Human disturbance and shifts in vertebrate community composition in a biodiversity hotspot.

Understanding how human modification of the landscape shapes vertebrate community composition is vital to understanding the current status and future trajectory of wildlife. Using a participatory approach, we deployed the largest camera-trap network in Mesoamerica to date to investigate how anthropogenic disturbance shapes the occupancy and co-occurrence of terrestrial vertebrate species in a tropical biodiversity hotspot: the Osa Peninsula, Costa Rica. We estimated species richness in different categories of land protection with rarefaction analysis and estimated the expected occupancy with a joint species distribution model that included covariates for anthropogenic disturbance, land protection, habitat quality, and habitat availability. Areas with the most stringent land-use protections (e.g., Corcovado National Park, 24 species [95% CI 23-25]) harbored significantly more species than unprotected areas (20 species [19.7-20.3]), mainly due to a reduced presence of large-bodied species of conservation concern in unprotected areas (e.g., jaguar Panthera onca and white-lipped peccary Tayassu pecari). Small-bodied generalist species, such as opossums (Didelphidae) and armadillos (Dasypus novemcinctus), in contrast, were more common at disturbed sites, resulting in a significant difference in vertebrate community composition between sites with low and high disturbance. Co-occurrence of species was also mainly associated with response to disturbance. Similar responses to disturbance create two groups of species, those whose site-level occupancy usually increased as anthropogenic disturbance increased and those whose estimated occupancy decreased. The absence of large-bodied species entails an important loss of ecological function in disturbed areas and can hinder forest development and maintenance. Efforts to protect and restore forested landscapes are likely having a positive effect on the abundance of some threatened species. These efforts, however, must be sustained and expanded to increase connectivity and ensure the long-term viability of the wildlife community.

Perturbaciones Humanas y Cambios en la Composición de la Comunidad de Vertebrados en un Punto Caliente de Biodiversidad Resumen El entendimiento de cómo las modificaciones humanas del paisaje conforman la composición de las comunidades de vertebrados es vital para entender el estado actual y la trayectoria futura de la fauna. Mediante una estrategia participativa, desplegamos la mayor red de cámaras trampa en Mesoamérica hasta la fecha para investigar cómo la perturbación antropogénica determina la ocupación y coocurrencia de las especies terrestres de vertebrados en un punto caliente de biodiversidad tropical: la Península de Osa, Costa Rica. Estimamos la riqueza de especies en diferentes categorías de protección de suelo con un análisis de rarefacción y estimamos la ocupación esperada con un modelo de distribución conjunta de especies que incluyó covariables para la perturbación antropogénica, la protección del suelo, la calidad del hábitat y la disponibilidad del hábitat. Las áreas con la protección más estricta de uso de suelo (p. ej.: Parque Nacional Corcovado, 24 especies [95% CI 23-25]) albergaron significativamente a más especies que las áreas desprotegidas (20 especies [19.7-20.3]), principalmente debido a la presencia reducida de especies de talla grande de interés para la conservación en las áreas desprotegidas (p. ej.: el jaguar Panthera onca, el pecarí de labios blancos, Tayassu pecari). Al contrario, las especies generalistas de talla pequeña, como las zarigüeyas (Didelphidae) y el armadillo (Dasypus novemcinctus) fueron más comunes en los sitios perturbados, lo que resulta en una diferencia significativa en la composición de las comunidades de vertebrados entre los sitios con una perturbación baja y alta. La coocurrencia de especies también estuvo asociada principalmente con la respuesta a la perturbación. Las respuestas similares a la perturbación crean dos grupos de especies: aquellas cuya ocupación a nivel de sitio generalmente incrementó conforme incrementó la perturbación antropogénica y aquellas cuya ocupación estimada disminuyó. La ausencia de especies de talla grande conlleva una pérdida importante de la función ecológica en las áreas perturbadas y puede dificultar el desarrollo y mantenimiento del bosque. Los esfuerzos para proteger y restaurar los paisajes forestales probablemente estén teniendo un efecto positivo sobre la abundancia de algunas especies amenazadas. Estos esfuerzos, sin embargo, deben ser sostenidos y expandidos para incrementar la conectividad y asegurar la viabilidad a largo plazo de la comunidad faunística.

A recovery network leads to the natural recolonization of an archipelago and a potential trailing edge refuge.

