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This study provides an original theological interpretation of Immanuel Kant’s (1724–1804) Transcendental Unity of Apperception in light of Nicholas of Cusa’s (1401–1464) apophaticism. The study will show that Kant’s Transcendental Unity contains a thoroughly anti-theological premise. This is namely that our own ‘I think’ grounds the distinction between the transcendent (that which we think but don’t know) and immanent (that which we know). This premise is then contrasted with Cusa’s conception whereby the distinction is grounded in the transcendent itself. Whilst for Kant our thought itself produces the distinction between ‘thinking’ (Denken) and ‘knowing’ (Erkennen), for Cusa the distinction between ‘knowing that’ (quia est), and ‘knowing what’ (quid est) is produced by God himself.

In drug discovery, computational methods are a key part of making informed design decisions and prioritising compounds. In particular, optimizing compound affinity is a central concern during the early stages of development. In the last 10 years, alchemical free energy (FE) calculations have transformed our ability to incorporate accurate in silico potency predictions in design decisions, and represent the 'gold standard' for augmenting experiment-driven drug discovery. However, relative FE calculations are complex to set up and require significant expert intervention to prepare the calculation and analyse the results. Additionally, some of the commonly employed simulation engines for FE calculation are provided as closed-source software, and do not allow for fine-grained control over the underlying settings. In this work, we introduce an end-to-end relative FE workflow based on the non-equilibrium switching approach that facilitates calculation of binding free energies starting from SMILES strings. The workflow is implemented using fully modular steps, allowing various components to be exchanged depending on licence availability. Open-source options for ligand parameterization, embedding, docking, perturbation map generation and FE calculation steps are available to make the workflow fully open-source. We further investigate the dependence of the calculated free energy accuracy on the initial ligand pose generated by various docking algorithms. We show that both commercial and open-source docking engines can be used to generate poses that lead to good correlation of free energies with experimental reference data. Incorporating experimental information about binding pose generally leads to a more accurate set of predictions, particularly in more challenging cases. The code is freely available at https://github.com/MolecularAI/Icolos, where several example relative FE workflows can be found in both commercial and open-source flavours.

Aim: The incidence of major fires is increasing globally, creating extraordinary challenges for governments, managers and conservation scientists. In 2019–2020, Australia experienced precedent-setting fires that burned over several months, affecting seven states and territories and causing massive biodiversity loss. Whilst the fires were still burning, the Australian Government convened a biodiversity Expert Panel to guide its bushfire response. A pressing need was to target emergency investment and management to reduce the chance of extinctions and maximise the chances of longer-term recovery. We describe the approach taken to rapidly prioritise fire-affected animal species. We use the experience to consider the organisational and data requirements for evidence-based responses to future ecological disasters. Location: Forested biomes of subtropical and temperate Australia, with lessons for other regions. Methods: We developed assessment frameworks to screen fire-affected species based on their pre-fire conservation status, the proportion of their distribution overlapping with fires, and their behavioural/ecological traits relating to fire vulnerability. Using formal and informal networks of scientists, government and non-government staff and managers, we collated expert input and data from multiple sources, undertook the analyses, and completed the assessments in 3 weeks for vertebrates and 8 weeks for invertebrates. Results: The assessments prioritised 92 vertebrate and 213 invertebrate species for urgent management response; another 147 invertebrate species were placed on a watchlist requiring further information. Conclusions: The priority species lists helped focus government and non-government investment, management and research effort, and communication to the public. Using multiple expert networks allowed the assessments to be completed rapidly using the best information available. However, the assessments highlighted substantial gaps in data availability and access, deficiencies in statutory threatened species listings, and the need for capacity-building across the conservation science and management sectors. We outline a flexible template for using evidence effectively in emergency responses for future ecological disasters.

In response to Australia’s current extinction crisis, substantial research efforts have been targeted towards some of the most imperilled species. One such species is the northern quoll (Dasyurus hallucatus), a marsupial predator that has recently suffered substantial declines in range and is now listed as Endangered. We conducted a systematic review of all literature relevant to the conservation and ecology of northern quolls. We reviewed 143 studies, including research articles, government and industry reports, theses, and books, and quantified research effort in terms of topic, location, and publication period. We then summarised research relevant to northern quoll taxonomy, genetics, distribution, habitat associations, diet, reproduction, movement, threats, management, and Indigenous knowledge. Research effort was higher between 2011 and 2020 than the previous four decades combined. Northern quolls in the Northern Territory were the most studied, followed by the Pilbara, the Kimberley, and Queensland populations. Most studies focused on northern quoll distribution and habitat, management, and threats – primarily cane toads, predation, and fire. We conclude with a non-exhaustive list of ten future research directions. If pursued, these future research directions should provide information critical to managing and conserving northern quolls.

