between receptor/ligand interaction and toxin activity. We describe the first example of folate conjugated to a protein via carbohydrate residues, using a novel SH-folate intermediate. The folate-gelonin conjugate retains over 99% of toxin activity in a cell-free translational assay compared with unmodified gelonin and is able to bind the FR at the same affinity as free folic acid (10(-10) m). Additionally, the conjugate exhibits prolonged inhibition of protein synthesis in FR positive cell lines in vitro. Folate linked to gelonin via amino conjugation exhibits the same affinity for FR as free folic acid but the toxin is 225-fold less active in a cell-free translational assay. The effect of different conjugation methods on toxin activity and the implications for folate targeting of other glycoproteins are discussed.     

Estimating Bat and Bird Mortality Occurring at Wind Energy Turbines from Covariates and Carcass Searches Using Mixture Models

Environmental impacts of wind energy facilities increasingly cause concern, a central issue being bats and birds killed by rotor blades. Two approaches have been employed to assess collision rates: carcass searches and surveys of animals prone to collisions. Carcass searches can provide an estimate for the actual number of animals being killed but they offer little information on the relation between collision rates and, for example, weather parameters due to the time of death not being precisely known. In contrast, a density index of animals exposed to collision is sufficient to analyse the parameters influencing the collision rate. However, quantification of the collision rate from animal density indices (e.g. acoustic bat activity or bird migration traffic rates) remains difficult. We combine carcass search data with animal density indices in a mixture model to investigate collision rates. In a simulation study we show that the collision rates estimated by our model were at least as precise as conventional estimates based solely on carcass search data. Furthermore, if certain conditions are met, the model can be used to predict the collision rate from density indices alone, without data from carcass searches. This can reduce the time and effort required to estimate collision rates. We applied the model to bat carcass search data obtained at 30 wind turbines in 15 wind facilities in Germany. We used acoustic bat activity and wind speed as predictors for the collision rate. The model estimates correlated well with conventional estimators. Our model can be used to predict the average collision rate. It enables an analysis of the effect of parameters such as rotor diameter or turbine type on the collision rate. The model can also be used in turbine-specific curtailment algorithms that predict the collision rate and reduce this rate with a minimal loss of energy production.     

Localized Modeling of Biochemical and Flow Interactions during Cancer Cell Adhesion

This work focuses on one component of a larger research effort to develop a simulation tool to model populations of flowing cells. Specifically, in this study a local model of the biochemical interactions between circulating melanoma tumor cells (TC) and substrate adherent polymorphonuclear neutrophils (PMN) is developed. This model provides realistic three-dimensional distributions of bond formation and attendant attraction and repulsion forces that are consistent with the time dependent Computational Fluid Dynamics (CFD) framework of the full system model which accounts local pressure, shear and repulsion forces. The resulting full dynamics model enables exploration of TC adhesion to adherent PMNs, which is a known participating mechanism in melanoma cell metastasis. The model defines the adhesion molecules present on the TC and PMN cell surfaces, and calculates their interactions as the melanoma cell flows past the PMN. Biochemical rates of reactions between individual molecules are determined based on their local properties. The melanoma cell in the model expresses ICAM-1 molecules on its surface, and the PMN expresses the β-2 integrins LFA-1 and Mac-1. In this work the PMN is fixed to the substrate and is assumed fully rigid and of a prescribed shear-rate dependent shape obtained from micro-PIV experiments. The melanoma cell is transported with full six-degrees-of-freedom dynamics. Adhesion models, which represent the ability of molecules to bond and adhere the cells to each other, and repulsion models, which represent the various physical mechanisms of cellular repulsion, are incorporated with the CFD solver. All models are general enough to allow for future extensions, including arbitrary adhesion molecule types, and the ability to redefine the values of parameters to represent various cell types. The model presented in this study will be part of a clinical tool for development of personalized medical treatment programs.     

Donation of embryos for human development and stem cell research

Using donated human embryos for scientific research raises ethical questions about the donation process. We describe a two-stage consent process designed to help couples make informed decisions about embryo disposition. This consent methodology minimizes conflict of interest, respects patient choice, and provides a much-needed resource to patients and the research community.     