Rapid environmental change is reshaping ecosystems and driving species loss globally. Carnivore populations have declined and retracted rapidly and have been the target of numerous translocation projects. Success, however, is complicated when these efforts occur in novel ecosystems. Identifying refuges, locations that are resistant to environmental change, within a translocation framework should improve population recovery and persistence. American martens (Martes americana) are the most frequently translocated carnivore in North America. As elsewhere, martens were extirpated across much of the Great Lakes region by the 1930s and, despite multiple translocations beginning in the 1950s, martens remain of regional conservation concern. Surprisingly, martens were rediscovered in 2014 on the Apostle Islands of Lake Superior after a putative absence of >40 yr. To identify the source of martens to the islands and understand connectivity of the reintroduction network, we collected genetic data on martens from the archipelago and from all regional reintroduction sites. In total, we genotyped 483 individual martens, 43 of which inhabited the Apostle Islands (densities 0.42-1.46 km-2 ). Coalescent analyses supported the contemporary recolonization of the Apostle Islands with progenitors likely originating from Michigan, which were sourced from Ontario. We also identified movements by a first-order relative between the Apostle Islands and the recovery network. We detected some regional gene flow, but in an unexpected direction: individuals moving from the islands to the mainland. Our findings suggest that the Apostle Islands were naturally recolonized by progeny of translocated individuals and now act as a source back to the reintroduction sites on the mainland. We suggest that the Apostle Islands, given its protection from disturbance, complex forest structure, and reduced carnivore competition, will act as a potential refuge for marten along their trailing range boundary and a central node for regional recovery. Our work reveals that translocations, even those occurring along southern range boundaries, can create recovery networks that function like natural metapopulations. Identifying refuges, locations that are resistant to environmental change, within these recovery networks can further improve species recovery, even within novel environments. Future translocation planning should a priori identify potential refuges and sources to improve short-term recovery and long-term persistence.

Impacts of lake breeze meteorology on ozone gradient observations along Lake Michigan Shorelines in Wisconsin.

Daytime onshore lake breezes are a critical factor controlling ozone abundance at coastal sites around Lake Michigan. Coastal counties along the western shore of Lake Michigan have historically observed high ozone episodes dating to the 1970s. We classified ozone episode days based on the extent or absence of the lake breeze (i.e., "inland", "near-shore" or "no" lake breeze) to establish a climatology of these events. This work demonstrated variable gradients in ozone abundances based on these different types of meteorology, with the sharpest ozone concentration gradients on days with a near-shore lake breeze. On 76-82% of days in which ozone reached 70 ppb for at least 1 hour, a lake breeze was present. Evidence of ozone gradients from multiple observation platforms during the 2017 Lake Michigan Ozone Study (LMOS 2017) are shown for two days with different depths of lake breezes.

A landscape of overlapping risks for wolf-human conflict in Wisconsin, USA.

Managing risk requires an adequate understanding of risk-factors that influence the likelihood of a particular event occurring in time and space. Risk maps can be valuable tools for natural resource managers, allowing them to better understand spatial characteristics of risk. Risk maps can also support risk-avoidance efforts by identifying which areas are relatively riskier than others. However, risks, such as human-carnivore conflict, can be diverse, multi-faceted, and overlapping in space. Yet, efforts to describe risk typically focus on only one aspect of risk. We examined wolf complaints investigated in Wisconsin, USA for the period of 1999-2011. We described the spatial patterns of four types of wolf-human conflict: livestock depredation, depredation on hunting hounds, depredation on non-hound dogs, and human health and safety concerns (HHSC). Using predictive landscape models and discriminant functions analysis, we visualized the landscape of risk as a continuous surface of overlapping risks. Each type of conflict had its own unique landscape signature; however, the probability of any type of conflict increased closer to the center of wolf pack territories and with increased forest cover. Hunting hound depredations tended to occur in areas considered to be highly suitable wolf habitat, while livestock depredations occurred more regularly in marginal wolf habitat. HHSC and non-hound dog depredations were less predictable spatially but tended to occur in areas with low housing density adjacent to large wildland areas. Similar to other research evaluating the risk of human-carnivore conflict, our data suggests that human-carnivore conflict is most likely to occur where humans or human property and large carnivores co-occur. However, identifying areas of co-occurrence is only marginally valuable from a conservation standpoint and could be described using spatially-explicit human and carnivore data without complex analytical approaches. These results challenge our traditional understanding of risk and the standard approach used in describing risk. We suggest that a more comprehensive understanding of the risk of human-carnivore conflict can be achieved by examining the spatial and non-spatial factors influencing risk within areas of co-occurrence and by describing the landscape of risk as a continuous surface of multiple overlapping risks.