Artificial refuges are human-made structures that aim to create safe places for animals to breed, hibernate, or take shelter in lieu of natural refuges. Artificial refuges are used across the globe to mitigate the impacts of a variety of threats on wildlife, such as habitat loss and degradation. However, there is little understanding of the science underpinning artificial refuges, and what comprises best practice for artificial refuge design and implementation for wildlife conservation. We address this gap by undertaking a systematic review of the current state of artificial refuge research for the conservation of wildlife. We identified 224 studies of artificial refuges being implemented in the field to conserve wildlife species. The current literature on artificial refuges is dominated by studies of arboreal species, primarily birds and bats. Threatening processes addressed by artificial refuges were biological resource use (26%), invasive or problematic species (20%), and agriculture (15%), yet few studies examined artificial refuges specifically for threatened (Vulnerable, Endangered, or Critically Endangered) species (7%). Studies often reported the characteristics of artificial refuges (i.e. refuge size, construction materials; 87%) and surrounding vegetation (35%), but fewer studies measured the thermal properties of artificial refuges (18%), predator activity (17%), or food availability (3%). Almost all studies measured occupancy of the artificial refuges by target species (98%), and over half measured breeding activity (54%), whereas fewer included more detailed measures of fitness, such as breeding productivity (34%) or animal body condition (4%). Evaluating the benefits and impacts of artificial refuges requires sound experimental design, but only 39% of studies compared artificial refuges to experimental controls, and only 10% of studies used a before-after-control-impact (BACI) design. As a consequence, few studies of artificial refuges can determine their overall effect on individuals or populations. We outline a series of key steps in the design, implementation, and monitoring of artificial refuges that are required to avoid perverse outcomes and maximise the chances of achieving conservation objectives. This review highlights a clear need for increased rigour in studies of artificial refuges if they are to play an important role in wildlife conservation.

Gamete fusion is a critical event of mammalian fertilization. A random one-bead one-compound combinatorial peptide library represented synthetic human egg mimics and identified a previously unidentified ligand as Fc receptor–like 3, named MAIA after the mythological goddess intertwined with JUNO. This immunoglobulin super family receptor was expressed on humanoolemmaand played a major role during sperm-egg adhesion and fusion. MAIA forms a highly stable interaction with the known IZUMO1/JUNO sperm-egg complex, permitting specific gamete fusion. The complexity of the MAIA isotype may offer a cryptic sexual selection mechanism to avoid genetic incompatibility and achieve favorable fitness outcomes.

Rocky environments host rich levels of biodiversity and provide vital habitat for specialised organisms, range-restricted species and a broad range of ectotherms adapted to saxicoline environments. In Australia, rock habitat is being destroyed during soil amelioration practices associated with agricultural intensification. Advances in rock crushing technology, developed to expand or increase crop yields and efficiency, pose an undocumented threat to global biodiversity, especially reptiles dependent on non-renewable rock habitat in agricultural landscapes. Rock removal is a legislated key threatening process in parts of Australia and will accelerate biodiversity loss if not mitigated. We estimated reptile species’ range overlap with dryland cropping and modified pastoral regions within the Australian wheat-sheep zone to assess the potential impacts of rock crushing practices. We examined species- and family-richness within the impact zone and across bioregions within the impact zone, to identify areas where rock removal has the greatest potential to impact terrestrial and fossorial squamates. Our analysis revealed that 159 potentially impacted reptile species occur within the study area, representing 16% of Australian terrestrial squamates. Fourteen of these species, including six threatened species, have more than 50% range overlap with areas of intensive agriculture, and include several endangered pygopodids, scincids and agamids. Bioregions rich in rock and burrow-dwelling reptiles include the Brigalow Belt South, Murray Darling Depression, Darling Riverine Plains, Eyre Yorke Block, Avon Wheatbelt, Nandewar, Flinders Lofty Block and New South Wales South Western Slopes. Synthesis and applications. The conservation of reptiles in agricultural landscapes requires appropriate management and retention of surface rocks. Potential yield increases from destroying rock habitat to intensify or expand cropland will not compensate for the net loss of reptile populations dependent on non-renewable resources. Financial incentives to prevent the expansion and transformation of non-arable landscapes to cropland are required to prevent the ongoing loss of biodiversity.

