Advancing stem cell technologies for conservation of wildlife biodiversity.

Wildlife biodiversity is essential for healthy, resilient and sustainable ecosystems. For biologists, this diversity also represents a treasure trove of genetic, molecular and developmental mechanisms that deepen our understanding of the origins and rules of life. However, the rapid decline in biodiversity reported recently foreshadows a potentially catastrophic collapse of many important ecosystems and the associated irreversible loss of many forms of life on our planet. Immediate action by conservationists of all stripes is required to avert this disaster. In this Spotlight, we draw together insights and proposals discussed at a recent workshop hosted by Revive & Restore, which gathered experts to discuss how stem cell technologies can support traditional conservation techniques and help protect animal biodiversity. We discuss reprogramming, in vitro gametogenesis, disease modelling and embryo modelling, and we highlight the prospects for leveraging stem cell technologies beyond mammalian species.

Phylogenomics of the extinct Heath Hen provides support for sex-biased introgression among extant prairie grouse.

Rapid divergence and subsequent reoccurring patterns of gene flow can complicate our ability to discern phylogenetic relationships among closely related species. To what degree such patterns may differ across the genome can provide an opportunity to extrapolate better how life history constraints may influence species boundaries. By exploring differences between autosomal and Z (or X) chromosomal-derived phylogenetic patterns, we can better identify factors that may limit introgression despite patterns of incomplete lineage sorting among closely related taxa. Here, using a whole-genome resequencing approach coupled with an exhaustive sampling of subspecies within the recently divergent prairie grouse complex (genus: Tympanuchus), including the extinct Heath Hen (T. cupido cupido), we show that their phylogenomic history differs depending on autosomal or Z-chromosome partitioned SNPs. Because the Heath Hen was allopatric relative to the other prairie grouse taxa, its phylogenetic signature should not be influenced by gene flow. In contrast, all the other extant prairie grouse taxa, except Attwater's Prairie-chicken (T. c. attwateri), possess overlapping contemporary geographic distributions and have been known to hybridize. After excluding samples that were likely translocated prairie grouse from the Midwest to the eastern coastal states or their resulting hybrids with mainland Heath Hens, species tree analyses based on autosomal SNPs consistently identified a paraphyletic relationship with regard to the Heath Hen with Lesser Prairie-chicken (T. pallidicinctus) sister to Greater Prairie-chicken (T. c. pinnatus) regardless of genic or intergenic partitions. In contrast, species trees based on the Z-chromosome were consistent with Heath Hen sister to a clade that included its conspecifics, Greater and Attwater's Prairie-chickens (T. c. attwateri). These results were further explained by historic gene flow, as shown with an excess of autosomal SNPs shared between Lesser and Greater Prairie-chickens but not with the Z-chromosome. Phylogenetic placement of Sharp-tailed Grouse (T. phasianellus), however, did not differ among analyses and was sister to a clade that included all other prairie grouse despite low levels of autosomal gene flow with Greater Prairie-chicken. These results, along with strong sexual selection (i.e., male hybrid behavioral isolation) and a lek breeding system (i.e., high variance in male mating success), are consistent with a pattern of female-biased introgression between prairie grouse taxa with overlapping geographic distributions. Additional study is warranted to explore how genomic components associated with the Z-chromosome influence the phenotype and thereby impact species limits among prairie grouse taxa despite ongoing contemporary gene flow.

Husbandry protocols for the Band-tailed pigeon, Patagioenas fasciata albilinea, at the WCS, Bronx Zoo for future conservation management programs.

