Human-Induced Range Expansions Result in a Recent Hybrid Zone between Sister Species of Ducks.

Landscapes are consistently under pressure from human-induced ecological change, often resulting in shifting species distributions. For some species, changing the geographical breadth of their niche space results in matching range shifts to regions other than those in which they are formally found. In this study, we employ a population genomics approach to assess potential conservation issues arising from purported range expansions into the south Texas Brush Country of two sister species of ducks: mottled (Anas fulvigula) and Mexican (Anas diazi) ducks. Specifically, despite being non-migratory, both species are increasingly being recorded outside their formal ranges, with the northeastward and westward expansions of Mexican and mottled ducks, respectively, perhaps resulting in secondary contact today. We assessed genetic ancestry using thousands of autosomal loci across the ranges of both species, as well as sampled Mexican- and mottled-like ducks from across overlapping regions of south Texas. First, we confirm that both species are indeed expanding their ranges, with genetically pure Western Gulf Coast mottled ducks confirmed as far west as La Salle county, Texas, while Mexican ducks recorded across Texas counties near the USA-Mexico border. Importantly, the first confirmed Mexican × mottled duck hybrids were found in between these regions, which likely represents a recently established contact zone that is, on average, ~100 km wide. We posit that climate- and land use-associated changes, including coastal habitat degradation coupled with increases in artificial habitats in the interior regions of Texas, are facilitating these range expansions. Consequently, continued monitoring of this recent contact event can serve to understand species' responses in the Anthropocene, but it can also be used to revise operational survey areas for mottled ducks.

Flood-irrigated agriculture mediates climate-induced wetland scarcity for summering sandhill cranes in western North America.

Information about species distributions is lacking in many regions of the world, forcing resource managers to answer complex ecological questions with incomplete data. Information gaps are compounded by climate change, driving ecological bottlenecks that can act as new demographic constraints on fauna. Here, we construct greater sandhill crane (Antigone canadensis tabida) summering range in western North America using movement data from 120 GPS-tagged individuals to determine how landscape composition shaped their distributions. Landscape variables developed from remotely sensed data were combined with bird locations to model distribution probabilities. Additionally, land-use and ownership were summarized within summer range as a measure of general bird use. Wetland variables identified as important predictors of bird distributions were evaluated in a post hoc analysis to measure long-term (1984-2022) effects of climate-driven surface water drying. Wetlands and associated agricultural practices accounted for 1.2% of summer range but were key predictors of occurrence. Bird distributions were structured by riparian floodplains that concentrated wetlands, and flood-irrigated agriculture in otherwise arid and semi-arid landscapes. Findings highlighted the role of private lands in greater sandhill crane ecology as they accounted for 78% of predicted distributions. Wetland drying observed in portions of the range from 1984 to 2022 represented an emerging ecological bottleneck that could limit future greater sandhill crane summer range. Study outcomes provide novel insight into the significance of ecosystem services provided by flood-irrigated agriculture that supported nearly 60% of wetland resources used by birds. Findings suggest greater sandhill cranes function as a surrogate species for agroecology and climate change adaptation strategies seeking to reduce agricultural water use through improved efficiency while also maintaining distinct flood-irrigation practices supporting greater sandhill cranes and other wetland-dependent wildlife. We make our wetland and sandhill crane summering distributions available as interactive web-based mapping tools to inform conservation design.

Analysis of SARS-CoV-2 Variant-Specific Serum Antibody Post-Vaccination Utilizing Immortalized Human Hepatocyte-Like Cells (HLC) to Assess Development of Immunity.

