Minimally Invasive Establishment of Murine Orthotopic Bladder Xenografts

Orthotopic bladder cancer xenografts are the gold standard to study molecular cellular manipulations and new therapeutic agents in vivo. Suitable cell lines are inoculated either by intravesical instillation (model of nonmuscle invasive growth) or intramural injection into the bladder wall (model of invasive growth). Both procedures are complex and highly time-consuming. Additionally, the superficial model has its shortcomings due to the lack of cell lines that are tumorigenic following instillation. Intramural injection, on the other hand, is marred by the invasiveness of the procedure and the associated morbidity for the host mouse.With these shortcomings in mind, we modified previous methods to develop a minimally invasive approach for creating orthotopic bladder cancer xenografts. Using ultrasound guidance we have successfully performed percutaneous inoculation of the bladder cancer cell lines UM-UC1, UM-UC3 and UM-UC13 into 50 athymic nude. We have been able to demonstrate that this approach is time efficient, precise and safe. With this technique, initially a space is created under the bladder mucosa with PBS, and tumor cells are then injected into this space in a second step. Tumor growth is monitored at regular intervals with bioluminescence imaging and ultrasound. The average tumor volumes increased steadily in in all but one of our 50 mice over the study period.In our institution, this novel approach, which allows bladder cancer xenograft inoculation in a minimally-invasive, rapid and highly precise way, has replaced the traditional model.     

Evaluation and optimisation of unnatural amino acid incorporation and bioorthogonal bioconjugation for site-specific fluorescent labelling of proteins expressed in mammalian cells

Many biophysical techniques that are available to study the structure, function and dynamics of cellular constituents require modification of the target molecules. Site-specific labelling of a protein is of particular interest for fluorescence-based single-molecule measurements including single-molecule FRET or super-resolution microscopy. The labelling procedure should be highly specific but minimally invasive to preserve sensitive biomolecules. The modern molecular engineering toolkit provides elegant solutions to achieve the site-specific modification of a protein of interest often necessitating the incorporation of an unnatural amino acid to introduce a unique reactive moiety. The Amber suppression strategy allows the site-specific incorporation of unnatural amino acids into a protein of interest. Recently, this approach has been transferred to the mammalian expression system. Here, we demonstrate how the combination of unnatural amino acid incorporation paired with current bioorthogonal labelling strategies allow the site-specific engineering of fluorescent dyes into proteins produced in the cellular environment of a human cell. We describe in detail which parameters are important to ensure efficient incorporation of unnatural amino acids into a target protein in human expression systems. We furthermore outline purification and bioorthogonal labelling strategies that allow fast protein preparation and labelling of the modified protein. This way, the complete eukaryotic proteome becomes available for single-molecule fluorescence assays.     

Lipid peroxidation and biochemical parameters in maternal pre-delivery and post-delivery plasma

Lipid peroxidation (LPX) can play an important role in development of functional and pathological changes of maternal tissues in the course of pregnancy and delivery. LPX products were measured as thiobarbituric acid reacting substances (TBARS), using malondialdehyde as the standard solution. Actual TBARS determined in maternal post-delivery plasma (2.71 ± 0.602 nmol/mL) were not statistically different from those determined in pre-delivery plasma (3.45 ± 0.530 nmol/mL). TBARS production was measured in vitro in the both incubated plasma (30 min, 37°C) with and without the added LPX activator (125 μM L-ascorbate plus 5 μM FeSO₄). A difference in the TBARS formation was found only in the post-delivery plasma, as a result of approximately twice higher (marginally significant) TBARS formation in the incubated plasma without the added LPX activator comparing with the actual TBARS levels in this plasma. These results suggest that changes in maternal tissues in the process of labour could create suitable conditions for activation of LPX in maternal plasma. On the other hand, all other analysed biochemical parameters (iron, total iron-binding capacity, uric acid, proteins, magnesium, calcium, phosphate, glucose, potassium, sodium, chlorides, alanine aminotransferase, aspartate aminotransferase, γ-glutamyltransferase, creatine kinase, lactate dehydrogenase, α-hydroxybutyrate dehydrogenase, α-amylase, alkaline phosphatase, acid phosphatase in the post-delivery plasma were not different from those analysed in the pre-delivery plasma.     

