Temperature dependence of collagen fluorescence.

Dermal collagens have several fluorescent moieties in the UV and visible spectral regions that may serve as molecular probes of collagen. We studied the temperature-dependence of a commercial calf skin collagen and acid-extracted Skh-1 hairless mouse collagen at temperatures from 9 degrees C to 60 degrees C for excitation/emission wavelengths 270/305 nm (tyrosine), 270/360 nm (excimer-like aggregated species), 325/400 nm (dityrosine) and 370/450 nm (glycation adduct). L-tyrosine (1 x 10(-5) M in 0.5 M HOAc) acted as a "reference compound" devoid of any collagen structural effects. In general, the fluorescence efficiency of these fluorophores decreases with increasing temperature. Assuming that rate constant for fluorescence deactivation has the form k(d)(T) = k(d) degrees exp (-DeltaE/RT), an Arrhenius plot of log[(1/Phi) - 1] vs. 1/T affords a straight line whose (negative) slope is proportional to the activation energy, DeltaE, of the radiationless process(es) that compete with fluorescence. Because it is difficult to accurately measure Phi(f) for collagen-bound fluorophores, we derived an approximate formula for an activation parameter, DeltaE*, evaluated from an Arrhenius-like plot of log 1/I(N)vs. 1/T, (1/I(N)vs. is the reciprocal normalized fluorescence intensity). Tyrosine in dilute solution affords a linear Arrhenius plot in both of the above cases. Using the known value of Phi(f) = 0.21 for free tyrosine at room temperature, we determined that DeltaE* is accurate to approximately 25% in the present instance. Collagen curves are non-linear, but they are quasi-linear below approximately 20 degrees C, where the helical form predominates. Values of DeltaE* determined from the data at T < 20 degrees C ranged from 6.2-8.4 kJ mol(-1) (1.5-2.0 kcal mol(-1)) for mouse collagen and 10.3-11.4 kJ mol(-1) (2.5-2.7 kcal mol(-1)) for calf skin collagen, consistent with collisional deactivation of the fluorescent state via thermally enhanced molecular vibrations and rotations. Above 20 degrees C, log 1/I(N)vs. 1/T plots from Skh-1 hairless mouse collagen are concave-downward, suggesting that fluorescence deactivation from the denatured coil has a significant temperature-independent component. For calf skin collagen, these plots are concave-upward, suggesting an increase in activation energy above Tm. These results suggest that collagen backbone and supramolecular structure can influence the temperature dependence of the bound fluorophores, indicating the future possibility of using activation data as a probe of supramolecular structure and conformation.     

Effects of DNase production, plasmid size, and restriction barriers on transformation of Vibrio cholerae by electroporation and osmotic shock

Attempts to transform wild type strains of V. cholerae with plasmid DNA by traditional osmotic shock methods were not successful. A mutant of V. cholerae that was deficient in extracellular DNase was transformed with plasmid DNA by osmotic shock, demonstrating directly that extracellular DNase is a major barrier to transformation of V. cholerae. Transformation of wild type and DNase-negative strains of V. cholerae was accomplished by electroporation. Efficiency of transformation by electroporation increased with field strength, decreased with plasmid size, and was relatively insensitive to changes in the electrolyte composition of the buffer as long as isotonic sucrose was present. Host-controlled modification/restriction systems also affected transformation efficiency in V. cholerae.     