Novel Feature of Mycobacterium avium subsp. paratuberculosis,Highlighted by Characterization of the Heparin-Binding Hemagglutinin Adhesin

Mycobacterium avium subsp. paratuberculosis comprises two genotypically defined groups, known as the cattle (C) and sheep (S) groups. Recent studies have reported phenotypic differences between M. avium subsp. paratuberculosis groups C and S, including growth rates, infectivity for macrophages, and iron metabolism. In this study, we investigated the genotypes and biological properties of the virulence factor heparin-binding hemagglutinin adhesin (HBHA) for both groups. In Mycobacterium tuberculosis, HBHA is a major adhesin involved in mycobacterium-host interactions and extrapulmonary dissemination of infection. To investigate HBHA in M. avium subsp. paratuberculosis, we studied hbhA polymorphisms by fragment analysis using the GeneMapper technology across a large collection of isolates genotyped by mycobacterial interspersed repetitive-unit–variable-number tandem-repeat (MIRU-VNTR) and IS900 restriction fragment length polymorphism (RFLP-IS900) analyses. Furthermore, we analyzed the structure-function relationships of recombinant HBHA proteins of types C and S by heparin-Sepharose chromatography and surface plasmon resonance (SPR) analyses. In silico analysis revealed two forms of HBHA, corresponding to the prototype genomes for the C and S types of M. avium subsp. paratuberculosis. This observation was confirmed using GeneMapper on 85 M. avium subsp. paratuberculosis strains, including 67 strains of type C and 18 strains of type S. We found that HBHAs from all type C strains contain a short C-terminal domain, while those of type S present a long C-terminal domain, similar to that produced by Mycobacterium avium subsp. avium. The purification of recombinant HBHA from M. avium subsp. paratuberculosis of both types by heparin-Sepharose chromatography highlighted a correlation between their affinities for heparin and the lengths of their C-terminal domains, which was confirmed by SPR analysis. Thus, types C and S of M. avium subsp. paratuberculosis may be distinguished by the types of HBHA they produce, which differ in size and adherence properties, thereby providing new evidence that strengthens the genotypic differences between the C and S types of M. avium subsp. paratuberculosis.     

The Critical Role of DNA Extraction for Detection of Mycobacteria in Tissues

Background

Nucleic acid-based methods offer promise for both targeted and exploratory investigations of microbes in tissue samples. As the starting material for such studies is a mixture of host and microbial DNA, we have critically evaluated the DNA extraction step to determine the quantitative and qualitative parameters that permit faithful molecular detection of mycobacteria in infected tissue. Specifically, we assessed: 1) tissue disruption procedures; 2) DNA extraction protocols; and 3) inhibition of bacterial PCR by host DNA.

Principal Findings

Regarding DNA extraction, we found that 1) grinding was not necessary if bead-beating is done, 2) the reference mycobacterial DNA extraction method recovered more pure DNA than commercial spin column kits, 3) lysozyme digestion of 1 hour was sufficient, and 4) repeated steps of phenol:chloroform:isoamyl alcohol offered minimal gain in DNA quality. By artificially mixing mycobacterial DNA with DNA extracted from uninfected mice, we found that bacterial real-time quantitative PCR was only reliable when the quantity of host DNA was < 3 µg in a final volume of 25 µl and the quality was high (260/280 nm ratio = 1.89±0.08). Findings from spiked DNA studies were confirmed using DNA extracted from mice infected with different intracellular pathogens (M. tuberculosis, M. avium subsp. paratuberculosis).

Conclusions

Our findings point to the most appropriate methods for extracting DNA from tissue samples for the purpose of detecting and quantifying mycobacteria. These data also inform on the limits of detection for two mycobacterial species and indicate that increasing the sample mass to improve analytic sensitivity comes at the cost of inhibition of PCR by host DNA.     