A novel chemopreventive mechanism for a traditional medicine: East Indian sandalwood oil induces autophagy and cell death in proliferating keratinocytes.

One of the primary components of the East Indian sandalwood oil (EISO) is α-santalol, a molecule that has been investigated for its potential use as a chemopreventive agent in skin cancer. Although there is some evidence that α-santalol could be an effective chemopreventive agent, to date, purified EISO has not been extensively investigated even though it is widely used in cultures around the world for its health benefits as well as for its fragrance and as a cosmetic. In the current study, we show for the first time that EISO-treatment of HaCaT keratinocytes results in a blockade of cell cycle progression as well as a concentration-dependent inhibition of UV-induced AP-1 activity, two major cellular effects known to drive skin carcinogenesis. Unlike many chemopreventive agents, these effects were not mediated through an inhibition of signaling upstream of AP-1, as EISO treatment did not inhibit UV-induced Akt or MAPK activity. Low concentrations of EISO were found to induce HaCaT cell death, although not through apoptosis as annexin V and PARP cleavage were not found to increase with EISO treatment. However, plasma membrane integrity was severely compromised in EISO-treated cells, which may have led to cleavage of LC3 and the induction of autophagy. These effects were more pronounced in cells stimulated to proliferate with bovine pituitary extract and EGF prior to receiving EISO. Together, these effects suggest that EISO may exert beneficial effects upon skin, reducing the likelihood of promotion of pre-cancerous cells to actinic keratosis (AK) and skin cancer.

Uncertainty and precaution in hunting wolves twice in a year: Reanalysis of Treves and Louchouarn.

Management of wolves is controversial in many jurisdictions where wolves live, which underscores the importance of rigor, transparency, and reproducibility when evaluating outcomes of management actions. Treves and Louchouarn 2022 (hereafter TL) predicted outcomes for various fall 2021 hunting scenarios following Wisconsin's judicially mandated hunting and trapping season in spring 2021, and concluded that even a zero harvest scenario could result in the wolf population declining below the population goal of 350 wolves specified in the 1999 Wisconsin wolf management plan. TL further concluded that with a fall harvest of > 16 wolves there was a "better than average possibility" that the wolf population size would decline below that 350-wolf threshold. We show that these conclusions are incorrect and that they resulted from mathematical errors and selected parameterizations that were consistently biased in the direction that maximized mortality and minimized reproduction (i.e., positively biased adult mortality, negatively biased pup survival, further halving pup survival to November, negatively biased number of breeding packs, and counting harvested wolves twice among the dead). These errors systematically exaggerated declines in predicted population size and resulted in erroneous conclusions that were not based on the best available or unbiased science. Corrected mathematical calculations and more rigorous parameterization resulted in predicted outcomes for the zero harvest scenario that more closely coincided with the empirical population estimates in 2022 following a judicially prevented fall hunt in 2021. Only in scenarios with simulated harvest of 300 or more wolves did probability of crossing the 350-wolf population threshold exceed zero. TL suggested that proponents of some policy positions bear a greater burden of proof than proponents of other positions to show that "their estimates are accurate, precise, and reproducible". In their analysis, TL failed to meet this standard that they demanded of others.

Species-specific responses to white-nose syndrome in the Great Lakes region.

