A model of replicating coupled oscillators generates naturally occurring cell networks.

When a founder cell and its progeny divide with incomplete cytokinesis, a network forms in which each intercellular bridge corresponds to a past mitotic event. Such networks are required for gamete production in many animals, and different species have evolved diverse final network topologies. Although mechanisms regulating network assembly have been identified in particular organisms, we lack a quantitative framework to understand network assembly and inter-species variability. Motivated by cell networks responsible for oocyte production in invertebrates, where the final topology is typically invariant within each species, we devised a mathematical model for generating cell networks, in which each node is an oscillator and, after a full cycle, the node produces a daughter to which it remains connected. These cell cycle oscillations are transient and coupled via diffusion over the edges of the network. By variation of three biologically motivated parameters, our model generates nearly all such networks currently reported across invertebrates. Furthermore, small parameter variations can rationalize cases of intra-species variation. Because cell networks outside of the ovary often form less deterministically, we propose model generalizations to account for sources of stochasticity.

Process analysis of pluripotent stem cell differentiation to megakaryocytes to make platelets applying European GMP.

Quality, traceability and reproducibility are crucial factors in the reliable manufacture of cellular therapeutics, as part of the overall framework of Good Manufacturing Practice (GMP). As more and more cellular therapeutics progress towards the clinic and research protocols are adapted to comply with GMP standards, guidelines for safe and efficient adaptation have become increasingly relevant. In this paper, we describe the process analysis of megakaryocyte manufacture from induced pluripotent stem cells with a view to manufacturing in vitro platelets to European GMP for transfusion. This process analysis has allowed us an overview of the entire manufacturing process, enabling us to pinpoint the cause and severity of critical risks. Risk mitigations were then proposed for each risk, designed to be GMP compliant. These mitigations will be key in advancing this iPS-derived therapy towards the clinic and have broad applicability to other iPS-derived cellular therapeutics, many of which are currently advancing towards GMP-compliance. Taking these factors into account during protocol design could potentially save time and money, expediting the advent of safe, novel therapeutics from stem cells.

Pleiotropy enables specific and accurate signaling in the presence of ligand cross talk.

Living cells sense their environment through the binding of extracellular molecular ligands to cell surface receptors. Puzzlingly, vast numbers of signaling pathways exhibit a high degree of cross talk between different signals whereby different ligands act through the same receptor or shared components downstream. It remains unclear how a cell can accurately process information from the environment in such cross-wired pathways. We show that a feature which commonly accompanies cross talk-signaling pleiotropy (the ability of a receptor to produce multiple outputs)-offers a solution to the cross-talk problem. In a minimal model we show that a single pleiotropic receptor can simultaneously identify and accurately sense the concentrations of arbitrary unknown ligands present individually or in a mixture. We calculate the fundamental limits of the signaling specificity and accuracy of such signaling schemes. The model serves as an elementary "building block" toward understanding more complex cross-wired receptor-ligand signaling networks.

Roles of phenotypic heterogeneity and microenvironment feedback in early tumor development.

The local microenvironment of a tumor plays an important and commonly observed role in cancer development and progression. Dynamic changes in the tissue microenvironment are thought to epigenetically disrupt healthy cellular phenotypes and drive cancer incidence. Despite the experimental work in this area there are no conceptual models to understand the interplay between the epigenetic dysregulation in the microenvironment of early tumors and the appearance of cancer driver mutations. Here, we develop a minimal model of the tissue microenvironment which considers three interacting subpopulations: healthy, phenotypically dysregulated, and mutated cancer cells. Healthy cells can epigenetically (reversibly) transition to the dysregulated phenotype, and from there to the cancer state. The epigenetic transition rates of noncancer cells can be influenced by the number of cancer cells in the microenvironment (termed microenvironment feedback). Our model delineates the regime in which microenvironment feedback accelerates the rate of cancer initiation. In addition, the model shows when and how microenvironment feedback may inhibit cancer progression. We discuss how our framework may provide resolution to some of the puzzling experimental observations of slow cancer progression.

Do the Quality and Quantity of Honey Bee-Collected Pollen Vary Across an Agricultural Land-Use Gradient?

