Degradation of Tyrosine in Anaerobically Stored Piggery Wastes and in Pig Feces

Radioactively labeled compounds that might be intermediates in the anaerobic degradation of tyrosine were added to pig feces and to stored piggery wastes. Changes in the compounds were followed by using thin-layer and gas chromatography. In feces, p-cresol and 3-phenylpropionic acid were the end products of tyrosine metabolism; in anaerobically stored mixed wastes, phenol, p-cresol, and minor quantities of phenylpropionic acid were formed. Schemes were proposed for the degradation of tyrosine in pig feces and in mixed wastes.     

CRISPR-based DNA and RNA detection with liquid-liquid phase separation

The ability to detect specific nucleic acid sequences allows for a wide range of applications such as the identification of pathogens, clinical diagnostics, and genotyping. CRISPR-Cas proteins Cas12a and Cas13a are RNA-guided endonucleases that bind and cleave specific DNA and RNA sequences, respectively. After recognition of a target sequence, both enzymes activate indiscriminate nucleic acid cleavage, which has been exploited for sequence-specific molecular diagnostics of nucleic acids. Here, we present a label-free detection approach that uses a readout based on solution turbidity caused by liquid-liquid phase separation (LLPS). Our approach relies on the fact that the LLPS of oppositely charged polymers requires polymers to be longer than a critical length. This length dependence is predicted by the Voorn-Overbeek model, which we describe in detail and validate experimentally in mixtures of polynucleotides and polycations. We show that the turbidity resulting from LLPS can be used to detect the presence of specific nucleic acid sequences by employing the programmable CRISPR-nucleases Cas12a and Cas13a. Because LLPS of polynucleotides and polycations causes solutions to become turbid, the detection of specific nucleic acid sequences can be observed with the naked eye. We furthermore demonstrate that there is an optimal polynucleotide concentration for detection. Finally, we provide a theoretical prediction that hints towards possible improvements of an LLPS-based detection assay. The deployment of LLPS complements CRISPR-based molecular diagnostic applications and facilitates easy and low-cost nucleotide sequence detection.     

Structure of the Shigella dysenteriae haem transport locus and its phylogenetic distribution in enteric bacteria

The ability to transport and use haemin as an iron source is frequently observed in clinical isolates of Shigella spp. and pathogenic Escherichia coli . We found that many of these haem-utilizing E. coli strains contain a gene that hybridizes at high stringency to the S. dysenteriae type 1 haem receptor gene, shuA . These shuA -positive strains belong to multiple phylogenetic groups and include clinical isolates from enteric, urinary tract and systemic infections. The distribution of shuA in these strains suggests horizontal transfer of the haem transport locus. Some haem-utilizing pathogenic E. coli strains did not hybridize with shuA , so at least one other haem transport system is present in this group. We also characterized the chromosomal region containing shuA in S. dysenteriae . The shuA gene is present in a discrete locus, designated the haem transport locus, containing eight open reading frames. Several of the proteins encoded in this locus participate with ShuA in haem transport, as a Salmonella typhimurium strain containing the entire haem transport locus used haem much more efficiently than the same strain containing only shuA . The haem transport locus is not present in E. coli K-12 strains, but the sequences flanking the haem transport locus in S. dysenteriae matched those at the 78.7 minute region of E. coli K-12. The junctions and flanking sequences in the shuA -positive pathogenic E. coli strains tested were nearly identical to those in S. dysenteriae , indicating that, in these strains, the haem transport locus has an organization similar to that in S. dysenteriae , and it is located in the same relative position on the chromosome.     

Immunodetection and immunolocalization of globulin storage proteins during zygotic and somatic embryo development in Zea mays

Globulins (GLB) are storage proteins that accumulate to high levels during zygotic embryo development of Zea mays L. We visualized the distribution of GLB during zygotic embryo development by immunolabelling of polyethylene glycol sections with a GLB-specific antiserum and a fluorescent secondary antibody. In sections of embryos at 10 days after pollimation (DAP), GLB were detected in the scutellar node only. Sections of embryos of 17 DAP showed, besides the presence of GLB in the scutellar node, the presence of a low amount of GLB in the coleoptile and the leaf primordia. In 30-DAP embryos GLB were localized in the root, the coleorhiza, the leaf primordia, the coleoptile and in all cells of the scutellum with the exception of the epidermis and the pro-vascular tissues. The subcellular location of GLB was visualized by immunolabelling of ultrathin sections with anti-GLB and a gold-conjugated secondary antibody. Scutellum cells and root cortex cells of 30-DAP embryos were packed with protein storage vacuoles (PSV), which differed in electron density. GLB were either evenly distributed throughout the PSV or were localized in electron-dense inclusions within the PSV. SDS-PAGE and immunoblot analysis of total protein extracts indicated the presence of a low amount of the GLB1 processing intermediate proGLB1' in globular as well as mature somatic embryos. After maturation on an ABA-containing medium, somatic embryos showed the additional presence of the next GLB1 processing intermediate GLB1'. By immuno-electron microscopy it was possible to localize GLB in globular deposits in PSV in scutellum cells of these somatic embryos.     