White-nose syndrome is a fungal disease that is threatening bat populations across North America. The disease primarily affects cave-hibernating bats by depleting fat reserves during hibernation and causing a range of other physiological consequences when immune responses are suppressed. Since it was first detected in 2006, the disease has killed millions of bats and is responsible for extensive local extinctions. To better understand the effects of white-nose syndrome on various bat species, we analyzed summer acoustic survey data collected from 2016 to 2020 at nine US National Parks within the Great Lakes region. We examined the effect that white-nose syndrome, time of the year relative to pup volancy, habitat type, and regional variation (i.e., park) have on the acoustic abundance (i.e., mean call abundance) of six bat species. As expected, little brown bat (Myotis lucifugus) and northern long-eared bat (Myotis septentrionalis), both hibernating species, experienced a significant decline in acoustic abundance following white-nose syndrome detection. We observed a significant increase in acoustic abundance as white-nose syndrome progressed for hoary bats (Lasiurus cinereus) and silver-haired bats (Lasionycteris noctivagans), both migratory species that are not impacted by the disease. Contrary to our predictions, we observed an increase in big brown bat (Eptesicus fuscus; hibernating) acoustic abundance and a decrease in eastern red bat (Lasiurus borealis; migratory) acoustic abundance following the detection of white-nose syndrome. We did not observe any significant changes after the onset of white-nose syndrome in the seasonal patterns of acoustic activity related to pup volancy, suggesting that production or recruitment of young may not be affected by the disease. Our results suggest that white-nose syndrome is affecting the acoustic abundance of certain species; however, these changes may not be a result of reduced reproductive success caused by the disease. In addition, species population dynamics may be indirectly affected by white-nose syndrome as a result of reduced competition or a foraging niche release. We also found that for parks located at higher latitudes, little brown bat and northern long-eared bat were more likely to experience greater declines in acoustic abundance as a result of white-nose syndrome. Our work provides insight into species-specific responses to white-nose syndrome at a regional scale and examines factors that may facilitate resistance or resiliency to the disease.

Sleeping Beauty-mediated correction of Fanconi anemia type C.

BACKGROUND: The Sleeping Beauty (SB) transposon system can insert defined sequences into chromosomes to direct the extended expression of therapeutic genes. Our goal is to develop the SB system for nonviral complementation of Fanconi anemia (FA), a rare autosomal recessive disorder accompanied by progressive bone marrow failure. METHODS: We used a CytoPulse electroporation system (CytoPulse, Glen Burnie, MD, USA) to introduce SB transposons into human lymphoblastoid cells (LCL) derived from both Fanconi anemia type C (FA-C) defective and normal patients. Correction of the FA-C defect was assessed by resistance to mitomycin C, a DNA-crosslinking agent. RESULTS: Culture of both cell types with the antioxidant N-acetyl- l-cysteine improved cell viability after electroporation. Co-delivery of enhanced green fluorescent protein (GFP) transposon with SB100X transposase-encoding plasmid supported a 50- to 90-fold increase in stable GFP expression compared to that observed in the absence of SB100X for normal LCL, but in FA-C defective LCL SB100X enhancement of stable GFP-expression was a more moderate five- to 13-fold. SB-mediated integration and expression of the FA-C gene was demonstrated by the emergence of a mitomycin C-resistant population bearing characteristic transposon-chromosome junction sequences and exhibiting a mitomycin dose response identical to that of normal LCL. CONCLUSIONS: The SB transposon system achieved stable expression of therapeutic FA-C genes, complementing the genetic defect in patient-derived cells by nonviral gene transfer.

p38 MAP kinase plays a functional role in UVB-induced mouse skin carcinogenesis.

UVB irradiation of epidermal keratinocytes results in the activation of the p38 mitogen-activated protein kinase (MAPK) pathway and subsequently activator protein-1 (AP-1) transcription factor activation and cyclooxygenase-2 (COX-2) expression. AP-1 and COX-2 have been shown to play functional roles in UVB-induced mouse skin carcinogenesis. In this study, the experimental approach was to express a dominant negative p38α MAPK (p38DN) in the epidermis of SKH-1 hairless mice and assess UVB-induced AP-1 activation, COX-2 expression, and the skin carcinogenesis response in these mice compared to wild-type littermates. We observed a significant inhibition of UVB-induced AP-1 activation and COX-2 expression in p38DN transgenic mice, leading to a significant reduction of UVB-induced tumor number and growth compared to wild-type littermates in a chronic UVB skin carcinogenesis model. A potential mechanism for this reduction in tumor number and growth rate is an inhibition of chronic epidermal proliferation, observed as reduced Ki-67 staining in p38DN mice compared to wild-type. Although we detected no difference in chronic apoptotic rates between transgenic and nontransgenic mice, analysis of acutely irradiated mice demonstrated that expression of the p38DN transgene significantly inhibited UVB-induced apoptosis of keratinocytes. These results counter the concerns that inhibition of p38 MAPK in a chronic situation could compromise the ability of the skin to eliminate potentially tumorigenic cells. Our data indicate that p38 MAPK is a good target for pharmacological intervention for UV-induced skin cancer in patients with sun damaged skin, and suggest that inhibition of p38 signaling reduces skin carcinogenesis by inhibiting COX-2 expression and proliferation of UVB-irradiated cells.

Vivid biofluorescence discovered in the nocturnal Springhare (Pedetidae).