Pollen is the source of protein for most bee species, yet the quality and quantity of pollen is variable across landscapes and growing seasons. Understanding the role of landscapes in providing nutritious forage to bees is important for pollinator health, particularly in areas undergoing significant land-use change such as in the Northern Great Plains (NGP) region of the United States where grasslands are being converted to row crops. We investigated how the quality and quantity of pollen collected by honey bees (Apis mellifera L. [Hymenoptera: Apidae]) changed with land use and across the growing season by sampling bee-collected pollen from apiaries in North Dakota, South Dakota, and Minnesota, USA, throughout the flowering season in 2015-2016. We quantified protein content and quantity of pollen to investigate how they varied temporally and across a land-use gradient of grasslands to row crops. Neither pollen weight nor crude protein content varied linearly across the land-use gradient; however, there were significant interactions between land use and sampling date across the season, particularly in grasslands. Generally, pollen protein peaked mid-July while pollen weight had two maxima in late-June and late-August. Results suggest that while land use itself may not correlate with the quality or quantity of pollen resources collected by honey bees among our study apiaries, the nutritional landscape of the NGP is seasonally dynamic, especially in certain land covers, and may impose seasonal resource limitations for both managed and native bee species. Furthermore, results indicate periods of qualitative and quantitative pollen dearth may not coincide.

Nutritional status of honey bee (Apis mellifera L.) workers across an agricultural land-use gradient.

Land use, habitat, and forage quality have emerged as critical factors influencing the health, productivity, and survival of honey bee colonies. However, characterization of the mechanistic relationship between differential land-use conditions and ultimate outcomes for honey bee colonies has been elusive. We assessed the physiological health of individual worker honey bees in colonies stationed across a gradient of agricultural land use to ask whether indicators of nutritional physiology including glycogen, total sugar, lipids, and protein were associated with land-use conditions over the growing season and colony population size the subsequent spring during almond pollination. Across the observed land-use gradient, we found that September lipid levels related to growing-season land use, with honey bees from apiaries surrounded by more favorable land covers such as grassland, pasture, conservation land, and fallow fields having greater lipid reserves. Further, we observed a significant relationship between total protein during September and population size of colonies during almond pollination the following February. We demonstrate and discuss the utility of quantifying nutritional biomarkers to infer land-use quality and predict colony population size.

The Economics of Honey Bee (Hymenoptera: Apidae) Management and Overwintering Strategies for Colonies Used to Pollinate Almonds.

Commercial honey bee (Apis mellifera L.) colonies significantly contribute to agricultural productivity through crop pollination. Almond production requires the most colonies because there are more than a million acres of orchards that require cross-pollination for nut set. With the rising costs of managing and transporting colonies to almond orchards combined with the high colony losses beekeepers routinely experience, we asked if renting colonies for almond pollination was profitable. We conducted a longitudinal study on 190 colonies from their establishment in April until they were placed in almond orchards 10 mo later. In the fall, equal numbers of colonies were placed either in cold storage (CS) facilities or in outdoor apiaries for the winter. We found that the cost of overwintering colonies in CS was lower than in apiaries, but CS did not reduce overwintering losses. A key finding from our study is that there is little or no profit in renting colonies for almond pollination once summer management and overwintering costs are considered. Our only profitable venture was honey production in the summer. We propose alternative management strategies to lower costs and make almond pollination profitable. We also developed a decision tool for selecting colonies to overwinter in CS and reduce expenditures on those that will not reach sufficient size for almond pollination. Our study exposes the unsustainable financial burden experienced by migratory beekeepers that is not included in estimates of yearly colony losses, and underscores the urgent need for forage plantings to generate revenue from honey and improve overwinter survival.

Nonsense-mediated mRNA decay efficiency varies in choroideremia providing a target to boost small molecule therapeutics.

Choroideremia (CHM) is an x-linked recessive chorioretinal dystrophy, with 30% caused by nonsense mutations in the CHM gene resulting in an in-frame premature termination codon (PTC). Nonsense-mediated mRNA decay (NMD) is the cell's natural surveillance mechanism that detects and destroys PTC-containing transcripts, with UPF1 being the central NMD modulator. NMD efficiency can be variable amongst individuals with some transcripts escaping destruction, leading to the production of a truncated non-functional or partially functional protein. Nonsense suppression drugs, such as ataluren, target these transcripts and read-through the PTC, leading to the production of a full length functional protein. Patients with higher transcript levels are considered to respond better to these drugs, as more substrate is available for read-through. Using Quantitative reverse transcription PCR (RT-qPCR), we show that CHM mRNA expression in blood from nonsense mutation CHM patients is 2.8-fold lower than controls, and varies widely amongst patients, with 40% variation between those carrying the same UGA mutation [c.715 C>T; p.(R239*)]. These results indicate that although NMD machinery is at work, efficiency is highly variable and not wholly dependent on mutation position. No significant difference in CHM mRNA levels was seen between two patients' fibroblasts and their induced pluripotent stem cell-derived retinal pigment epithelium. There was no correlation between CHM mRNA expression and genotype, phenotype or UPF1 transcript levels. NMD inhibition with caffeine was shown to restore CHM mRNA transcripts to near wild-type levels. Baseline mRNA levels may provide a prognostic indicator for response to nonsense suppression therapy, and caffeine may be a useful adjunct to enhance treatment efficacy where indicated.