From 2015 to 2016 we determined the husbandry protocols involved in the captive rearing of the Band-tailed Pigeon (BTPI), Patagioenas fascinate albilinea, for use as a tool in the future management of like extant and extinct avian taxa. Current and historical ex-situ conservation management of BTPIs and the closely related Passenger Pigeon, Ectopistes migratorius, is limited in scope and required further examination. Focus on the BTPI within zoos and private aviculture facilities is currently lacking. New pressures on the wild populations and future examination of the parameters involved in the possible restoration of the Passenger Pigeon may rely on a complete understanding of these conservation management techniques. Here we report on the establishment of a colony of BTPIs, at the Wildlife Conservation Society (WCS), and detail the progress attained. A confiscated group of BTPIs was presented to WCS and allowed us to set up the colony, document the husbandry involved, and monitor neonatal development and the factors that influence that development. The information has provided a better understanding of the BTPI and has implications for the future conservation management of this and like species.

Complete mitochondrial genomes of living and extinct pigeons revise the timing of the columbiform radiation.

BACKGROUND: Pigeons and doves (Columbiformes) are one of the oldest and most diverse extant lineages of birds. However, the nature and timing of the group's evolutionary radiation remains poorly resolved, despite recent advances in DNA sequencing and assembly and the growing database of pigeon mitochondrial genomes. One challenge has been to generate comparative data from the large number of extinct pigeon lineages, some of which are morphologically unique and therefore difficult to place in a phylogenetic context. RESULTS: We used ancient DNA and next generation sequencing approaches to assemble complete mitochondrial genomes for eleven pigeons, including the extinct Ryukyu wood pigeon (Columba jouyi), the thick-billed ground dove (Alopecoenas salamonis), the spotted green pigeon (Caloenas maculata), the Rodrigues solitaire (Pezophaps solitaria), and the dodo (Raphus cucullatus). We used a Bayesian approach to infer the evolutionary relationships among 24 species of living and extinct pigeons and doves. CONCLUSIONS: Our analyses indicate that the earliest radiation of the Columbidae crown group most likely occurred during the Oligocene, with continued divergence of major clades into the Miocene, suggesting that diversification within the Columbidae occurred more recently than has been reported previously.

A Road Map for 21st Century Genetic Restoration: Gene Pool Enrichment of the Black-Footed Ferret.

Interspecies somatic cell nuclear transfer (iSCNT) could benefit recovery programs of critically endangered species but must be weighed with the risks of failure. To weigh the risks and benefits, a decision-making process that evaluates progress is needed. Experiments that evaluate the efficiency and efficacy of blastocyst, fetal, and post-parturition development are necessary to determine the success or failure or species-specific iSCNT programs. Here, we use the black-footed ferret (Mustela nigripes) as a case study for evaluating this emerging biomedical technology as a tool for genetic restoration. The black-footed ferret has depleted genetic variation yet genome resource banks contain genetic material of individuals not currently represented in the extant lineage. Thus, genetic restoration of the species is in theory possible and could help reduce the persistent erosion of genetic diversity from drift. Extensive genetic, genomic, and reproductive science tools have previously been developed in black-footed ferrets and would aid in the process of developing an iSCNT protocol for this species. Nonetheless, developing reproductive cloning will require years of experiments and a coordinated effort among recovery partners. The information gained from a well-planned research effort with the goal of genetic restoration via reproductive cloning could establish a 21st century model for evaluating and implementing conservation breeding that would be applicable to other genetically impoverished species.

Whole-genome survey reveals extensive variation in genetic diversity and inbreeding levels among peregrine falcon subspecies.

In efforts to prevent extinction, resource managers are often tasked with increasing genetic diversity in a population of concern to prevent inbreeding depression or improve adaptive potential in a changing environment. The assumption that all small populations require measures to increase their genetic diversity may be unwarranted, and limited resources for conservation may be better utilized elsewhere. We test this assumption in a case study focused on the peregrine falcon (Falco peregrinus), a cosmopolitan circumpolar species with 19 named subspecies. We used whole-genome resequencing to generate over two million single nucleotide polymorphisms (SNPs) from multiple individuals of all peregrine falcon subspecies. Our analyses revealed extensive variation among subspecies, with many island-restricted and nonmigratory populations possessing lower overall genomic diversity, elevated inbreeding coefficients (F ROH)-among the highest reported, and extensive runs of homozygosity (ROH) compared to mainland and migratory populations. Similarly, the majority of subspecies that are either nonmigratory or restricted to islands show a much longer history of low effective population size (N e). While mutational load analyses indicated an increased proportion of homozygous-derived deleterious variants (i.e., drift load) among nonmigrant and island populations compared to those that are migrant or reside on the mainland, no significant differences in the proportion of heterozygous deleterious variants (i.e., inbreeding load) was observed. Our results provide evidence that high levels of inbreeding may not be an existential threat for some populations or taxa. Additional factors such as the timing and severity of population declines are important to consider in management decisions about extinction potential.

