Use of Multilocus Sequence Typing To Investigate the Association between the Presence of Campylobacter spp. in Broiler Drinking Water and Campylobacter Colonization in Broilers

The presence of campylobacters in broiler chickens and throughout the broiler water delivery systems of 12 farms in northeastern Scotland was investigated by sensitive enrichment methods and large-volume filtration. Campylobacter presence was independent of the water source and whether the water was treated. The genotypes of Campylobacter jejuni isolates recovered from chickens and various locations within the water delivery systems were compared by multilocus sequence typing. Matching strains in shed header tanks and birds were found at 1 of the 12 farms investigated. However, the sequence of contamination or whether the source was within or outside the shed was not determined. Nevertheless, these data provide evidence that drinking water could be associated with broiler infection by campylobacters.     

The Invasion of Barred Owls and its Potential Effect on the Spotted Owl: a Conservation Conundrum

The spotted owl (Strix occidentalis) is a threatened species in many areas of its western North American range. Concomitant with its decline has been a rapid invasion of its range and habitat by barred owls (Strix varia), a native species that was restricted, until relatively recently, to eastern North America. We assess the theoretical potential for negative interactions between these two owls by examining size dimorphism and ecological relationships within various owl assemblages throughout the world. We then review the anecdotal, natural history, modeling, and experimental evidence that suggest barred owls may negatively affect spotted owls with at least a potential for the competitive exclusion of spotted owls by barred owls throughout all or part of the former’2019;s range. While it is widely accepted that barred owls are either causing or exacerbating declines of spotted owl populations, there are confounding factors, such as habitat loss and bad weather that also may contribute to declines of spotted owls. Both theory and empirical information suggest that barred owls are likely to have negative effects on spotted owl range and density, but the degree of the impact is not predictable. There is a conservation conundrum here, in that the barred owl is a native species that has expanded its range westwards, either naturally or with a degree of human facilitation, and now constitutes a major threat to the viability of another native species, the threatened spotted owl. We propose that only through carefully designed experiments involving removal of barred owls will we be able to determine if recent declines in spotted owl populations are caused by barred owls or by other factors. It is rare in conservation science that replicate study areas exist for which we also have long-standing demographic information, as is the case with the spotted owl. Removal experiments would take advantage of the wealth of data on spotted owls, and allow ecologists to assess formally the impacts of an invasive species on a threatened species, as well as to suggest mitigation measures.     

Effects of helicopter net-gunning on survival of bighorn sheep

Wildlife capture, and the data collection associated with it, has led to major advancements in ecology that are integral to decision making pertaining to wildlife conservation. Capturing wildlife, however, can cause lethal and non-lethal risks to animals. Understanding the factors that contribute to the level of risk involved in wildlife capture is therefore important for the development and implementation of the safest and most effective methodologies. We used data from 736 animal captures of 389 individuals for 2 subspecies of female bighorn sheep (Rocky Mountain bighorn [Ovis canadensis canadensis], Sierra Nevada bighorn sheep [O. c. sierrae]) in Wyoming and California, USA, in 2002–2020 to evaluate the degree and extent of time that capture via helicopter net-gunning affects survival. We compared pre- and post-capture survival during a 10-week window centered on a capture event, and post-capture survival between captured animals and animals that were monitored but not captured during the 10-week window. Additionally, we evaluated the effects of handling techniques (number of times captured, season of capture event, handling time, chase time, and body temp) and biological factors (age and nutritional condition) on probability of capture mortality. Mean daily survival was 0.9992 during a 5-week pre-capture window, dropped to 0.9864 on the day of capture, and rebounded within 3 days of capture to pre-capture levels and that of sheep that were not captured. Overall, direct mortality resulting from capture was 1.36%, with 0.54% mortality occurring within the 3 days following a capture event for an overall 1.90% capture-related mortality. The only handling and biological metrics that influenced the probability of capture mortality were rectal temperature and nutritional condition; high initial rectal temperatures and poor body condition were associated with increased risk of mortality in the days following capture. Overall, helicopter net-gunning imposed low and short-term risk to survival of female bighorn sheep. To reduce bias in survival estimates, we recommend using a 3-day censorship window for post-capture mortalities as opposed to the common practice of a 2–5-week censor window. Helicopter net-gunning, including annual or seasonal recaptures, remains an effective and comparatively safe technique for capture and associated data collection of bighorn sheep.     