Research on the impacts of anthropogenic habitat fragmentation has dominated landscape ecology for decades, yet our understanding of what drives species’ distributions in naturally fragmented landscapes remains limited. We aimed to (i) determine whether rocky patches embedded within a ‘matrix’ of fire prone grasslands act as naturally fragmented landscapes for an endangered marsupial predator, the northern quoll (Dasyurus hallucatus), and (ii) reveal the extent to which within-patch, patch, landscape variables, and matrix condition drive the occurrence of northern quolls. We deployed remote sensing cameras for a total of 200 nights, at 230 sites spanning rocky and grassland habitats across 6000 km2 of the Pilbara bioregion of Western Australia. We examined the influence of within-patch, patch, landscape variables, and matrix condition on northern quolls using Generalised Linear Mixed Models. We found strong evidence that northern quoll habitat is naturally fragmented, observing higher occurrence and abundance of quolls in rocky patches than the surrounding grassland matrix. Within rocky patches, quolls were more likely to use patches with higher vegetation cover and den availability (within-patch), lower amounts of edge habitat relative to patch area (patch), and larger amounts of surrounding rocky habitat (landscape). When quolls entered the matrix, they tended to remain in areas with high vegetation cover, close to rocky patches. Species occurrence in naturally fragmented landscapes is influenced by factors operating at multiple scales. Rocky habitats are naturally fragmented and vital to the conservation of a range of taxa around the world, including the northern quoll.

This study argues that the early philosophy of Semyon Liudvigovich Frank (1877–1950) exhibits significant intellectual correlations with nineteenth century German Idealist philosophy. The idealists in question are Immanuel Hermann Fichte (1796–1879), G.W.F. Hegel (1770–1831) and F.W.J. Schelling (1775–1854). It will be suggested that the critical tension of Frank’s early philosophy is precisely a tension between his Hegelian and Schellingian tendencies. The paper will first introduce Frank’s theory of a “personal absolute”, exploring its surprising parallels with the religious philosophy of I. H. Fichte. The analysis then addresses the self-dispersal of Hegel’s absolute, before finally turning to Schelling’s immediate intuition of subject-object identity.

Summary The enteric nervous system (ENS) is derived primarily from the vagal neural crest, a migratory multipotent cell population emerging from the dorsal neural tube between somites 1 and 7. Defects in the development and function of the ENS cause a range of enteric neuropathies, including Hirschsprung disease. Little is known about the signals that specify early ENS progenitors, limiting progress in the generation of enteric neurons from human pluripotent stem cells (hPSCs) to provide tools for disease modeling and regenerative medicine for enteric neuropathies. We describe the efficient and accelerated generation of ENS progenitors from hPSCs, revealing that retinoic acid is critical for the acquisition of vagal axial identity and early ENS progenitor specification. These ENS progenitors generate enteric neurons in vitro and, following in vivo transplantation, achieved long-term colonization of the ENS in adult mice. Thus, hPSC-derived ENS progenitors may provide the basis for cell therapy for defects in the ENS., Highlights • Retinoic acid alters the axial identity of hPSC-derived neural crest cells • ENS progenitor markers are upregulated in response to RA • ENS progenitors are capable of generating enteric neurons in vitro • hPSC ENS progenitors colonize the ENS of mice following long-term transplantation, In this article, Frith and colleagues show that retinoic acid (RA) signaling alters the axial identity of hPSC-derived neural crest cells in a time- and dose-dependent manner. They utilized this to derive enteric nervous system (ENS) progenitors from hSPCs, which can differentiate to enteric neurons in vitro and colonize the ENS of adult mice following long-term transplantation.

A selective Pd-catalyzed C(3)–H cis-functionalization of piperidine and tetrahydropyran carboxylic acids is achieved using a C(4) aminoquinoline amide auxiliary. High mono- and cis-selectivity is attained by using mesityl carboxylic acid as an additive. Conditions are developed with significantly lower reaction temperatures (≤50 °C) than other reported heterocycle C(sp3)–H functionalization reactions, which is facilitated by a DoE optimization. A one-pot C–H functionalization-epimerization procedure provides the trans-3,4-disubstituted isomers directly. Divergent aminoquinoline removal is accomplished with the installation of carboxylic acid, alcohol, amide and nitrile functional groups. Overall fragment compounds suitable for screening are generated in 3–4 steps from readily-available heterocyclic carboxylic acids.

Slide deck summarising research carried out during Master's research project

AIM: Species range contractions are increasingly common globally. The niche reduction hypothesis posits that geographic range contractions are often patterned across space owing to heterogeneity in threat impacts and tolerance. We applied the niche reduction hypothesis to the decline of a threatened marsupial predator across northern Australia, the northern quoll (Dasyurus hallucatus). LOCATION: Northern Australia. METHODS: We assembled a database containing 3,178 historic and contemporary records for northern quolls across the extent of their distribution dating between 1778 and 2019. Based on these records, we estimated changes in the geographic range of the northern quoll using α‐hulls across four main populations. We then examined how range contractions related to factors likely to mediate the exposure, susceptibility, or tolerance of northern quolls to threats. RESULT: The extent of range contractions showed an east–west gradient, most likely reflecting the timing of spread of introduced cane toads (Rhinella marina). There were clear changes in environmental characteristics within the contemporary compared to the historic geographic range, with the most substantial occurring in populations that have suffered the greatest range contractions. The contemporary range is comprised of higher quality habitats (measured using environmental niche models), characterized by higher topographical ruggedness and annual rainfall, and reduced distance to water, compared to the historic range. MAIN CONCLUSIONS: Changes to range and niche likely reflect the capacity of complex habitats to ameliorate threats (namely predation and altered fire regimes), and access to resources that increase threat tolerance. This study highlights the multivariate nature of ecological refuges and the importance of high‐quality habitats for the persistence of species exposed to multiple threats. Our methods provide a useful framework which can be applied across taxa in providing valuable insight to management.