Past role and future outlook of the Conservation Reserve Program for supporting honey bees in the Great Plains.

Human dependence on insect pollinators continues to grow even as pollinators face global declines. The Northern Great Plains (NGP), a region often referred to as America's last honey bee (Apis mellifera) refuge, has undergone rapid land-cover change due to cropland expansion and weakened land conservation programs. We conducted a trend analysis and estimated conversion rates of Conservation Reserve Program (CRP) enrollments around bee apiaries from 2006 to 2016 and developed models to identify areas of habitat loss. Our analysis revealed that NGP apiaries lost over 53% of lands enrolled in the CRP, and the rate of loss was highest in areas of high apiary density. We estimated over 163,000 ha of CRP lands in 2006 within 1.6 km of apiaries was converted to row crops by 2012. We also evaluated how alternative scenarios of future CRP acreage caps may affect habitat suitability for supporting honey bee colonies. Our scenario revealed that a further reduction in CRP lands to 7.7 million ha nationally would reduce the number of apiaries in the NGP that meet defined forage criteria by 28% on average. Alternatively, increasing the national cap to 15 million ha would increase the number of NGP apiaries that meet defined forage criteria by 155%. Our scenarios also show that strategic placement of CRP lands near existing apiaries increased the number of apiaries that meet forage criteria by 182%. Our research will be useful for informing the potential consequences of future US farm bill policy and land management in the epicenter of the US beekeeping industry.

Rescue of the MERTK phagocytic defect in a human iPSC disease model using translational read-through inducing drugs.

Inherited retinal dystrophies are an important cause of blindness, for which currently there are no effective treatments. In order to study this heterogeneous group of diseases, adequate disease models are required in order to better understand pathology and to test potential therapies. Induced pluripotent stem cells offer a new way to recapitulate patient specific diseases in vitro, providing an almost limitless amount of material to study. We used fibroblast-derived induced pluripotent stem cells to generate retinal pigment epithelium (RPE) from an individual suffering from retinitis pigmentosa associated with biallelic variants in MERTK. MERTK has an essential role in phagocytosis, one of the major functions of the RPE. The MERTK deficiency in this individual results from a nonsense variant and so the MERTK-RPE cells were subsequently treated with two translational readthrough inducing drugs (G418 & PTC124) to investigate potential restoration of expression of the affected gene and production of a full-length protein. The data show that PTC124 was able to reinstate phagocytosis of labeled photoreceptor outer segments at a reduced, but significant level. These findings represent a confirmation of the usefulness of iPSC derived disease specific models in investigating the pathogenesis and screening potential treatments for these rare blinding disorders.

Mechanism and evidence of nonsense suppression therapy for genetic eye disorders.

Between 5 and 70% of genetic disease is caused by in-frame nonsense mutations, which introduce a premature termination codon (PTC) within the disease-causing gene. Consequently, during translation, non-functional or gain-of-function truncated proteins of pathological significance, are formed. Approximately 50% of all inherited retinal disorders have been associated with PTCs, highlighting the importance of novel pharmacological or gene correction therapies in ocular disease. Pharmacological nonsense suppression of PTCs could delineate a therapeutic strategy that treats the mutation in a gene- and disease-independent manner. This approach aims to suppress the fidelity of the ribosome during protein synthesis so that a near-cognate aminoacyl-tRNA, which shares two of the three nucleotides of the PTC, can be inserted into the peptide chain, allowing translation to continue, and a full-length functional protein to be produced. Here we discuss the mechanisms and evidence of nonsense suppression agents, including the small molecule drug ataluren (or PTC124) and next generation 'designer' aminoglycosides, for the treatment of genetic eye disease.

Opposing effects of cationic antimicrobial peptides and divalent cations on bacterial lipopolysaccharides.

The permeability of the bacterial outer membrane, enclosing Gram-negative bacteria, depends on the interactions of the outer, lipopolysaccharide (LPS) layer, with surrounding ions and molecules. We present a coarse-grained model for describing how cationic amphiphilic molecules (e.g., antimicrobial peptides) interact with and perturb the LPS layer in a biologically relevant medium, containing monovalent and divalent salt ions (e.g., Mg^{2+}). In our approach, peptide binding is driven by electrostatic and hydrophobic interactions and is assumed to expand the LPS layer, eventually priming it for disruption. Our results suggest that in parameter ranges of biological relevance (e.g., at micromolar concentrations) the antimicrobial peptide magainin 2 effectively disrupts the LPS layer, even though it has to compete with Mg^{2+} for the layer. They also show how the integrity of LPS is restored with an increasing concentration of Mg^{2+}. Using the approach, we make a number of predictions relevant for optimizing peptide parameters against Gram-negative bacteria and for understanding bacterial strategies to develop resistance against cationic peptides.