Absence of Endochondral Ossification and Craniosynostosis in Posterior Frontal Cranial Sutures of Axin2?/? Mice

During the first month of life, the murine posterior-frontal suture (PF) of the cranial vault closes through endochondral ossification, while other sutures remain patent. These processes are tightly regulated by canonical Wnt signaling. Low levels of active canonical Wnt signaling enable endochondral ossification and therefore PF-suture closure, whereas constitutive activation of canonical Wnt causes PF-suture patency. We therefore sought to test this concept with a knockout mouse model. PF-sutures of Axin2^−/− mice, which resemble a state of constantly activated canonical Wnt signaling, were investigated during the physiological time course of PF-suture closure and compared in detail with wild type littermates. Histological analysis revealed that the architecture in Axin2^−/− PF-sutures was significantly altered in comparison to wild type. The distance between the endocranial layers was dramatically increased and suture closure was significantly delayed. Moreover, physiological endochondral ossification did not occur, rather an ectopic cartilage appeared between the endocranial and ectocranial bone layers at P7 which eventually involutes at P13. Quantitative PCR analysis showed the lack of Col10α1 upregulation in Axin2^−/− PF-suture. Immunohistochemistry and gene expression analysis also revealed high levels of type II collagen as compared to type I collagen and absence of Mmp-9 in the cartilage of Axin2^−/− PF-suture. Moreover, TUNEL staining showed a high percentage of apoptotic chondrocytes in Axin2^−/− PF-sutures at P9 and P11 as compared to wild type. These data indicated that Axin2^−/− PF-sutures lack physiological endochondral ossification, contain ectopic cartilage and display delayed suture closure.     

Standardization of computer-assisted semen analysis using an e-learning application

Computer-assisted semen analysis (CASA) is primarily used to obtain accurate and objective kinetic sperm measurements. Additionally, AI centers use computer-assessed sperm concentration in the sample as a basis for calculating the number of insemination doses available from a given ejaculate. The reliability of data is often limited and results can vary even when the same CASA systems with identical settings are used. The objective of the present study was to develop a computer-based training module for standardized measurements with a CASA system and to evaluate its training effect on the quality of the assessment of sperm motility and concentration. A digital versatile disc (DVD) has been produced showing the standardization of sample preparation and analysis with the CASA system SpermVision™ version 3.0 (Minitube, Verona, WI, USA) in words, pictures, and videos, as well as the most probable sources of error. Eight test persons educated in spermatology, but with different levels of experience with the CASA system, prepared and assessed 10 aliquots from one prediluted bull ejaculate using the same CASA system and laboratory equipment before and after electronic learning (e-learning). After using the e-learning application, the coefficient of variation was reduced on average for the sperm concentration from 26.1% to 11.3% (P ≤ 0.01), and for motility from 5.8% to 3.1% (P ≤ 0.05). For five test persons, the difference in the coefficient of variation before and after use of the e-learning application was significant (P ≤ 0.05). Individual deviations of means from the group mean before e-learning were reduced compared with individual deviations from the group mean after e-learning. According to a survey, the e-learning application was highly accepted by users. In conclusion, e-learning presents an effective, efficient, and accepted tool for improvement of the precision of CASA measurements. This study provides a model for the standardization of other laboratory procedures using e-learning.     

Structures of 5-methylthioribose kinase reveal substrate specificity and unusual mode of nucleotide binding

The methionine salvage pathway is ubiquitous in all organisms, but metabolic variations exist between bacteria and mammals. 5-Methylthioribose (MTR) kinase is a key enzyme in methionine salvage in bacteria and the absence of a mammalian homolog suggests that it is a good target for the design of novel antibiotics. The structures of the apo-form of Bacillus subtilis MTR kinase, as well as its ADP, ADP-PO(4), AMPPCP, and AMPPCP-MTR complexes have been determined. MTR kinase has a bilobal eukaryotic protein kinase fold but exhibits a number of unique features. The protein lacks the DFG motif typically found at the beginning of the activation loop and instead coordinates magnesium via a DXE motif (Asp(250)-Glu(252)). In addition, the glycine-rich loop of the protein, analogous to the "Gly triad" in protein kinases, does not interact extensively with the nucleotide. The MTR substrate-binding site consists of Asp(233) of the catalytic HGD motif, a novel twin arginine motif (Arg(340)/Arg(341)), and a semi-conserved W-loop, which appears to regulate MTR binding specificity. No lobe closure is observed for MTR kinase upon substrate binding. This is probably because the enzyme lacks the lobe closure/inducing interactions between the C-lobe of the protein and the ribosyl moiety of the nucleotide that are typically responsible for lobe closure in protein kinases. The current structures suggest that MTR kinase has a dissociative mechanism.