Funder: Medical Research Council; doi: http://dx.doi.org/10.13039/501100000265, Funder: Biotechnology and Biological Sciences Research Council; doi: http://dx.doi.org/10.13039/501100000268, Funder: Engineering and Physical Sciences Research Council; doi: http://dx.doi.org/10.13039/501100000266, Background: A major challenge for the clinical use of human pluripotent stem cells is the development of safe, robust and controlled differentiation protocols. Adaptation of research protocols using reagents designated as research-only to those which are suitable for clinical use, often referred to as good manufacturing practice (GMP) reagents, is a crucial and laborious step in the translational pipeline. However, published protocols to assist this process remain very limited. Methods: We adapted research-grade protocols for the derivation and differentiation of long-term neuroepithelial stem cell progenitors (lt-NES) to GMP-grade reagents and factors suitable for clinical applications. We screened the robustness of the protocol with six clinical-grade hESC lines deposited in the UK Stem Cell Bank. Results: Here, we present a new GMP-compliant protocol to derive lt-NES, which are multipotent, bankable and karyotypically stable. This protocol resulted in robust and reproducible differentiation of several clinical-grade embryonic stem cells from which we derived lt-NES. Furthermore, GMP-derived lt-NES demonstrated a high neurogenic potential while retaining the ability to be redirected to several neuronal sub-types. Conclusions: Overall, we report the feasibility of derivation and differentiation of clinical-grade embryonic stem cell lines into lt-NES under GMP-compliant conditions. Our protocols could be used as a flexible tool to speed up translation-to-clinic of pluripotent stem cells for a variety of neurological therapies or regenerative medicine studies.

One technique used to combat the growing global species extinction crisis has been to create artificial refuges—human‐made replacements for natural refuges destroyed during habitat modification. However, there is limited knowledge of how closely artificial refuges replicate the natural refuges they seek to replace. Mining threatens many species worldwide through large‐scale habitat modification, and artificial refuges have been proposed as a method to offset the resulting habitat loss. Here, we examined the microclimatic, physical, and biotic characteristics of natural dens occupied by the northern quoll (Dasyurus hallucatus)—an endangered marsupial threatened by habitat loss—and compared these to (a) superficially similar unoccupied crevices, and (b) artificial dens created by mining companies for northern quolls. Northern quolls occupied natural dens that were cooler and deeper than unoccupied crevices, likely to avoid lethal air temperatures as well as predators. Artificial dens provided similar thermal properties to occupied dens, but lacked key characteristics in having shallower den cavities, less complex surrounding habitat, increased feral cat visitation, and less small mammal prey compared to occupied dens. This study highlights the need to consider multiple facets when constructing artificial refuges, in order to avoid perverse outcomes, such as inadequate shelter, increased predation, and food shortages.

Background\ud \ud Few potentially modifiable risk factors of male infertility have been identified, and while different diets and food groups have been associated with male infertility, evidence linking dietary factors including phytoestrogens and semen quality is limited and contradictory.\ud \ud \ud \ud Objectives\ud \ud To study the associations between phytoestrogen intake and other dietary factors and semen quality.\ud \ud \ud \ud Materials and Methods\ud \ud A case‐referent study was undertaken of the male partners, of couples attempting conception with unprotected intercourse for 12 months or more without success, recruited from 14 UK assisted reproduction clinics. A total of 1907 participants completed occupational, lifestyle and dietary questionnaires before semen quality (concentration, motility and morphology) were assessed. Food intake was estimated by a 65‐item food frequency questionnaire (FFQ) covering the 12 months prior to recruitment. Analyses of dietary risk factors for low motile sperm concentration (MSC: 3 times/wk.\ud \ud \ud \ud Discussion\ud \ud In this case‐referent study of men attending an infertility clinic for fertility diagnosis, we have identified that low MSC is inversely associated with daidzein intake. In contrast, daidzein intake was not associated with PM but eating red milk and drinking whole milk were protective.\ud \ud \ud \ud Conclusions\ud \ud Dietary factors associated with semen quality were identified, suggesting that male fertility might be improved by dietary changes.