Mislocalisation of BEST1 in iPSC-derived retinal pigment epithelial cells from a family with autosomal dominant vitreoretinochoroidopathy (ADVIRC).

Autosomal dominant vitreoretinochoroidopathy (ADVIRC) is a rare, early-onset retinal dystrophy characterised by distinct bands of circumferential pigmentary degeneration in the peripheral retina and developmental eye defects. ADVIRC is caused by mutations in the Bestrophin1 (BEST1) gene, which encodes a transmembrane protein thought to function as an ion channel in the basolateral membrane of retinal pigment epithelial (RPE) cells. Previous studies suggest that the distinct ADVIRC phenotype results from alternative splicing of BEST1 pre-mRNA. Here, we have used induced pluripotent stem cell (iPSC) technology to investigate the effects of an ADVIRC associated BEST1 mutation (c.704T > C, p.V235A) in patient-derived iPSC-RPE. We found no evidence of alternate splicing of the BEST1 transcript in ADVIRC iPSC-RPE, however in patient-derived iPSC-RPE, BEST1 was expressed at the basolateral membrane and the apical membrane. During human eye development we show that BEST1 is expressed more abundantly in peripheral RPE compared to central RPE and is also expressed in cells of the developing retina. These results suggest that higher levels of mislocalised BEST1 expression in the periphery, from an early developmental stage, could provide a mechanism that leads to the distinct clinical phenotype observed in ADVIRC patients.

Land-use change reduces habitat suitability for supporting managed honey bee colonies in the Northern Great Plains.

Human reliance on insect pollination services continues to increase even as pollinator populations exhibit global declines. Increased commodity crop prices and federal subsidies for biofuel crops, such as corn and soybeans, have contributed to rapid land-use change in the US Northern Great Plains (NGP), changes that may jeopardize habitat for honey bees in a part of the country that supports >40% of the US colony stock. We investigated changes in biofuel crop production and grassland land covers surrounding ∼18,000 registered commercial apiaries in North and South Dakota from 2006 to 2014. We then developed habitat selection models to identify remotely sensed land-cover and land-use features that influence apiary site selection by Dakota beekeepers. Our study demonstrates a continual increase in biofuel crops, totaling 1.2 Mha, around registered apiary locations in North and South Dakota. Such crops were avoided by commercial beekeepers when selecting apiary sites in this region. Furthermore, our analysis reveals how grasslands that beekeepers target when selecting commercial apiary locations are becoming less common in eastern North and South Dakota, changes that may have lasting impact on pollinator conservation efforts. Our study highlights how land-use change in the NGP is altering the landscape in ways that are seemingly less conducive to beekeeping. Our models can be used to guide future conservation efforts highlighted in the US national pollinator health strategy by identifying areas that support high densities of commercial apiaries and that have exhibited significant land-use changes.

Functional rescue of REP1 following treatment with PTC124 and novel derivative PTC-414 in human choroideremia fibroblasts and the nonsense-mediated zebrafish model.

Choroideremia (CHM) is an X-linked chorioretinal dystrophy that is caused by mutations within a single gene, CHM Currently no effective treatment exists for these patients. Since over 30% of patients harbour nonsense mutations in CHM, nonsense suppression therapy using translational readthrough inducing drugs may provide functional rescue of REP1, thus attenuating progressive sight loss. Here, we employed two CHM model systems to systematically test the efficacy and safety of ataluren (PTC124) and its novel analog PTC-414: (1) the chmru848 zebrafish, the only nonsense mutation animal model of CHM harbouring a TAA nonsense mutation, and (2) a primary human fibroblast cell line from a CHM patient harbouring a TAG nonsense mutation. PTC124 or PTC-414 treatment of chmru848 embryos led to a ∼2.0-fold increase in survival, prevented the onset of retinal degeneration with reduced oxidative stress and apoptosis, increased rep1 protein by 23.1% (PTC124) and 17.2% (PTC-414) and restored biochemical function as confirmed through in vitro prenylation assays (98 ± 2% [PTC124] and 68 ± 5% [PTC-414]). In CHMY42X/y fibroblasts, there was a recovery of prenylation activity following treatment with either PTC124 (42 ± 5%) or PTC-414 (36 ± 11%), although an increase in REP1 protein was not detected in these cells, in contrast to the zebrafish model. This comprehensive study on the use of PTC124 and PTC-414 as successful nonsense suppression agents for the treatment of CHM highlights the translational potential of these drugs for inherited retinal disease.