Positional Information,Positional Error,and Readout Precision in Morphogenesis: A Mathematical Framework

The concept of positional information is central to our understanding of how cells determine their location in a multicellular structure and thereby their developmental fates. Nevertheless, positional information has neither been defined mathematically nor quantified in a principled way. Here we provide an information-theoretic definition in the context of developmental gene expression patterns and examine the features of expression patterns that affect positional information quantitatively. We connect positional information with the concept of positional error and develop tools to directly measure information and error from experimental data. We illustrate our framework for the case of gap gene expression patterns in the early Drosophila embryo and show how information that is distributed among only four genes is sufficient to determine developmental fates with nearly single-cell resolution. Our approach can be generalized to a variety of different model systems; procedures and examples are discussed in detail.     

Novel anti-bacterial activities of β-defensin 1 in human platelets: suppression of pathogen growth and signaling of neutrophil extracellular trap formation

Human β-defensins (hBD) are antimicrobial peptides that curb microbial activity. Although hBD's are primarily expressed by epithelial cells, we show that human platelets express hBD-1 that has both predicted and novel antibacterial activities. We observed that activated platelets surround Staphylococcus aureus (S. aureus), forcing the pathogens into clusters that have a reduced growth rate compared to S. aureus alone. Given the microbicidal activity of β-defensins, we determined whether hBD family members were present in platelets and found mRNA and protein for hBD-1. We also established that hBD-1 protein resided in extragranular cytoplasmic compartments of platelets. Consistent with this localization pattern, agonists that elicit granular secretion by platelets did not readily induce hBD-1 release. Nevertheless, platelets released hBD-1 when they were stimulated by α-toxin, a S. aureus product that permeabilizes target cells. Platelet-derived hBD-1 significantly impaired the growth of clinical strains of S. aureus. hBD-1 also induced robust neutrophil extracellular trap (NET) formation by target polymorphonuclear leukocytes (PMNs), which is a novel antimicrobial function of β-defensins that was not previously identified. Taken together, these data demonstrate that hBD-1 is a previously-unrecognized component of platelets that displays classic antimicrobial activity and, in addition, signals PMNs to extrude DNA lattices that capture and kill bacteria.     

A simplified method for the generation of human osteoclasts in vitro

Osteoclasts are large, multinucleated cells responsible for the resorption of mineralized bone matrix. These cells are critical players in the bone turnover involved in bone homeostasis. Osteoclast activity is connected to the establishment and expansion of skeletal metastases from a number of primary neoplasms. Thus, the formation and activation of osteoclasts is an area of research with many potential avenues for clinical translation. Past studies of osteoclast biology have utilized primary murine cells cultured in vitro. Recently, techniques have been described that involve the generation of osteoclasts from human precursor cells. However, these protocols are often time-consuming and insufficient for generating large numbers of osteoclasts. We therefore developed a simplified protocol by which human osteoclasts may be easily and reliably generated in large numbers in vitro. In this study, osteoclasts were differentiated from bone marrow cells that had been aliquotted and frozen. Cells were generated by culture with recombinant macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL). Both human and murine RANKL were shown to efficiently generate osteoclasts, although higher concentrations of murine RANKL were required. Formation of osteoclasts was demonstrated qualitatively by tartrate-resistant acid phosphatase (TRAP) staining. These cells were fully functional, as confirmed by their ability to form resorption pits on cortical bone slices. Functional human osteoclasts can be difficult to generate in vitro by current protocols. We have demonstrated a simplified system for the generation of human osteoclasts in vitro that allows for large numbers of osteoclasts to be obtained from a single donor.     