The occurrence of repetitive genomic changes that provide a selective growth advantage in pluripotent stem cells is of concern for their clinical application. However, the effect of different culture conditions on the underlying mutation rate is unknown. Here we show that the mutation rate in two human embryonic stem cell lines derived and banked for clinical application is low and not substantially affected by culture with Rho Kinase inhibitor, commonly used in their routine maintenance. However, the mutation rate is reduced by >50% in cells cultured under 5% oxygen, when we also found alterations in imprint methylation and reversible DNA hypomethylation. Mutations are evenly distributed across the chromosomes, except for a slight increase on the X-chromosome, and an elevation in intergenic regions suggesting that chromatin structure may affect mutation rate. Overall the results suggest that pluripotent stem cells are not subject to unusually high rates of genetic or epigenetic alterations., Nature Communications, 11 (1), ISSN:2041-1723

If the field of regenerative medicine is to deliver therapies, rapid expansion and delivery over considerable distances to large numbers of patients is needed. This will demand efficient stabilization and shipment of cell products. However, cryopreservation science is poorly understood by life-scientists in general and in recent decades only limited progress has been made in the technology of preservation and storage of cells. Rapid translation of new developments to a broader range of cell types will be vital, as will assuring a deeper knowledge of the fundamental cell biology relating to successful preservation and recovery of cell cultures. This report presents expert consensus on these and other issues which need to be addressed for more efficient delivery of cell therapies.

Background: Cell banking initial outgrowths from newly derived human embryonic stem cells (hESCs) requires an efficient freezing method. Vitrification is used for the preservation of gametes and early embryos in assisted reproduction techniques (ART). Moreover, vitrification was applied for cryopreservation of hESCs using open pulled straws. Objective: To derive and characterize new hESC lines and then use Cryotech and Cryowin tools for their vitrification. Materials and Methods: Human ESC lines were generated in a microdrop culture system using mouse embryonic fibroblasts (MEFs) as the feeder layer; this was later scaled up using both MEFs and Yazd human foreskin fibroblasts batch 8 (YhFF#8). To bank the cell lines, master cell banks of 100 Cryotech and Cryowin tools were produced for each individual cell line using the vitrification method; flasks of hESC lines were also cryopreserved using a conventional slow-freezing method. Results: The pluripotency of cell lines was assessed by their expression of pluripotency-associated genes (OCT4/POU5F1, NANOG, and SOX2) and markers such as SSEA4, TRA-1-60, and TRA-2-49. Their in vitro capacity to differentiate into germ layers and germ cells using embryoid body (EB) formation and monolayer culture was assessed by screening the expression of differentiation-associated genes. The chromosomal constitution of each hESC line was assessed by G-banding karyotyping. Conclusion: Cryotech and Cryowin tools used to vitrify new hESCs at an early stage of derivation is an efficient means of preserving hESCs. Key words: Derivation, Human embryonic stem cells, Human foreskin fibroblast, Microdrop, Vitrification.

1,3-Dinitrobenzene (mDNB) is a widely used intermediate in commercial products and causes testicular injury. However, genotoxic effects upon low-level exposure are poorly understood. The present study evaluated the effects of very low-chronic doses of mDNB on sperm nuclear integrity. Male hamsters were treated with 1.5 mg/kg/d/4 wks (group A), 1.5 mg/kg/mDNB/d/week/4 weeks (group B), 1.0 mg/kg/mDNB/3 d/wk/4 wks (group C), or polyethylene glycol 600 (control). Nuclear integrity of distal cauda epididymal sperm was determined using the sperm chromatin structure assay and acridine orange staining (AOS). The germ cell nuclear integrity was assessed by the comet assay. Testicular histopathology was conducted to evaluate the sensitive stages. The comet assay revealed denatured nuclear DNA in group A (in diploid and polyploid cells from weeks 2-5); respectively at week 4 and weeks 3 to 4 in groups B and C. According to AOS, only group A animals exhibited denatured sperm DNA (weeks 1 and 3). The effective sperm count declined from weeks 1 to 6. Mean sperm DNA denaturation extent, percentage cells outside the main population, and standard deviation indicated altered sperm nuclear integrity in group A. Same animals exhibited progressive disruption of the Sertoli cells, while groups B and C exhibited damages on germ cells. The results suggest that mDNB affects sperm nuclear integrity at very low chronic doses targeting cell-specific testicular damage.