Identification and pharmacological modification of resistance mechanisms to protoporphyrin-mediated photodynamic therapy in human cutaneous squamous cell carcinoma cell lines.

BACKGROUND: Photodynamic therapy (PDT) is clinically approved to treat neoplastic skin diseases such as precursors of cutaneous squamous cell carcinoma (cSCC). In PDT, 5-aminolevulinic acid (5-ALA) drives the selective formation of the endogenous photosensitizer protoporphyrin IX (PpIX). Although 5-ALA PDT is clinically highly effective, resistance might occur due to decreased accumulation of PpIX in certain tumors. Such resistance may be caused by any fundamental step of PpIX accumulation: 5-ALA uptake, PpIX synthesis and PpIX efflux. METHODS: We investigated PpIX accumulation and photodynamically induced cell death in PDT refractory SCC-13, PDT susceptible A431, and normal human epidermal keratinocytes (NHEK). Expression of genes associated with cellular PpIX kinetics was investigated on mRNA and protein level. PpIX accumulation and cell death upon illumination were pharmacologically manipulated using drugs targeting 5-ALA uptake, PpIX synthesis or efflux. RESULTS: The experiments indicate that taurine transporter (SLC6A6) is the major pathway for 5-ALA uptake in cSCC cells, while being less important in NHEK. Downregulation of PpIX synthesis enzymes in SCC-13 was counteracted by methotrexate (MTX) treatment, which restored PpIX formation and cell death. PpIX efflux inhibitors targeting ABC transporters led to significantly increased PpIX accumulation in SCC-13, thereby fully overcoming resistance. CONCLUSIONS: The results indicate a conserved threshold for PpIX accumulation with respect to PDT-resistance. Cells showed increased viability after PDT at PpIX concentrations below 1.5 nM. Selective uptake of 5-ALA via taurine transporter SLC6A6 in cutaneous tumor cells is novel but unrelated to resistance. MTX can partially abrogate resistance by PpIX synthesis enzyme induction, while efflux mechanisms via ABC transporters seem the main driving force and promising drug targets.

Clinical Pharmacokinetics and Safety of a 10% Aminolevulinic Acid Hydrochloride Nanoemulsion Gel (BF-200 ALA) in Photodynamic Therapy of Patients Extensively Affected With Actinic Keratosis: Results of 2 Maximal Usage Pharmacokinetic Trials.

The nanoemulsion-based 10% aminolevulinic acid (ALA) hydrochloride gel BF-200 ALA optimizes epidermal penetration of its active ingredient and is approved for topical photodynamic therapy (PDT) for the treatment of actinic keratosis in the United States and Europe. To characterize systemic absorption from dermal application during PDT, ALA and its key active metabolite protoporphyrin IX (PpIX) were analyzed in 2 maximal usage pharmacokinetic trials (MUsT) in patients severely affected with actinic keratosis. The primary objective of both MUsTs was to assess baseline-adjusted plasma concentration-time curves for ALA and PpIX after a single PDT treatment applying either 2 g (1 tube) of BF-200 ALA on the face (MUsT-1) or applying 6 g (3 tubes) of BF-200 ALA on the face/scalp or body periphery (MUsT-2), to 20 or 60 cm2 , respectively. All PDTs were performed using red light at around 635 nm wavelength. Safety and tolerability were documented along with pharmacokinetics. In both MUsTs, ALA plasma concentrations were transiently increased to a maximum concentration at about 2.5 to 3.3 times above endogenous baseline with time to maximum concentration at ≈3 hours after dosing. Plasma levels subsequently returned to baseline within 10 hours after dosing. Overall baseline-adjusted mean area under the baseline-adjusted plasma concentration-time curve from time zero to the last sampling time point at which the concentration was at or above the lower limit of quantification ranged from 142.8 to 146.2, indicating that a similar, minor fraction of topical ALA is systemically absorbed under both dosing regimens. Systemic PpIX exposure after administration of either dose of BF-200 ALA was equally minimal. Application site skin reactions were treatment area size-related, albeit transient and consistent with the known safety profile of BF-200 ALA.