Efficient and error-free fluorescent gene tagging in human organoids without double-strand DNA cleavage

CRISPR-associated nucleases are powerful tools for precise genome editing of model systems, including human organoids. Current methods describing fluorescent gene tagging in organoids rely on the generation of DNA double-strand breaks (DSBs) to stimulate homology-directed repair (HDR) or non-homologous end joining (NHEJ)-mediated integration of the desired knock-in. A major downside associated with DSB-mediated genome editing is the required clonal selection and expansion of candidate organoids to verify the genomic integrity of the targeted locus and to confirm the absence of off-target indels. By contrast, concurrent nicking of the genomic locus and targeting vector, known as in-trans paired nicking (ITPN), stimulates efficient HDR-mediated genome editing to generate large knock-ins without introducing DSBs. Here, we show that ITPN allows for fast, highly efficient, and indel-free fluorescent gene tagging in human normal and cancer organoids. Highlighting the ease and efficiency of ITPN, we generate triple fluorescent knock-in organoids where 3 genomic loci were simultaneously modified in a single round of targeting. In addition, we generated model systems with allele-specific readouts by differentially modifying maternal and paternal alleles in one step. ITPN using our palette of targeting vectors, publicly available from Addgene, is ideally suited for generating error-free heterozygous knock-ins in human organoids.

A major downside of double-strand break-mediated genome editing is the need to verify the genomic integrity of the targeted locus and confirm the absence of off-target indels. This study shows that in-trans paired nicking is a mutation-free CRISPR strategy to introduce precise knock-ins into human organoids; its genomic fidelity allows all knock-in cells to be pooled, accelerating the establishment of new organoid models.     

Taxonomic and functional homogenization of an endemic desert fish fauna

Aim The highly endemic fishes of the arid Southwest USA have been heavily impacted by human activities resulting in one of the most threatened fish faunas in the world. The aim of this study was to examine the patterns and drivers of taxonomic and functional beta diversity of freshwater fish in the Lower Colorado River Basin across the 20th century. Location Lower Colorado River Basin (LCRB). Methods The taxonomic and functional similarities of watersheds were quantified to identify patterns of biotic homogenization or differentiation over the period 1900–1999. Path analysis was used to identify the relative influence of dam density, urban land use, precipitation regimes and non‐native species richness on observed changes in fish faunal composition. Results The fish fauna of the LCRB has become increasingly homogenized, both taxonomically (1.1% based on β_sim index) and functionally (6.2% based on Bray–Curtis index), over the 20th century. The rate of homogenization varied substantially; range declines of native species initially caused taxonomic differentiation (?7.9% in the 1960s), followed by marginal homogenization (observed in the 1990s) in response to an influx of non‐native species introductions. By contrast, functional homogenization of the basin was evident considerably earlier (in the 1950s) because of the widespread introduction of non‐native species sharing similar suites of biological traits. Path analysis revealed that both taxonomic and functional homogenization were positively related to the direct and indirect (facilitation by dams and urbanization) effects of non‐native species richness. Main conclusions Our study simultaneously examines rates of change in multiple dimensions of the homogenization process. For the endemic fish fauna of the LCRB, we found that the processes of taxonomic and functional homogenization are highly dynamic over time, varying both in terms of the magnitude and rate of change over the 20th century.     

Identification and characterization of the aspartate chemosensory receptor of Campylobacter jejuni

Campylobacter jejuni is a highly motile bacterium that responds via chemotaxis to environmental stimuli to migrate towards favourable conditions. Previous in silico analysis of the C. jejuni strain NCTC11168 genome sequence identified 10 open reading frames, tlp1‐10, that encode putative chemosensory receptors. We describe the characterization of the role and specificity of the Tlp1 chemoreceptor (Cj1506c). In vitro and in vivo models were used to determine if Tlp1 had a role in host colonization. The tlp1^‐ isogenic mutant was more adherent in cell culture, however, showed reduced colonization ability in chickens. Specific interactions between the purified sensory domain of Tlp1 and l ‐aspartate were identified using an amino acid array and saturation transfer difference nuclear magnetic resonance spectroscopy. Chemotaxis assays showed differences between migration of wild‐type C. jejuni cells and that of a tlp1^‐ isogenic mutant, specifically towards aspartate. Furthermore, using yeast two‐hybrid and three‐hybrid systems for analysis of protein–protein interactions, the cytoplasmic signalling domain of Tlp1 was found to preferentially interact with CheV, rather than the CheW homologue of the chemotaxis signalling pathway; this interaction was confirmed using immune precipitation assays. This is the first identification of an aspartate receptor in bacteria other than Escherichia coli and Salmonella enterica serovar Typhimurium.     