Human stem cells have the potential to transform medicine. However, hurdles remain to ensure that manufacturing processes produce safe and effective products. A thorough understanding of the biological processes occurring during manufacture is fundamental to assuring these qualities and thus, their acceptability to regulators and clinicians. Leaders in both human pluripotent and somatic stem cells, were brought together with experts in clinical translation, bio-manufacturing and regulation, to discuss key issues in assuring appropriate manufacturing conditions for delivery of effective and safe products from these cell types. This report summarizes the key issues discussed and records consensus reached by delegates and emphasizes the need for accurate language and nomenclature in the scientific discourse around stem cells. Lay abstract Novel therapies derived from different kinds of precursor cells and stem cells are increasingly moving to clinical trials to restore tissue function in patients who have suffered injury or disease. The manufacture of these new therapies is unusually complex, which means that the manufacturing processes require great attention to assure they are safe and effective. This paper describes a conversation amongst experts in the field who are exploring therapeutic applications of two different kinds of stem cells (pluripotent stem cells and tissue-derived stem/precursor cells). It considers critical issues in developing the manufacturing process for each of these quite different cells types.

Summary Genetic changes in human pluripotent stem cells (hPSCs) gained during culture can confound experimental results and potentially jeopardize the outcome of clinical therapies. Particularly common changes in hPSCs are trisomies of chromosomes 1, 12, 17, and 20. Thus, hPSCs should be regularly screened for such aberrations. Although a number of methods are used to assess hPSC genotypes, there has been no systematic evaluation of the sensitivity of the commonly used techniques in detecting low-level mosaicism in hPSC cultures. We have performed mixing experiments to mimic the naturally occurring mosaicism and have assessed the sensitivity of chromosome banding, qPCR, fluorescence in situ hybridization, and digital droplet PCR in detecting variants. Our analysis highlights the limits of mosaicism detection by the commonly employed methods, a pivotal requirement for interpreting the genetic status of hPSCs and for setting standards for safe applications of hPSCs in regenerative medicine., Highlights • hPSCs conform to random sampling rules used for karyotyping • Excluding mosaicism at 500 metaphases • qPCR is a rapid assay for detection of commonly amplified regions in hPSCs • Cultures scored as normal by commonly used methods could harbor up to 10% variants, Barbaric and colleagues tested how many metaphases need to be scored to detect different levels of mosaicism in hPSC cultures. They also devised qPCR assays as a rapid means of detecting common chromosomal abnormalities. Testing of the sensitivity of qPCR, digital droplet PCR, and FISH revealed that these methods can miss as many as 10% abnormal cells in the population.

© 2016 Future Medicine Ltd.This paper summarizes the proceedings of a workshop held at Trinity Hall, Cambridge to discuss comparability and includes additional information and references to related information added subsequently to the workshop. Comparability is the need to demonstrate equivalence of product after a process change; a recent publication states that this may be difficult for cell-based medicinal products. Therefore a well-managed change process is required which needs access to good science and regulatory advice and developers are encouraged to seek help early. The workshop shared current thinking and best practice and allowed the definition of key research questions. The intent of this report is to summarize the key issues and the consensus reached on each of these by the expert delegates.

STUDY QUESTION As Syncytin 1 (human endogenous retrovirus (HERV-W)) is crucial for human embryo placentation is it expressed during preimplantation embryo development? SUMMARY ANSWER Syncytin 1 was expressed mainly in trophoblast cells of the blastocyst particularly in cells underlying the inner cell mass (ICM). WHAT IS KNOWN ALREADY Syncytin 1 (along with HERV-FRD or Syncytin 2) is expressed in first-trimester placenta and required for cell-cell fusion to enable formation of syncytiotrophoblast and effective placentation. STUDY DESIGN, SIZE AND DURATION Preimplantation human embryos donated for research were cultured in vitro and protein expression of Syncytin 1 at the blastocyst stage of development investigated. Comparisons were made with protein (Syncytin 1) and mRNA (Syncytin 1 and 2) expression in human embryonic stem cells (hESCs) undergoing differentiation to trophoblast-like cells in vitro. In total, 10 blastocysts (×3 or 4 replicates) were analysed and 4 hESC lines. The study was terminated after consistent observations of embryos were made. PARTICIPANTS/MATERIALS, SETTING, METHODS Donated embryos were thawed and cultured to blastocyst, fixed with 4% w/v paraformaldehyde. Syncytin 1 protein expression was determined by immunofluorescent localisation and confocal microscopy. Additionally, hESCs were differentiated to trophoblast-like cells in standard and conditioned culture medium with growth factors (bone morphogenetic protein 4 (BMP4) or fibroblast growth factor 4 (FGF4) and assessed for mRNA (Syncytin 1 and 2) by quantitative polymerase chain reaction (qPCR) and protein expression by immunolocalization and western blot. MAIN RESULTS AND ROLE OF CHANCE Syncytin 1 was expressed in cytoplasm and on the cell surface of some trophoblast cells, and consistently the trophectoderm underlying the ICM of the blastocyst. There was weak but consistent expression of Syncytin 1 in cells on the periphery of the ICM also displaying pluripotency antibody marker (Tra-1-60). Three-dimensional reconstruction of confocal slice data provided good visualization of expression. The time course of expression of Syncytin 1 was replicated in hESCs differentiated in vitro confirming the embryo observations and providing statistically significant differences in protein and mRNA level (P= 0.002) and (P< 0.05), respectively. LIMITATION, REASONS FOR CAUTION Culture of a limited number of embryos to blastocyst in vitro may not replicate the range and quality of development in situ. Probes (antibodies, PCR) were tested for specificity, but might have non-specific reactions. WIDER IMPLICATIONS OF FINDINGS Syncytin expression is a prerequisite for embryo implantation and placentation. Understanding when expression first occurs during embryo development may be informative for understanding conditions of abnormal gestations such as pre-clampsia. STUDY FUNDING/COMPETING INTERESTS The study was supported partly by an ERASMUS training grant and grant G0801059 from the Medical Research Council, U.K. There were no competing interests.