Transforming Ocean Conservation: Applying the Genetic Rescue Toolkit.

Although oceans provide critical ecosystem services and support the most abundant populations on earth, the extent of damage impacting oceans and the diversity of strategies to protect them is disconcertingly, and disproportionately, understudied. While conventional modes of conservation have made strides in mitigating impacts of human activities on ocean ecosystems, those strategies alone cannot completely stem the tide of mounting threats. Biotechnology and genomic research should be harnessed and developed within conservation frameworks to foster the persistence of viable ocean ecosystems. This document distills the results of a targeted survey, the Ocean Genomics Horizon Scan, which assessed opportunities to bring novel genetic rescue tools to marine conservation. From this Horizon Scan, we have identified how novel approaches from synthetic biology and genomics can alleviate major marine threats. While ethical frameworks for biotechnological interventions are necessary for effective and responsible practice, here we primarily assessed technological and social factors directly affecting technical development and deployment of biotechnology interventions for marine conservation. Genetic insight can greatly enhance established conservation methods, but the severity of many threats may demand genomic intervention. While intervention is controversial, for many marine areas the cost of inaction is too high to allow controversy to be a barrier to conserving viable ecosystems. Here, we offer a set of recommendations for engagement and program development to deploy genetic rescue safely and responsibly.

Cretaceous dinosaur bone contains recent organic material and provides an environment conducive to microbial communities.

Fossils were thought to lack original organic molecules, but chemical analyses show that some can survive. Dinosaur bone has been proposed to preserve collagen, osteocytes, and blood vessels. However, proteins and labile lipids are diagenetically unstable, and bone is a porous open system, allowing microbial/molecular flux. These 'soft tissues' have been reinterpreted as biofilms. Organic preservation versus contamination of dinosaur bone was examined by freshly excavating, with aseptic protocols, fossils and sedimentary matrix, and chemically/biologically analyzing them. Fossil 'soft tissues' differed from collagen chemically and structurally; while degradation would be expected, the patterns observed did not support this. 16S rRNA amplicon sequencing revealed that dinosaur bone hosted an abundant microbial community different from lesser abundant communities of surrounding sediment. Subsurface dinosaur bone is a relatively fertile habitat, attracting microbes that likely utilize inorganic nutrients and complicate identification of original organic material. There exists potential post-burial taphonomic roles for subsurface microorganisms.

The chances of establishing a real-world Jurassic Park are slim. During the fossilization process, biological tissues degrade over millions of years, with some types of molecules breaking down faster than others. However, traces of biological material have been found inside some fossils. While some researchers believe these could be the remains of ancient proteins, blood vessels, and cells, traditionally thought to be among the least stable components of bone, others think that they have more recent sources. One hypothesis is that they are in fact biofilms formed by bacteria. To investigate the source of the biological material in fossil bone, Saitta et al. performed a range of analyses on the fossilized bones of a horned dinosaur called Centrosaurus. The bones were carefully excavated in a manner to reduce contamination, and the sediment the bones had been embedded in was also tested for comparison. Saitta et al. found no evidence of ancient dinosaur proteins. However, the fossils contained more organic carbon, DNA, and certain amino acids than the sediment surrounding them. Most of these appeared to have a very recent source. Sequencing the genetic material revealed that the fossil had become a habitat for an unusual community of microbes that is not found in the surrounding sediment or above ground. These buried microbes may have evolved unique ways to thrive inside fossils. Future work could investigate how these unusual organisms live and whether the communities vary in different parts of the world.