Background: Our previous studies demonstrated that SARS-CoV-2 spike protein could bind to primary hepatocytes and immortalized Hepatocyte-like cells (HLC) via the asialoglycoprotein receptor-1 (ASGR-1). The binding of biotinylated spike protein could be inhibited by Spike-neutralizing monoclonal antibodies, anti-ASGR-1 antibodies and unlabeled spike protein. The cells were unable to bind Spike S1 and Spike S1 was incapable of blocking labeled Spike protein, suggesting that the Receptor Binding Domain (RBD) was not involved in the binding event. This study was done to investigate the utility of these cells and immortalized alveolar type 2-like (AT-2) cells in studying the development of variant-specific antibodies post-vaccination. Methods: Serum was collected from 10 individuals pre- and post-vaccination with the J&J, Moderna or Pfizer vaccines. The serum samples were quantified for variant-specific antibodies in a flow cytometry-based immunofluorescent assay utilizing beads coated with biotinylated variant spike proteins. Inhibition of spike protein binding to HLC and AT-2 cells by donor serum was analyzed by immunofluorescent confocal analysis. Results: All variant spike proteins bound to HLC and AT-2 cells. Post-vaccination serum samples demonstrated increases of SARS-CoV-2 antibody levels from 2 weeks to 2.5 months post-vaccination with associated increased spike-blocking capacity. It was also demonstrated that vaccination with all the available vaccines stimulated antibodies that inhibited binding of all the available variant spike proteins to both HLC and AT-2 cells. Conclusion: HLC, along with AT-2 cells, provides a useful platform to study the development of neutralizing antibodies post-vaccination. Vaccination with the 3 available vaccines all elicited neutralizing serum antibodies that inhibited binding of each of the variant spike proteins to both AT-2 and HLC cells. This study suggests that inhibition of spike binding to target cells may be a more useful technique to assess immunity than gross quantitation of antibody.

Joint use of location and acceleration data reveals influences on transitions among habitats in wintering birds.

Quantifying relationships between animal behavior and habitat use is essential to understanding animal decision-making. High-resolution location and acceleration data allows unprecedented insights into animal movement and behavior. These data types allow researchers to study the complex linkages between behavioral plasticity and habitat distribution. We used a novel Markov model in a Bayesian framework to quantify the influence of behavioral state frequencies and environmental variables on transitions among landcover types through joint use of location and tri-axial accelerometer data. Data were collected from 56 greater white-fronted geese (Anser albifrons frontalis) across seven ecologically distinct winter regions over two years in midcontinent North America. We showed that goose decision-making varied across landcover types, ecoregions, and abiotic conditions, and was influenced by behavior. We found that time spent in specific behaviors explained variation in the probability of transitioning among habitats, revealing unique behavioral responses from geese among different habitats. Combining GPS and acceleration data allowed unique study of potential influences of an ongoing large-scale range shift in the wintering distribution of a migratory bird across midcontinent North America. We anticipate that behavioral adaptations among variable landscapes is a likely mechanism explaining goose use of highly variable ecosystems during winter in ways which optimize their persistence.

Caught in the act: Monitoring OO bond cleavage in Acylperoxoferric cytochrome P450cam to form compound I in real time.

While monitoring the reaction of ferric cytochrome P450cam (Cyp101) with substituted peroxybenzoic acids using rapid-scanning, stopped-flow (RSSF) spectroscopy, an intermediate appears en route to formation of the high-valent moiety known as Compound I [Fe(IV)=O/porphyrin radical cation] that is thought to be the key catalytic species for O-atom transfer to substrate. We have previously suggested (Spolitak, T., Dawson, J.H., Ballou, D.P., J. Biol. Chem.2005, 280, 20,300-20,309) that this species is an acylperoxo-ferric heme adduct that subsequently undergoes OO bond cleavage to generate Compound I. Singular value decomposition analysis of the RSSF data for formation of this intermediate shows that the energy of its Soret absorption peak is sensitive to the electron donor properties of the aryl substituents on the peracid. A linear Hammett correlation plot is seen for the energy of the Soret absorption peak vs. the Hammett σ constant. This correlation requires that the aryl substituents remain as part of the ligand bound to the heme iron, providing direct evidence that the adduct is indeed a ferric acylperoxo derivative. Linear Hammett correlation plots are also seen for both the rate of formation of the intermediate as well as for its conversion to Compound I. It is proposed that the electron donating/withdrawing properties of the aryl-bound substituents affect the electrophilic nature for binding substrate, changing the observed rate of formation for the acylperoxo intermediate, as well as the propensity and stability of the substituted benzoic acid to serve as the leaving group during OO bond cleavage yielding Compound I.