Oxysterol-binding protein (OSBP) enhances replication of intracellular Salmonella and binds the Salmonella SPI-2 effector SseL via its N-terminus

Effectors translocated into the host cell by Salmonella enterica serovar Typhimurium are critical for bacterial virulence. For many effectors, the mechanisms of their interactions with host pathways are not yet understood. We have recently found an interaction between the SPI-2 effector SseL and oxysterol-binding protein (OSBP). We show here that SseL binds the N-terminus of OSBP and that S. Typhimurium infection results in redistribution of OSBP. We furthermore demonstrate that OSBP is required for efficient replication of intracellular S. Typhimurium. This suggests that S. Typhimurium hijacks OSBP-dependent pathways to benefit its intracellular life-style, possibly by SseL- and OSBP-mediated manipulation of host lipid metabolism.     

Generalist predator's niche shifts reveal ecosystem changes in an experimentally fragmented landscape

Habitat fragmentation can alter the trophic structure of communities and environmental conditions, thus driving changes in biodiversity and ecosystem functions. Quantifying niches of generalist predators can reveal how fragmentation alters ecosystems. In a habitat fragmentation experiment, we used stable isotopes of a generalist predator skink to test predictions from spatial theory on trophic structure and to quantify abiotic changes associated with fragmentation among continuous forest, fragments, and matrix habitats. We predicted that in fragments and the matrix, isotopic niches would shift due to decreases in skink trophic positions (δ¹⁵N) from reductions in trophic structure of arthropod food webs and abiotic changes over time (δ¹³C) relative to continuous forest. Contrary to theoretical predictions, we did not find evidence of reductions in trophic structure with fragmentation. In fact, skink δ¹⁵N values were higher in the matrix and fragments than continuous forest, likely due to changes in distributions of a detritivorous prey species. In addition, δ¹³C values in the matrix decreased over years since fragmentation due to abiotic changes associated with matrix tree maturation. We show how isotopic niches are influenced by fragmentation via shifts in biotic and abiotic processes. The potential for either or both spatial and abiotic effects of fragmentation present a challenge for theory to better predict ecological changes in fragmented landscapes.     

Characterization of the soluble nanoparticles formed through Coulombic interaction of bovine serum albumin with anionic graft copolymers at low pH

A static light scattering (SLS) study of bovine serum albumin (BSA) mixtures with two anionic graft copolymers of poly(sodium acrylate-co-sodium 2-acrylamido-2-methyl-1-propanesulphonate)-graft-poly(N,N-dimethylacrylamide), with a high composition in poly(N,N-dimethylacrylamide) (PDMAM) side chains, revealed the formation of oppositely charged complexes, at pH lower than 4.9, the isoelectric point of BSA. The core-corona nanoparticles formed at pH = 3.00 were characterized. Their molecular weight and radius of gyration were determined by SLS, while their hydrodynamic radius was determined by dynamic light scattering. Small angle neutron scattering measurements were used to determine the radius of the insoluble complexes, comprising the core of the particles. The values obtained indicated that their size and aggregation number of the nanoparticles were smaller when the content of the graft copolymers in neutral PDMAM side chains was higher. Such particles should be interesting drug delivery candidates, if the gastrointestinal tract was to be used.     

Mapping protein-protein interactions by mass spectrometry

Mass spectrometry is currently at the forefront of technologies for mapping protein-protein interactions, as it is a highly sensitive technique that enables the rapid identification of proteins from a variety of biological samples. When used in combination with affinity purification and/or chemical cross-linking, whole or targeted protein interaction networks can be elucidated. Several methods have recently been introduced that display increased specificity and a reduced occurrence of false-positives. In the future, information gained from human protein interaction studies could lead to the discovery of novel pathway associations and therapeutic targets.