De-Extinction.

De-extinction projects for species such as the woolly mammoth and passenger pigeon have greatly stimulated public and scientific interest, producing a large body of literature and much debate. To date, there has been little consistency in descriptions of de-extinction technologies and purposes. In 2016, a special committee of the International Union for the Conservation of Nature (IUCN) published a set of guidelines for de-extinction practice, establishing the first detailed description of de-extinction; yet incoherencies in published literature persist. There are even several problems with the IUCN definition. Here I present a comprehensive definition of de-extinction practice and rationale that expounds and reconciles the biological and ecological inconsistencies in the IUCN definition. This new definition brings together the practices of reintroduction and ecological replacement with de-extinction efforts that employ breeding strategies to recover unique extinct phenotypes into a single "de-extinction" discipline. An accurate understanding of de-extinction and biotechnology segregates the restoration of certain species into a new classification of endangerment, removing them from the purview of de-extinction and into the arena of species' recovery. I term these species as "evolutionarily torpid species"; a term to apply to species falsely considered extinct, which in fact persist in the form of cryopreserved tissues and cultured cells. For the first time in published literature, all currently active de-extinction breeding programs are reviewed and their progress presented. Lastly, I review and scrutinize various topics pertaining to de-extinction in light of the growing body of peer-reviewed literature published since de-extinction breeding programs gained public attention in 2013.

Advancing a New Toolkit for Conservation: From Science to Policy.

Climate change and non-native wildlife diseases are exacerbating persistent challenges to biodiversity such as habitat destruction, invasive species and over-harvesting. With these increasing threats there is a pressing need to expand the conservationists' toolbox. CRISPR-Cas9 genome editing (GE) offers a precise and potentially transformative tool to confront these challenges. Researchers, regulators, conservationists and the public are all needed to engage proactively in the thoughtful and responsible development and application of these new tools.

Daylight photodynamic therapy for field cancerization: lessons from molecular biology.

Actinic keratoses (AKs) represent in-situ squamous cell carcinomas that potentially invade subepidermal structures and may metastasize. Until now, it is unpredictable to determine which AK lesions show this aggressive behavior. As AKs usually occur in large sun exposed areas, field-directed treatments have become the standard treatment regimen. Among these, conventional photodynamic therapy (cPDT) with 5-aminolaevulinic acid (ALA) or methyl-aminolevulinate (MAL) using red light is particularly effective in the treatment of AKs, but acceptance of the therapy is impaired by severe pain during treatment. Daylight PDT (dPDT) has demonstrated to be an equally effective alternative treatment option which is less painful. Recent attempts to determine the risk of AKs that demonstrate particular aggressive biological behavior by implementation of clinical and histological characteristics of AKs have not lead to conclusive results. Therefore, a look at the molecular biology of AKs could serve as a useful tool to develop a risk profiling for separation of those patients that are of particular risk to develop invasive tumor and, by this, to facilitate a more effective and adapted treatment option.

Creating Disease Resistant Chickens: A Viable Solution to Avian Influenza?

Influenza A virus (IAV) represents an ongoing threat to human and animal health worldwide. The generation of IAV-resistant chickens through genetic modification and/or selective breeding may help prevent viral spread. The feasibility of creating genetically modified birds has already been demonstrated with the insertion of transgenes that target IAV into the genomes of chickens. This approach has been met with some success in minimising the spread of IAV but has limitations in terms of its ability to prevent the emergence of disease. An alternate approach is the use of genetic engineering to improve host resistance by targeting the antiviral immune responses of poultry to IAV. Harnessing such resistance mechanisms in a "genetic restoration" approach may hold the greatest promise yet for generating disease resistant chickens. Continuing to identify genes associated with natural resistance in poultry provides the opportunity to identify new targets for genetic modification and/or selective breeding. However, as with any new technology, economic, societal, and legislative barriers will need to be overcome before we are likely to see commercialisation of genetically modified birds.