Differentiation of immortalized human multi-lineage progenitor to alveolar type 2-like cells: angiotensin-converting enzyme 2 expression and binding of severe acute respiratory syndrome coronavirus 2 spike and spike 1 proteins.

Along with the nasal epithelium, the lung epithelium is a portal of entry for sudden acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and many other respiratory viruses. In the case of SARS-CoV-2, the virus surface spike proteins bind to the angiotensin-converting enzyme 2 (ACE-2) receptor to facilitate entry into the respiratory epithelium. Alveolar type 2 (AT2) cells are committed respiratory progenitor cells responsible for the integrity and regeneration of the respiratory epithelium and production of respiratory surfactant proteins. AT2 cells express high levels of surface ACE-2 and thus are a leading target for primary infection by SARS-CoV-2. This study describes a method for directly differentiating telomerase reverse transcriptase-immortalized human cord blood-derived multi-lineage progenitor cells (MLPCs) to AT2-like cells for the purpose of generating an in vitro cellular platform for viral studies. Differentiation was confirmed with the acquisition of AT2 and absence of alveolar type 1 (AT1) specific markers by confocal microscopy. Expression of the ACE-2 receptor was confirmed by immunofluorescence antibody staining, quantitative reverse transcription polymerase chain reaction and binding of biotinylated SARS-CoV-2 spike and spike 1 proteins. The binding of biotinylated spike proteins was specifically blocked by unlabeled spike proteins and neutralizing antibodies. Additionally, it was demonstrated that the spike protein was internalized after binding to the surface membrane of the cells. The authors defined the culture conditions that enabled AT2-like cells to be repeatedly passaged and cryopreserved without further differentiation to AT1. The authors' method provides a stable and renewable source of AT2 cells for respiratory viral binding, blocking and uptake studies.

Binding of the SARS-CoV-2 Spike Protein to the Asialoglycoprotein Receptor on Human Primary Hepatocytes and Immortalized Hepatocyte-Like Cells by Confocal Analysis.

BACKGROUND: The SARS-CoV-2 virus may have direct or indirect effects on other human organs beyond the respiratory system and including the liver, via binding of the spike protein. This study investigated the potential direct interactions with the liver by comparing the binding of SARS-CoV-2 spike proteins to human AT2-like cells, primary human hepatocytes and immortalized hepatocyte-like hybrid cells. Receptors with binding specificity for SARS-CoV-2 spike protein on AT2 cells and hepatocytes were identified. METHODS: The specific binding of biotinylated spike and spike 1 proteins to undifferentiated human E12 MLPC (E12), E12 differentiated alveolar type 2 (AT2) cells, primary human hepatocytes (PHH) and E12 human hepatocyte-like hybrid cells (HLC) was studied by confocal microscopy. We investigated the expression of ACE-2, binding of biotinylated spike protein, biotinylated spike 1 and inhibition of binding by unlabeled spike protein, two neutralizing antibodies and an antibody directed against the hepatocyte asialoglycoprotein receptor 1 (ASGr1). RESULTS: E12 MLPC did not express ACE-2 and did not bind either of spike or spike 1 proteins. AT2-like cells expressed ACE-2 and bound both spike and spike 1. Both PHH and HLC did not express ACE-2 and did not bind spike 1 protein. However, both PHH and HLC actively bound the spike protein. Biotinylated spike protein binding was inhibited by unlabeled spike but not spike 1 protein on PHH and HLC. Two commercial neutralizing antibodies blocked the binding of the spike to PHH and HLC but only one blocked binding to AT2. An antibody to the hepatocyte ASGr1 blocked the binding of the spike protein to PHH and HLC. CONCLUSION: The absence of ACE-2 receptors and inhibition of spike binding by an antibody to the ASGr1 on both PHH and HLC suggested that the spike protein interacts with the ASGr1. The differential antibody blocking of spike binding to AT2, PHH and HLC indicated that neutralizing activity of SARS-CoV-2 binding might involve additional mechanisms beyond RBD binding to ACE-2.