Autophagy is an important conserved cellular process, both constitutively as a recycling pathway for long lived proteins and as an upregulated stress response. Recent findings suggest a fundamental role for autophagic processes in the maintenance of pluripotent stem cell function. In human embryonic stem cells (hESCS), autophagy was investigated by transfection of LC3-GFP to visualize autophagosomes and with an antibody to LC3B protein. The presence of the primary cilium (PC) in hESCs as the site of recruitment of autophagy-related proteins was also assessed. HESCs (mShef11) in vitro displayed basal autophagy which was upregulated in response to deprivation of culture medium replacement. Significantly higher levels of autophagy were exhibited on spontaneous differentiation of hESCs in vitro. The PC was confirmed to be present in hESCs and therefore may serve to coordinate autophagy function.

Human male germ-line stem cells (hmGSCs) and human testis-derived embryonic stem cell-like (htESC-like) cells are claimed to be in vitro pluripotent counterparts of spermatogonial stem cells (SSCs), but the origin and pluripotency of human testis-derived cell cultures are still under debate. The aim of this study was to generate putative pluripotent stem cells in vitro from human testicular sperm-extracted (TESE) samples of infertile men, and to assess their pluripotency and capacity to differentiate. TESE samples were minced, enzymatically disaggregated and dispersed into single-cell or cluster suspensions, and then cultured. Initially, cell clusters resembled those described for hmGSCs and htESC-like cells, and were positive for markers such as OCT4/POU5F1, NANOG, and TRA-2-54. Prolonged propagation of cell clusters expressing pluripotency markers did not thrive; instead, the cells that emerged possessed characteristics of mesenchymal stromal cells (MSCs) such as STRO-1, CD105/EGLN1, CD13/ANPEP, SOX9, vimentin, and fibronectin. KIT, SOX2, and CD44 were not expressed by these MSCs. The multipotential differentiation capacity of these cells was confirmed using Oil Red-O and Alizarin Red staining after induction with specific culture conditions. It is therefore concluded that pluripotent stem cells could not be derived using the conditions previously reported to be successful for TESE samples.

Abstract ContextEstimating animal abundance often relies on being able to identify individuals; however, this can be challenging, especially when applied to large animals that are difficult to trap and handle. Camera traps have provided a non-invasive alternative by using natural markings to individually identify animals within image data. Although camera traps have been used to individually identify mammals, they are yet to be widely applied to other taxa, such as reptiles. AimsWe assessed the capacity of camera traps to provide images that allow for individual identification of the world’s fourth-largest lizard species, the perentie (Varanus giganteus), and demonstrate other basic morphological and behavioural data that can be gleaned from camera-trap images. MethodsVertically orientated cameras were deployed at 115 sites across a 10000km2 area in north-western Australia for an average of 216 days. We used spot patterning located on the dorsal surface of perenties to identify individuals from camera-trap imagery, with the assistance of freely available spot ID software. We also measured snout-to-vent length (SVL) by using image-analysis software, and collected image time-stamp data to analyse temporal activity patterns. ResultsNinety-two individuals were identified, and individuals were recorded moving distances of up to 1975m. Confidence in identification accuracy was generally high (91%), and estimated SVL measurements varied by an average of 6.7% (min=1.8%, max=21.3%) of individual SVL averages. Larger perenties (SVL of >45cm) were detected mostly between dawn and noon, and in the late afternoon and early evening, whereas small perenties (SVL of

A new record of an albino marsupial, the northern quoll (Dasyurus hallucatus), is described and placed in the context of 10 records since 1874 from all four Australian quoll species. Of the 10 previous records, one was D. hallucatus, seven are likely to be D. viverrinus, one D. maculatus and one unknown. The recent record comprises the live capture of a healthy adult female northern quoll from the Pilbara region of Western Australia. Despite the rarity and likely deleterious nature of the albino condition, this animal appeared to be in good health, carrying eight pouch young, and was released at location of capture following tissue sampling for DNA analysis.