Autophagy differentially controls plant basal immunity to biotrophic and necrotrophic pathogens

In plants, autophagy has been assigned 'pro-death' and 'pro-survival' roles in controlling programmed cell death associated with microbial effector-triggered immunity. The role of autophagy in basal immunity to virulent pathogens has not been addressed systematically, however. Using several autophagy-deficient (atg) genotypes, we determined the function of autophagy in basal plant immunity. Arabidopsis mutants lacking ATG5, ATG10 and ATG18a develop spreading necrosis upon infection with the necrotrophic fungal pathogen, Alternaria brassicicola, which is accompanied by the production of reactive oxygen intermediates and by enhanced hyphal growth. Likewise, treatment with the fungal toxin fumonisin B1 causes spreading lesion formation in atg mutant genotypes. We suggest that autophagy constitutes a 'pro-survival' mechanism that controls the containment of host tissue-destructive microbial infections. In contrast, atg plants do not show spreading necrosis, but exhibit marked resistance against the virulent biotrophic phytopathogen, Pseudomonas syringae pv. tomato. Inducible defenses associated with basal plant immunity, such as callose production or mitogen-activated protein kinase activation, were unaltered in atg genotypes. However, phytohormone analysis revealed that salicylic acid (SA) levels in non-infected and bacteria-infected atg plants were slightly higher than those in Col-0 plants, and were accompanied by elevated SA-dependent gene expression and camalexin production. This suggests that previously undetected moderate infection-induced rises in SA result in measurably enhanced bacterial resistance, and that autophagy negatively controls SA-dependent defenses and basal immunity to bacterial infection. We infer that the way in which autophagy contributes to plant immunity to different pathogens is mechanistically diverse, and thus resembles the complex role of this process in animal innate immunity.     

Small population size and extremely low levels of genetic diversity in island populations of the platypus, Ornithorhynchus anatinus

Genetic diversity generally underpins population resilience and persistence. Reductions in population size and absence of gene flow can lead to reductions in genetic diversity, reproductive fitness, and a limited ability to adapt to environmental change increasing the risk of extinction. Island populations are typically small and isolated, and as a result, inbreeding and reduced genetic diversity elevate their extinction risk. Two island populations of the platypus, Ornithorhynchus anatinus, exist; a naturally occurring population on King Island in Bass Strait and a recently introduced population on Kangaroo Island off the coast of South Australia. Here we assessed the genetic diversity within these two island populations and contrasted these patterns with genetic diversity estimates in areas from which the populations are likely to have been founded. On Kangaroo Island, we also modeled live capture data to determine estimates of population size. Levels of genetic diversity in King Island platypuses are perilously low, with eight of 13 microsatellite loci fixed, likely reflecting their small population size and prolonged isolation. Estimates of heterozygosity detected by microsatellites (H(E)= 0.032) are among the lowest level of genetic diversity recorded by this method in a naturally outbreeding vertebrate population. In contrast, estimates of genetic diversity on Kangaroo Island are somewhat higher. However, estimates of small population size and the limited founders combined with genetic isolation are likely to lead to further losses of genetic diversity through time for the Kangaroo Island platypus population. Implications for the future of these and similarly isolated or genetically depauperate populations are discussed.     

A sulfurtransferase is essential for activity of formate dehydrogenases in Escherichia coli

l-Cysteine desulfurases provide sulfur to several metabolic pathways in the form of persulfides on specific cysteine residues of an acceptor protein for the eventual incorporation of sulfur into an end product. IscS is one of the three Escherichia coli l-cysteine desulfurases. It interacts with FdhD, a protein essential for the activity of formate dehydrogenases (FDHs), which are iron/molybdenum/selenium-containing enzymes. Here, we address the role played by this interaction in the activity of FDH-H (FdhF) in E. coli. The interaction of IscS with FdhD results in a sulfur transfer between IscS and FdhD in the form of persulfides. Substitution of the strictly conserved residue Cys-121 of FdhD impairs both sulfur transfer from IscS to FdhD and FdhF activity. Furthermore, inactive FdhF produced in the absence of FdhD contains both metal centers, albeit the molybdenum cofactor is at a reduced level. Finally, FdhF activity is sulfur-dependent, as it shows reversible sensitivity to cyanide treatment. Conclusively, FdhD is a sulfurtransferase between IscS and FdhF and is thereby essential to yield FDH activity.