CRISPR/Cas9-Based Lateral Flow and Fluorescence Diagnostics.

Clustered regularly interspaced short palindromic repeat (CRISPR/Cas) proteins can be designed to bind specified DNA and RNA sequences and hold great promise for the accurate detection of nucleic acids for diagnostics. We integrated commercially available reagents into a CRISPR/Cas9-based lateral flow assay that can detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) sequences with single-base specificity. This approach requires minimal equipment and represents a simplified platform for field-based deployment. We also developed a rapid, multiplex fluorescence CRISPR/Cas9 nuclease cleavage assay capable of detecting and differentiating SARS-CoV-2, influenza A and B, and respiratory syncytial virus in a single reaction. Our findings provide proof-of-principle for CRISPR/Cas9 point-of-care diagnosis as well as a scalable fluorescent platform for identifying respiratory viral pathogens with overlapping symptomology.

Winter fidelity, movements, and energy expenditure of Midcontinent Greater White-fronted Geese.

BACKGROUND: Animal movement patterns are the result of both environmental and physiological effects, and the rates of movement and energy expenditure of given movement strategies are influenced by the physical environment an animal inhabits. Greater white-fronted geese in North America winter in ecologically distinct regions and have undergone a large-scale shift in wintering distribution over the past 20 years. White-fronts continue to winter in historical wintering areas in addition to contemporary areas, but the rates of movement among regions, and energetic consequences of those decisions, are unknown. Additionally, linkages between wintering and breeding regions are generally unknown, and may influence within-winter movement rates. METHODS: We used Global Positioning System and acceleration data from 97 white-fronts during two winters to elucidate movement characteristics, model regional transition probabilities using a multistate model in a Bayesian framework, estimate regional energy expenditure, and determine behavior time-allocation influences on energy expenditure using overall dynamic body acceleration and linear mixed-effects models. We assess the linkages between wintering and breeding regions by evaluating the winter distributions for each breeding region. RESULTS: White-fronts exhibited greater daily movement early in the winter period, and decreased movements as winter progressed. Transition probabilities were greatest towards contemporary winter regions and away from historical wintering regions. Energy expenditure was up to 55% greater, and white-fronts spent more time feeding and flying, in contemporary wintering regions compared to historical regions. White-fronts subsequently summered across their entire previously known breeding distribution, indicating substantial mixing of individuals of varying breeding provenance during winter. CONCLUSIONS: White-fronts revealed extreme plasticity in their wintering strategy, including high immigration probability to contemporary wintering regions, high emigration from historical wintering regions, and high regional fidelity to western regions, but frequent movements among eastern regions. Given that movements of white-fronts trended toward contemporary wintering regions, we anticipate that a wintering distribution shift eastward will continue. Unexpectedly, greater energy expenditure in contemporary wintering regions revealed variable energetic consequences of choice in wintering region and shifting distribution. Because geese spent more time feeding in contemporary regions than historical regions, increased energy expenditure is likely balanced by increased energy acquisition in contemporary wintering areas.

In vitro Differentiation of Human TERT-Transfected Multi-Lineage Progenitor Cells (MLPC) into Immortalized Hepatocyte-Like Cells.