Stressful colours: corticosterone concentrations in a free-living songbird vary with the spectral composition of experimental illumination

Organisms have evolved under natural daily light/dark cycles for millions of years. These cycles have been disturbed as night-time darkness is increasingly replaced by artificial illumination. Investigating the physiological consequences of free-living organisms in artificially lit environments is crucial to determine whether nocturnal lighting disrupts circadian rhythms, changes behaviour, reduces fitness and ultimately affects population numbers. We make use of a unique, large-scale network of replicated field sites which were experimentally illuminated at night using lampposts emanating either red, green, white or no light to test effect on stress hormone concentrations (corticosterone) in a songbird, the great tit (Parus major). Adults nesting in white-light transects had higher corticosterone concentrations than in the other treatments. We also found a significant interaction between distance to the closest lamppost and treatment type: individuals in red light had higher corticosterone levels when they nested closer to the lamppost than individuals nesting farther away, a decline not observed in the green or dark treatment. Individuals with high corticosterone levels had fewer fledglings, irrespective of treatment. These results show that artificial light can induce changes in individual hormonal phenotype. As these effects vary considerably with light spectrum, it opens the possibility to mitigate these effects by selecting street lighting of specific spectra.     

Effects of temperature on circadian clock and chronotype: an experimental study on a passerine bird

Daily schedules of many organisms, including birds, are thought to affect fitness. Timing in birds is based on circadian clocks that have a heritable period length, but fitness consequences for individuals in natural environments depend on the scheduling of entrained clocks. This chronotype, i.e., timing of an individual relative to a zeitgeber, results from interactions between the endogenous circadian clock and environmental factors, including light conditions and ambient temperature. To understand contributions of these factors to timing, we studied daily activity patterns of a captive songbird, the great tit (Parus major), under different temperature and light conditions. Birds were kept in a light (L)-dark (D) cycle (12.5?L:11.5 D) at either 8°C or 18°C with ad libitum access to food and water. We assessed chronotype and subsequently tested birds at the same temperature under constant dim light (LL(dim)) to determine period length of their circadian clock. Thermal conditions were then reversed so that period length was measured under both temperatures. We found that under constant dim light conditions individuals lengthened their free-running period at higher temperatures by 5.7?±?2.1?min (p?=?.002). Under LD, birds kept at 18°C started activity later and terminated it much earlier in the day than those kept under 8°C. Overall, chronotype was slightly earlier under higher temperature, and duration of activity was shorter. Furthermore, individuals timed their activities consistently on different days under LD and over the two test series under LL(dim) (repeatability from .38 to .60). Surprisingly, period length and chronotype did not show the correlation that had been previously found in other avian species. Our study shows that body clocks of birds are precise and repeatable, but are, nonetheless, affected by ambient temperature. (Author correspondence: marina.lehmann@uni-konstanz.de ).     

Deciphering the oxygen isotope composition of nitrous oxide produced by nitrification