Biofluorescence has been detected in several nocturnal-crepuscular organisms from invertebrates to birds and mammals. Biofluorescence in mammals has been detected across the phylogeny, including the monotreme duck-billed platypus (Ornithorhyncus anatinus), marsupial opossums (Didelphidae), and New World placental flying squirrels (Gluacomys spp.). Here, we document vivid biofluorescence of springhare (Pedetidae) in both museum specimens and captive individuals-the first documented biofluorescence of an Old World placental mammal. We explore the variation in biofluorescence across our sample and characterize its physical and chemical properties. The striking visual patterning and intensity of color shift was unique relative to biofluorescence found in other mammals. We establish that biofluorescence in springhare likely originates within the cuticle of the hair fiber and emanates, at least partially, from several fluorescent porphyrins and potentially one unassigned molecule absent from our standard porphyrin mixture. This discovery further supports the hypothesis that biofluorescence may be ecologically important for nocturnal-crepuscular mammals and suggests that it may be more broadly distributed throughout Mammalia than previously thought.

Cardiac myofibroblast differentiation is attenuated by alpha(3) integrin blockade: potential role in post-MI remodeling.

Cardiac fibroblasts and myofibroblasts are responsible for post-MI remodeling which occurs via regulation of extracellular matrix (ECM). Accelerated post-MI remodeling leads to excessive ECM deposition and fibrosis, contributing to impaired contractile function, arrhythmias, and heart failure. We have previously reported that type VI collagen induces myofibroblast differentiation in cultured cardiac fibroblasts, and that type VI collagen and myofibroblast content were both elevated in the myocardium 20 weeks post-MI. The purpose of this study was to determine the expression patterns of type VI collagen and myofibroblast content in early post-myocardial infarction (MI) remodeling to gain insight into whether type VI collagen induces in vivo myofibroblast differentiation via specific matrix-receptor interactions. Adult male Sprague-Dawley rats were anesthetized and left coronary arteries were permanently ligated. Histological tissue sections and whole tissue protein lysates were obtained from infarcted and non-infarcted areas of MI hearts and sham operated controls. At 3 days post-MI, we observed a significant increase in alpha(3) integrin expression (2.02+/-0.18 fold); at 7 days post-infarction both type VI collagen (2.27+/-0.18 fold) and myofibroblast (4.65+/-0.6 fold) content increased. By 14 days myofibroblast content returned to sham control levels, although type VI collagen (2.42+/-0.11 fold) was still elevated. In vitro cross-linking confirmed that the alpha(3) integrin interacts with type VI collagen, and alpha(3) integrin function blocking antibodies inhibited the differentiation of isolated cardiac fibroblasts. Collectively, our in vitro results indicate that the alpha(3) integrin receptor interacts with type VI collagen to promote myofibroblast differentiation, and that this interaction may impact in vivo post-MI remodeling.

Transgene Expression in Dogs After Liver-Directed Hydrodynamic Delivery of Sleeping Beauty Transposons Using Balloon Catheters.

The Sleeping Beauty transposon system has been extensively tested for integration of reporter and therapeutic genes in vitro and in vivo in mice. Dogs were used as a large animal model for human therapy and minimally invasive infusion of DNA solutions. DNA solutions were delivered into the entire liver or the left side of the liver using balloon catheters for temporary occlusion of venous outflow. A peak intravascular pressure between 80 and 140 mmHg supported sufficient DNA delivery in dog liver for detection of secretable reporter proteins. Secretable reporters allowed monitoring of the time course of gene products detectable in the circulation postinfusion. Canine secreted alkaline phosphatase reporter protein levels were measured in plasma, with expression detectable for up to 6 weeks, while expression of canine erythropoietin was detectable for 7-10 days. All animals exhibited a transient increase in blood transaminases that normalized within 10 days; otherwise the treated animals were clinically normal. These results demonstrate the utility of a secreted reporter protein for real-time monitoring of gene expression in the liver in a large animal model but highlight the need for improved delivery in target tissues to support integration and long-term expression of Sleeping Beauty transposons.

Prolonged Expression of Secreted Enzymes in Dogs After Liver-Directed Delivery of Sleeping Beauty Transposons: Implications for Non-Viral Gene Therapy of Systemic Disease.