BACKGROUND: Research directed towards drug development, metabolism, and liver functions often utilize primary hepatocytes (PH) for preliminary in vitro studies. Variability in the in vitro functionality of PH and the unsuitability of hepatocarcinoma cells for these studies have driven researchers to look to ESC, iPS, and other stem cell types using differentiation protocols to provide more reliable and available cells. This study describes the development of hepatocyte-like cells through the in vitro differentiation of human TERT-immortalized cord blood-derived multi-lineage progenitor cells (MLPC). The E12 clonal cell line derived from polyclonal TERT-transfected cells was used throughout the study. METHODS: E12 MLPC were subjected to a three-step differentiation protocol using alternating combinations of growth factors, cytokines, and maturational factors. Cells at various stages of differentiation were analyzed for consistency with PH by morphology, immunohistochemistry, urea production, and gene expression. RESULTS: E12 MLPC were shown to significantly change morphology with each stage of differentiation. Coincidental with the morphological changes in the cells, immunohistochemistry data documented the differentiation to committed endoderm by the expression of SOX-17 and GATA-4; the progression to committed hepatocyte-like cells by the expression of a large number of markers including α-fetoprotein and albumin; and the final differentiation by the expression of nuclear and cytoplasmic HNF4. Fully differentiated cells demonstrated gene expression, urea production, and immunohistochemistry consistent with PH. A methodology and medium formulation to continuously expand the E12-derived hepatocyte-like cells is described. CONCLUSION: The availability of immortalized hepatocyte-like cell lines could provide a consistent tool for the study of hepatic diseases, drug discovery, and the development of cellular therapies for liver disorders. Utilization of these techniques could provide a basis for the development of bridge therapies for liver failure patients awaiting transplant.

Development of immortalized human hepatocyte-like hybrid cells by fusion of multi-lineage progenitor cells with primary hepatocytes.

Human primary hepatocytes (PHs) are critical to studying liver functions, drug metabolism and toxicity. PHs isolated from livers that are unacceptable for transplantation have limited expansion and culture viability in vitro, in addition to rapidly deteriorating enzymatic functions. The unsuitability of immortalized hepato-carcinoma cell lines for this function has prompted studies to develop hepatocyte-like cells from alternative sources like ESC, iPS, and other stem cell types using differentiation protocols. This study describes a novel technique to produce expandable and functional hepatocyte-like cells from the fusion of an immortalized human umbilical cord blood derived cell line (E12 MLPC) to normal human primary hepatocytes. Multi-lineage progenitor cells (MLPC) comprise a small subset of mesenchymal-like cells isolated from human umbilical cord blood. MLPC are distinguishable from other mesenchymal-like cells by their extended expansion capacity (up to 80 cell doublings before senescence) and the ability to be differentiated into cells representative of endo-, meso- and ectodermal origins. Transfection of MLPC with the gene for telomerase reverse transcriptase (TERT) resulted in clonal cell lines that were capable of differentiation to different cellular outcomes while maintaining their functional immortality. A methodology for the development of immortalized hepatocyte-like hybrid cells by the in vitro fusion of human MLPC with normal human primary hepatocytes is reported. The resultant hybrid cells exhibited homology with hepatocytes by morphology, immunohistochemistry, urea and albumin production and gene expression. A medium that allows stable long-term expansion of hepatocyte-like fusion cells is described.

Hepatic Differentiation of Marmoset Embryonic Stem Cells and Functional Characterization of ESC-Derived Hepatocyte-Like Cells.