© 2018 Cambridge Philosophical Society Movement is a trait of fundamental importance in ecosystems subject to frequent disturbances, such as fire-prone ecosystems. Despite this, the role of movement in facilitating responses to fire has received little attention. Herein, we consider how animal movement interacts with fire history to shape species distributions. We consider how fire affects movement between habitat patches of differing fire histories that occur across a range of spatial and temporal scales, from daily foraging bouts to infrequent dispersal events, and annual migrations. We review animal movements in response to the immediate and abrupt impacts of fire, and the longer-term successional changes that fires set in train. We discuss how the novel threats of altered fire regimes, landscape fragmentation, and invasive species result in suboptimal movements that drive populations downwards. We then outline the types of data needed to study animal movements in relation to fire and novel threats, to hasten the integration of movement ecology and fire ecology. We conclude by outlining a research agenda for the integration of movement ecology and fire ecology by identifying key research questions that emerge from our synthesis of animal movements in fire-prone ecosystems.

The success of percutaneous coronary intervention (PCI) has been limited by restenosis and stent thrombosis. Delayed or incomplete endothelial regeneration is believed to be a key factor responsible for these events. Developing a stent with an accelerated healing profile may be of benefit. We aimed to evaluate the feasibility and safety of seeding a bare metal stent (BMS) with human trophoblastic endovascular progenitor cells (hTEC) derived from human embryonic stem cells. A porcine coronary artery model was used to compare the rate and extent of endothelial regeneration and the degree of neointimal proliferation. Characterisation of hTEC confirmed a mixed progenitor and endothelial cell phenotype. The biodistribution and fate of hTEC were studied using radiolabelled 111Indium oxine and fluorescent in situ hybridisation. Scanning electron microscopy showed earlier endothelial coverage in hTEC-seeded stents as compared to similar BMS. hTEC-seeded BMS achieved complete stent coverage in three days. Quantitative coronary angiography, intravascular ultrasound assessment and histomorphometry showed no difference in neointimal hyperplasia between hTEC-seeded and control BMS. hTEC seeding of coronary stents is a novel and safe approach to accelerate endothelial regeneration without increasing neointimal proliferation.

Common methods for the generation of human embryonic-derived neural stem cells (hNSCs) result in cells with potentially compromised safety profiles due to maintenance of cells in conditions containing non-human proteins (e.g. in bovine serum or on mouse fibroblast feeders). Additionally, sufficient expansion of resulting hNSCs for scaling out or up in a clinically relevant time frame has proven to be difficult. Here, we report a strategy that produces hNSCs in completely "Xeno-Free" culture conditions. Furthermore, we have enriched the hNSCs for the cell surface marker CD133 via magnetic sorting, which has led to an increase in the expansion rate and neuronal fate specification of the hNSCs in vitro. Critically, we have also confirmed neural lineage specificity upon sorted hNSC transplantation into the immunodeficient NOD- scid mouse brain. The future use or adaptation of these protocols has the potential to better facilitate the advancement of pre-clinical strategies from the bench to the bedside. © 2014.

Objective We examined occupational exposures and sperm morphology to establish whether exposures implicated differed from those affecting motile sperm concentration. Methods Computer aided sperm morphometric assessment was undertaken on morphology slides obtained as part of a multi-centre study in 1999–2002 of occupational factors in male infertility. Men attending 14 fertility clinics across the UK were recruited and gave a semen sample. Before results of the semen analysis were known, the men completed detailed questionnaires about their employment and lifestyle. Occupational exposures were assessed by occupational hygienists. Data were analysed using an unmatched case-referent design, allowing for clustering and for confounders. Three case definitions were used: poor morphology (normal morphology 6 ) and either condition. Results Morphology results were available for 1861/2011 men employed at the time of recruitment. Of these 1861, 296 (15.9%) had poor morphology; of the 2011with sperm count, 453 (22.5%) had low MSC; 654/1981 (33.0%) had either condition. Poor morphology, adjusted for confounding, was related to self-reported lifetime exposure to lead (OR=1.33; 95% CI 1.00 to 1.75). Low MSC was also related to self-reported lead and to hygienist-assessed glycol ether exposure. Self-reported use of paint stripper (OR=1.47; 95% CI 1.07 to 2.03) and lead, but not glycol ether, were significantly related to the combined case definition. Conclusions While this study did not identify any occupational exposure uniquely related to sperm morphology, the capacity of the study to detect risk was increased by including morphology with sperm concentration and motility.