c-Type cytochrome-dependent formation of U(IV) nanoparticles by Shewanella oneidensis

Modern approaches for bioremediation of radionuclide contaminated environments are based on the ability of microorganisms to effectively catalyze changes in the oxidation states of metals that in turn influence their solubility. Although microbial metal reduction has been identified as an effective means for immobilizing highly-soluble uranium(VI) complexes in situ, the biomolecular mechanisms of U(VI) reduction are not well understood. Here, we show that c-type cytochromes of a dissimilatory metal-reducing bacterium, Shewanella oneidensis MR-1, are essential for the reduction of U(VI) and formation of extracellular UO(2) nanoparticles. In particular, the outer membrane (OM) decaheme cytochrome MtrC (metal reduction), previously implicated in Mn(IV) and Fe(III) reduction, directly transferred electrons to U(VI). Additionally, deletions of mtrC and/or omcA significantly affected the in vivo U(VI) reduction rate relative to wild-type MR-1. Similar to the wild-type, the mutants accumulated UO(2) nanoparticles extracellularly to high densities in association with an extracellular polymeric substance (EPS). In wild-type cells, this UO(2)-EPS matrix exhibited glycocalyx-like properties and contained multiple elements of the OM, polysaccharide, and heme-containing proteins. Using a novel combination of methods including synchrotron-based X-ray fluorescence microscopy and high-resolution immune-electron microscopy, we demonstrate a close association of the extracellular UO(2) nanoparticles with MtrC and OmcA (outer membrane cytochrome). This is the first study to our knowledge to directly localize the OM-associated cytochromes with EPS, which contains biogenic UO(2) nanoparticles. In the environment, such association of UO(2) nanoparticles with biopolymers may exert a strong influence on subsequent behavior including susceptibility to oxidation by O(2) or transport in soils and sediments.     

New insights into DHFR interactions: analysis of Pneumocystis carinii and mouse DHFR complexes with NADPH and two highly potent 5-(omega-carboxy(alkyloxy) trimethoprim derivatives reveals conformational correlations with activity and novel parallel ring stacking interactions

Structural data are reported for two highly potent antifolates, 2,4-diamino-5-[3',4'-dimethoxy-5'-(5-carboxy-1-pentynyl)]benzylpyrimidine (PY1011), with 5000-fold selectivity for Pneumocystis carinii dihydrofolate reductase (pcDHFR), relative to rat liver DHFR, and 2,4-diamino-5-[2-methoxy-5-(4-carboxybutyloxy)benzyl]pyrimidine (PY957), that has 80-fold selectivity for pcDHFR. Crystal structures are reported for NADPH ternary complexes with PY957 and pcDHFR, refined to 2.2 A resolution; with PY1011 and pcDHFR, refined to 2.0 A resolution; and with PY1011 and mouse DHFR (mDHFR), refined to 2.2 A resolution. These results reveal that the carboxylate of the omega-carboxyalkyloxy side chain of these inhibitors form ionic interactions with the conserved Arg in the substrate binding pocket of DHFR. These data suggest that the enhanced inhibitory activity of PY1011 compared with PY957 is, in part, due to the favorable contacts with Phe69 of pcDHFR by the methylene carbons of the inhibitor side chain that are oriented by the triple bond of the 1-pentynyl side chain. These contacts are not present in the PY957 pcDHFR complex, or in the PY1011 mDHFR complex. In the structure of mDHFR the site of Phe69 in pcDHFR is occupied by Asn64. These data also revealed a preference for an unusual parallel ring stacking interaction between Tyr35 of the active site helix and Phe199 of the C-terminal beta sheet in pcDHFR and by Tyr33 and Phe179 in mDHFR that is independent of bound ligand. A unique His174-His187 parallel ring stacking interaction was also observed only in the structure of pcDHFR. These ring stacking interactions are rarely found in any other protein families and may serve to enhance protein stability.