BACKGROUND: Primary human hepatocytes (PHHs) are the ideal candidates for studying critical liver functions such as drug metabolism and toxicity. However, as they are isolated from discarded livers that are unsuitable for transplantation, they possess limited expansion ability in vitro and their enzymatic functions deteriorate rapidly because they are often of poor quality. Therefore, there is a compelling reason to find reliable alternative sources of hepatocytes. METHODS: In this study, we report on efficient and robust differentiation of embryonic stem cells (ESC) from the common marmoset Callithrix jacchus into functional hepatocyte-like cells (HLC) using a simple, and reproducible three-step procedure. ESC-derived HLCs were examined by morphological analysis and tested for their expression of hepatocyte-specific markers using a combination of immunohistochemistry, RT-PCR, and biochemical assays. Primary human hepatocytes were used as controls. RESULTS: ESC-derived HLCs expressed each of the hepatocyte-specific markers tested, including albumin; α-fetoprotein; asialoglycoprotein receptor 1; α-1 antitrypsin; hepatocyte nuclear factors 1α and 4; cytokeratin 18; hepatocyte growth factor receptor; transferrin; tyrosine aminotransferase; alkaline phosphatase; c-reactive protein; cytochrome P450 enzymes CYP1A2, CYP2E1 and CYP3A4; and coagulation factors FVII and FIX. They were functionally competent as demonstrated by biochemical assays in addition to producing urea. CONCLUSION: Our data strongly suggest that marmoset HLCs possess characteristics similar to those of PHHs. They could, therefore, be invaluable for studies on drug metabolism and cell transplantation therapy for a variety of liver disorders. Because of the similarities in the anatomical and physiological features of the common marmoset to that of humans, Callithrix jacchus is an appropriate animal model to study human disease conditions and cellular functions.

Climate and human water use diminish wetland networks supporting continental waterbird migration.

Migrating waterbirds moving between upper and lower latitudinal breeding and wintering grounds rely on a limited network of endorheic lakes and wetlands when crossing arid continental interiors. Recent drying of global endorheic water stores raises concerns over deteriorating migratory pathways, yet few studies have considered these effects at the scale of continental flyways. Here, we investigate the resiliency of waterbird migration networks across western North America by reconstructing long-term patterns (1984-2018) of terminal lake and wetland surface water area in 26 endorheic watersheds. Findings were partitioned regionally by snowmelt- and monsoon-driven hydrologies and combined with climate and human water-use data to determine their importance in predicting surface water trends. Nonlinear patterns of lake and wetland drying were apparent along latitudinal flyway gradients. Pervasive surface water declines were prevalent in northern snowmelt watersheds (lakes -27%, wetlands -47%) while largely stable in monsoonal watersheds to the south (lakes -13%, wetlands +8%). Monsoonal watersheds represented a smaller proportion of total lake and wetland area, but their distribution and frequency of change within highly arid regions of the continental flyway increased their value to migratory waterbirds. Irrigated agriculture and increasing evaporative demands were the most important drivers of surface water declines. Underlying agricultural and wetland relationships however were more complex. Approximately 7% of irrigated lands linked to flood irrigation and water storage practices supported 61% of all wetland inundation in snowmelt watersheds. In monsoonal watersheds, small earthen dams, meant to capture surface runoff for livestock watering, were a major component of wetland resources (67%) that supported networks of isolated wetlands surrounding endorheic lakes. Ecological trends and human impacts identified herein underscore the importance of assessing flyway-scale change as our model depictions likely reflect new and emerging bottlenecks to continental migration.

Land Use, anthropogenic disturbance, and riverine features drive patterns of habitat selection by a wintering waterbird in a semi-arid environment.

River ecosystems in semi-arid environments provide an array of resources that concentrate biodiversity, but also attract human settlement and support economic development. In the southwestern United States, land-use change, drought, and anthropogenic disturbance are compounding factors which have led to departures from historical conditions of river ecosystems, consequently affecting wildlife habitat, including important wintering areas for migratory birds. The Rio Grande (River) in central New Mexico is the lifeblood of the Middle Rio Grande Valley (MRGV), maintaining large urban and agricultural centers and riparian and wetland resources, which disproportionately support a diversity of wildlife. The MRGV has been identified as the most important wintering area for the Rocky Mountain Population of greater sandhill cranes (Antigone canadensis tabida). Presently, however, changes in the hydrogeomorphology of the Rio Grande and landscape modification by humans have reshaped the MRGV and winter habitat for sandhill cranes. To evaluate these impacts, we investigated how land-use practices, anthropogenic disturbance, and river morphology influenced patterns of diurnal and roosting habitat selection by sandhill cranes. During the diurnal period, sandhill cranes relied heavily on managed public lands selecting agriculture crops, such as corn fields, and wetlands for foraging and loafing while avoiding areas with increasing densities of human structures. Sandhill cranes selected areas for roosting in the Rio Grande characterized by shallower water interspersed with sandbars, wide channel width, low bank vegetation, and farther away from disturbances associated with bridges. Our results establish and identify the central processes driving patterns of diel habitat selection by wintering sandhill cranes. Land use and riverine trends have likely gradually reduced winter habitat to managed public lands and limited reaches of the Rio Grande, underscoring the importance of natural resources agencies in supporting migratory birds and challenges involved when managing for wildlife in highly pressured semi-arid environments.