Natural selection shaped the rise and fall of passenger pigeon genomic diversity.

The extinct passenger pigeon was once the most abundant bird in North America, and possibly the world. Although theory predicts that large populations will be more genetically diverse, passenger pigeon genetic diversity was surprisingly low. To investigate this disconnect, we analyzed 41 mitochondrial and 4 nuclear genomes from passenger pigeons and 2 genomes from band-tailed pigeons, which are passenger pigeons' closest living relatives. Passenger pigeons' large population size appears to have allowed for faster adaptive evolution and removal of harmful mutations, driving a huge loss in their neutral genetic diversity. These results demonstrate the effect that selection can have on a vertebrate genome and contradict results that suggested that population instability contributed to this species's surprisingly rapid extinction.

Potency of different red light sources in photodynamic induction of cell death in a squamous cell carcinoma cell line.

LED illumination systems were found to be more efficacious than broad spectrum lamps in recent phase III trials on photodynamic treatment of actinic keratosis. However, a detailed comparison of the light doses emitted at the appropriate spectral range and its correlation to photodynamic effects is thus far not available for the most frequently used devices. Here, we compared the spectral emissions of three different PDT lamps with their potency of inducing cell death in ALA-loaded A431 cells, including a new system equipped with more advanced LEDs matching the photosensitizer absorption peak more precisely and emitting more homogeneous light over time. Cells were exposed to two different ALA concentrations, incubated for 1 or 3h and then illuminated by one of two different LED or a broad-spectrum system at four different light doses, whereupon viability was assessed. Maximal doses were selected in accordance to clinically applied light doses in recent phase III studies and the manufacturers' recommendations. The data gathered here clearly demonstrate that the two LED systems were significantly more effective in inducing cell death than the broad spectrum system. Most efficient was the newer LED system, in agreement with emission parameters that more accurately corresponded to the photosensitizer's absorption peak.

Epidermal penetration and protoporphyrin IX formation of two different 5-aminolevulinic acid formulations in ex vivo human skin.

BACKGROUND: Photosensitizer formation and epidermal penetration depth represent basic predictors of drug efficacy in dermatological Photodynamic Therapy (PDT). Different drug formulations and application standards are used to perform PDT in clinical practice. METHODS: Thus, we developed a human ex vivo model suitable to explore drug permeation in human skin and compared in 10 patients the penetration of nanoemulsion formulation (BF-200 ALA) with that of a 20% ALA cream formulation frequently used in clinical practice. Protoporphyrin IX (PpIX) formation was assessed according to different durations of incubation with both preparations. RESULTS: BF-200 ALA led to more intense PpIX fluorescence than the 20% ALA cream formulation as assessed by fluorescence microscopy: after 12h of incubation, total measured fluorescence was at 101,995 fluorescence units with BF-200 ALA and 40,960 fluorescence units with 20% ALA cream, respectively. This could be reproduced using quantitative fluorimetric measurements in tissue lysates. After the clinically relevant incubation time of 3h the PpIX concentration induced by BF-200 ALA was more than three-fold higher than that induced by the 20% ALA formulation (7.1±5.5 and 1.9±1.8nmol/l, p<0.05, Mann-Whitney U-test) and four-fold higher after 12h (30.0±4.6 and 6.7±2.0nmol/l, p<0.01, Mann-Whitney U-test). CONCLUSIONS: In spite of the 50% lower ALA content BF-200 ALA triggers significantly higher PpIX concentrations than the 20% ALA formulation, indicating that clinical efficacy with BF-200 ALA may be higher. Moreover, the ex vivo eyelid skin model may represent a useful tool to investigate drug permeation in human skin.