The ability to use δ¹⁸O values of nitrous oxide (N₂O) to apportion environmental emissions is currently hindered by a poor understanding of the controls on δ¹⁸O–N₂O from nitrification (hydroxylamine oxidation to N₂O and nitrite reduction to N₂O). In this study fertilized agricultural soils and unfertilized temperate forest soils were aerobically incubated with different ¹⁸O/¹⁶O waters, and conceptual and mathematical models were developed to systematically explain the δ¹⁸O–N₂O formed by nitrification. Modeling exercises used a set of defined input parameters to emulate the measured soil δ¹⁸O–N₂O data (Monte Carlo approach). The Monte Carlo simulations implied that abiotic oxygen (O) exchange between nitrite (NO₂^?) and H₂O is important in all soils, but that biological, enzyme‐controlled O‐exchange does not occur during the reduction of NO₂^? to N₂O (nitrifier‐denitrification). Similarly, the results of the model simulations indicated that N₂O consumption is not characteristic of aerobic N₂O formation. The results of this study and a synthesis of the published literature data indicate that δ¹⁸O–N₂O formed in aerobic environments is constrained between +13‰ and +35‰ relative to Vienna Standard Mean Ocean Water (VSMOW). N₂O formed via hydroxylamine oxidation and nitrifier‐denitrification cannot be separated using δ¹⁸O unless ¹⁸O tracers are employed. The natural range of nitrifier δ¹⁸O–N₂O is discussed and explained in terms of our conceptual model, and the major and minor controls that define aerobically produced δ¹⁸O–N₂O are identified. Despite the highly complex nature of δ¹⁸O–N₂O produced by nitrification this δ¹⁸O range is narrow. As a result, in many situations δ¹⁸O values may be used in conjunction with δ¹⁵N–N₂O data to apportion nitrifier‐ and denitrifier‐derived N₂O. However, when biological O‐exchange during denitrification is high and N₂O consumption is low, there may be too much overlap in δ¹⁸O values to distinguish N₂O formed by these pathways.     

Tracing the Sources of Exported Nitrate in the Turkey Lakes Watershed Using 15N/14N and 18O/16O isotopic ratios

Nitrate produced by bacterially mediated nitrification in soils is isotopically distinct from atmospheric nitrate in precipitation. ¹⁵N/¹⁴N and ¹⁸O/¹⁶O isotopic ratios of nitrate can therefore be used to distinguish between these two sources of nitrate in surface waters and groundwaters. Two forested catchments in the Turkey Lakes Watershed (TLW) near Sault Ste. Marie, Ontario, Canada were studied to determine the relative contributions of atmospheric and microbial nitrate to nitrate export. The TLW is reasonably undisturbed and receives a moderate amount of inorganic nitrogen bulk deposition (8.7 kg N · ha⁻¹· yr⁻¹) yet it exhibits unusually low inorganic nitrogen retention (average = 65% of deposition). The measured isotopic ratios for nitrate in precipitation ranged from +35 to +59‰ (VSMOW) for δ¹⁸O and −4 to +0.8‰ (AIR) for δ¹⁵N. Nitrate produced from nitrification at the TLW is expected to have an average isotope value of approximately −1.0‰ for δ¹⁸O and a value of about 0 to +6‰ for δ¹⁵N, thus, the isotopic separation between atmospheric and soil sources of nitrate is substantial. Nitrate produced by nitrification of ammonium appears to be the dominant source of the nitrate exported in both catchments, even during the snowmelt period. These whole catchment results are consistent with the results of small but intensive plot scale studies that have shown that the majority of the nitrate leached from these catchments is microbial in origin. The isotopic composition of stream nitrate provides information about N-cycling in the forested upland and riparian zones on a whole catchment basis. Received 5 October 1999; accepted 18 August 2000     

Biodistribution and Clearance of Intra-articular Liposomes in a Large Animal Model Using a Radiographic Marker

The intra-articular (IA) route of administration in treating arthritis has potential for targeting drug delivery to affected tissues, thereby minimising the attendant side-effects of systemically administered drugs. The ultra-structure of the synovium however facilitates rapid drug efflux from the joint; effectively the IA route is equivalent to other non-IV parenteral routes with regards absorption and redistribution into the systemic circulation. The aim of this study was to extend the drug residence time within the knee joint by using a liposome formulation. DPPC-based liposomes were prepared with the radio contrast agent iohexol as a drug marker. 8 sheep had their right knees injected IA with iohexol liposomes and the contralateral joints with either free iohexol or empty liposomes. Joints were radiographed at multiple time points up to 16 days post-injection. Iohexol-mediated radiopacity was quantified by densitometer. Sheep were sacrificed at the end of the study for microscopy of synovial tissues.

Good visualization of iohexol-mediated radiopacity with fine anatomical definition was possible throughout the experiment. Also evident on the films was extra-articular radiopacity with liposomes tracking along muscle facial planes. Cellular and tissue localization with light microscopy was possible through use of frozen sections and because of the large liposome size.

Residence of encapsulated iohexol within the knee joint was greatly prolonged.

Liposomal iohexol declined bi-exponentially with a terminal elimination half-life of 134 hours. In contrast, free iohexol was undetectable @ 3 hours post-injection.