Heme Binding by Corynebacterium diphtheriae HmuT: Function and Heme Environment.

The heme uptake pathway (hmu) of Corynebacterium diphtheriae utilizes multiple proteins to bind and transport heme into the cell. One of these proteins, HmuT, delivers heme to the ABC transporter HmuUV. In this study, the axial ligation of the heme in ferric HmuT is probed by examination of wild-type (WT) HmuT and a series of conserved heme pocket residue mutants, H136A, Y235A, and M292A. Characterization by UV-visible, resonance Raman, and magnetic circular dichroism spectroscopies indicates that H136 and Y235 are the axial ligands in ferric HmuT. Consistent with this assignment of axial ligands, ferric WT and H136A HmuT are difficult to reduce while Y235A is reduced readily in the presence of dithionite. The FeCO Raman shifts in WT, H136A, and Y235A HmuT-CO complexes provide further evidence of the axial ligand assignments. Additionally, these frequencies provide insight into the nonbonding environment of the heme pocket. Ferrous Y235A and the Y235A-CO complex reveal that the imidazole of H136 exists in two forms, one neutral and one with imidazolate character, consistent with a hydrogen bond acceptor on the H136 side of the heme. The ferric fluoride complex of Y235A reveals the presence of at least one hydrogen bond donor on the Y235 side of the heme. Hemoglobin utilization assays showed that the axial Y235 ligand is required for heme uptake in HmuT.

Redox-dependent stability, protonation, and reactivity of cysteine-bound heme proteins.

Cysteine-bound hemes are key components of many enzymes and biological sensors. Protonation (deprotonation) of the Cys ligand often accompanies redox transformations of these centers. To characterize these phenomena, we have engineered a series of Thr78Cys/Lys79Gly/Met80X mutants of yeast cytochrome c (cyt c) in which Cys78 becomes one of the axial ligands to the heme. At neutral pH, the protonation state of the coordinated Cys differs for the ferric and ferrous heme species, with Cys binding as a thiolate and a thiol, respectively. Analysis of redox-dependent stability and alkaline transitions of these model proteins, as well as comparisons to Cys binding studies with the minimalist heme peptide microperoxidase-8, demonstrate that the protein scaffold and solvent interactions play important roles in stabilizing a particular Cys-heme coordination. The increased stability of ferric thiolate compared with ferrous thiol arises mainly from entropic factors. This robust cyt c model system provides access to all four forms of Cys-bound heme, including the ferric thiol. Protein motions control the rates of heme redox reactions, and these effects are amplified at low pH, where the proteins are less stable. Thermodynamic signatures and redox reactivity of the model Cys-bound hemes highlight the critical role of the protein scaffold and its dynamics in modulating redox-linked transitions between thiols and thiolates.

Cavity Enhancement of Single Quantum Dot Emission in the Blue.

Cavity-enhanced single-photon emission in the blue spectral region was measured from single InGaN/GaN quantum dots. The low-Q microcavities used were characterized using micro-reflectance spectroscopy where the source was the enhanced blue output from a photonic crystal fibre. Micro-photoluminescence was observed from several cavities and found to be ~10 times stronger than typical InGaN quantum dot emission without a cavity. The measurements were performed using non-linear excitation spectroscopy in order to suppress the background emission from the underlying wetting layer.