Linking Measures of Colony and Individual Honey Bee Health to Survival among Apiaries Exposed to Varying Agricultural Land Use.

We previously characterized and quantified the influence of land use on survival and productivity of colonies positioned in six apiaries and found that colonies in apiaries surrounded by more land in uncultivated forage experienced greater annual survival, and generally more honey production. Here, detailed metrics of honey bee health were assessed over three years in colonies positioned in the same six apiaries. The colonies were located in North Dakota during the summer months and were transported to California for almond pollination every winter. Our aim was to identify relationships among measures of colony and individual bee health that impacted and predicted overwintering survival of colonies. We tested the hypothesis that colonies in apiaries surrounded by more favorable land use conditions would experience improved health. We modeled colony and individual bee health indices at a critical time point (autumn, prior to overwintering) and related them to eventual spring survival for California almond pollination. Colony measures that predicted overwintering apiary survival included the amount of pollen collected, brood production, and Varroa destructor mite levels. At the individual bee level, expression of vitellogenin, defensin1, and lysozyme2 were important markers of overwinter survival. This study is a novel first step toward identifying pertinent physiological responses in honey bees that result from their positioning near varying landscape features in intensive agricultural environments.

Engineering efficient retinal pigment epithelium differentiation from human pluripotent stem cells.

Human embryonic stem cells (hESCs) are a promising source of retinal pigment epithelium (RPE) cells: cells that can be used for the treatment of common and incurable forms of blindness, such as age-related macular degeneration. Although most hESC lines will produce a number of clusters of pigmented RPE cells within 30-50 days when allowed to spontaneously differentiate, the timing and efficiency of differentiation is highly variable. This could prove problematic in the design of robust processes for the large scale production of RPE cells for cell therapy. In this study we sought to identify, quantify, and reduce the sources of variability in hESC-RPE differentiation. By monitoring the emergence of pigmented cells over time, we show how the cell line, passaging method, passage number, and seeding density have a significant and reproducible effect on the RPE yield. To counter this variability, we describe the production of RPE cells from two cell lines in feeder-free, density controlled conditions using single cell dissociation and seeding that is more amenable to scaled up production. The efficacy of small molecules in directing differentiation toward the RPE lineage was tested in two hESC lines with divergent RPE differentiation capacities. Neural induction by treatment with a bone morphogenetic protein inhibitor, dorsomorphin, significantly enhanced the RPE yield in one cell line but significantly reduce it in another, generating instead a Chx10 positive neural progenitor phenotype. This result underlines the necessity to tailor differentiation protocols to suit the innate properties of different cell lines.

Neural retinal regeneration with pluripotent stem cells.

Retinal degeneration represents a huge burden of blinding disease, and currently there are no effective treatments that reverse the most common causes of neural retinal degeneration. Stem cell biology has the potential to significantly ease this burden, not only through the development of disease models of retinal degeneration but also in the manufacture of a replacement for the neural retinal tissue. This review summarizes the major advancements in the last decade in the field of neural retinal regeneration with an emphasis on the differentiation of embryonic and induced pluripotent stem cells into cells with retinal and specifically photoreceptor characteristics.

Stem cells in retinal regeneration: past, present and future.

Stem cell therapy for retinal disease is under way, and several clinical trials are currently recruiting. These trials use human embryonic, foetal and umbilical cord tissue-derived stem cells and bone marrow-derived stem cells to treat visual disorders such as age-related macular degeneration, Stargardt's disease and retinitis pigmentosa. Over a decade of analysing the developmental cues involved in retinal generation and stem cell biology, coupled with extensive surgical research, have yielded differing cellular approaches to tackle these retinopathies. Here, we review these various stem cell-based approaches for treating retinal diseases and discuss future directions and challenges for the field.

Development of human embryonic stem cell therapies for age-related macular degeneration.

Age-related macular degeneration (AMD) is the leading cause of vision loss in older adults and ultimately leads to the death of photoreceptor cells in the macular area of the neural retina. Currently, treatments are only available for patients with the wet form of AMD. In this review, we describe recent approaches to develop cell-based therapies for the treatment of AMD. Recent research has focused on replacing the retinal pigment epithelium (RPE), a monolayer of cells vital to photoreceptor cell health. We discuss the various methods used to differentiate and purify RPE from human embryonic stem cells (HESC), and describe the surgical approaches being used to transplant these cells in existing and forthcoming clinical trials.