The non-viral, integrating Sleeping Beauty (SB) transposon system is efficient in treating systemic monogenic disease in mice, including hemophilia A and B caused by deficiency of blood clotting factors and mucopolysaccharidosis types I and VII caused by α-L-iduronidase (IDUA) and ß-glucuronidase (GUSB) deficiency, respectively. Modified approaches of the hydrodynamics-based procedure to deliver transposons to the liver in dogs were recently reported. Using the transgenic canine reporter secreted alkaline phosphatase (cSEAP), transgenic protein in the plasma was demonstrated for up to 6 weeks post infusion. This study reports that immunosuppression of dogs with gadolinium chloride (GdCl3) prolonged the presence of cSEAP in the circulation up to 5.5 months after a single vector infusion. Transgene expression declined gradually but appeared to stabilize after about 2 months at approximately fourfold baseline level. Durability of transgenic protein expression in the plasma was inversely associated with transient increase of liver enzymes alanine transaminase and aspartate transaminase in response to the plasmid delivery procedure, which suggests a deleterious effect of hepatocellular toxicity on transgene expression. GdCl3 treatment was ineffective for repeat vector infusions. In parallel studies, dogs were infused with potentially therapeutic transposons. Activities of transgenic IDUA and GUSB in plasma peaked at 50-350% of wildtype, but in the absence of immunosuppression lasted only a few days. Transposition was detectable by excision assay only when the most efficient transposase, SB100X, was used. Dogs infused with transposons encoding canine clotting factor IX (cFIX) were treated with GdCl3 and showed expression profiles similar to those in cSEAP-infused dogs, with expression peaking at 40% wt (2 µg/mL). It is concluded that GdCl3 can support extended transgene expression after hydrodynamic introduction of SB transposons in dogs, but that alternative regimens will be required to achieve therapeutic levels of transgene products.

A Broad Range of Dose Optima Achieve High-level, Long-term Gene Expression After Hydrodynamic Delivery of Sleeping Beauty Transposons Using Hyperactive SB100x Transposase.

The Sleeping Beauty (SB) transposon system has been shown to enable long-term gene expression by integrating new sequences into host cell chromosomes. We found that the recently reported SB100x hyperactive transposase conferred a surprisingly high level of long-term expression after hydrodynamic delivery of luciferase-encoding reporter transposons in the mouse. We conducted dose-ranging studies to determine the effect of varying the amount of SB100x transposase-encoding plasmid (pCMV-SB100x) at a set dose of luciferase transposon and of varying the amount of transposon-encoding DNA at a set dose of pCMV-SB100x in hydrodynamically injected mice. Animals were immunosuppressed using cyclophosphamide in order to prevent an antiluciferase immune response. At a set dose of transposon DNA (25 µg), we observed a broad range of pCMV-SB100x doses (0.1-2.5 µg) conferring optimal levels of long-term expression (>10(11) photons/second/cm(2)). At a fixed dose of 0.5 µg of pCMV-SB100x, maximal long-term luciferase expression (>10(10) photons/second/cm(2)) was achieved at a transposon dose of 5-125 µg. We also found that in the linear range of transposon doses (100 ng), co-delivering the CMV-SB100x sequence on the same plasmid was less effective in achieving long-term expression than delivery on separate plasmids. These results show marked flexibility in the doses of SB transposon plus pCMV-SB100x that achieve maximal SB-mediated gene transfer efficiency and long-term gene expression after hydrodynamic DNA delivery to mouse liver.

Inhibition of Akt Enhances the Chemopreventive Effects of Topical Rapamycin in Mouse Skin.

The PI3Kinase/Akt/mTOR pathway has important roles in cancer development for multiple tumor types, including UV-induced nonmelanoma skin cancer. Immunosuppressed populations are at increased risk of aggressive cutaneous squamous cell carcinoma (SCC). Individuals who are treated with rapamycin (sirolimus, a classical mTOR inhibitor) have significantly decreased rates of developing new cutaneous SCCs compared with those that receive traditional immunosuppression. However, systemic rapamycin use can lead to significant adverse events. Here, we explored the use of topical rapamycin as a chemopreventive agent in the context of solar-simulated light (SSL)-induced skin carcinogenesis. In SKH-1 mice, topical rapamycin treatment decreased tumor yields when applied after completion of 15 weeks of SSL exposure compared with controls. However, applying rapamycin during SSL exposure for 15 weeks, and continuing for 10 weeks after UV treatment, increased tumor yields. We also examined whether a combinatorial approach might result in more significant tumor suppression by rapamycin. We validated that rapamycin causes increased Akt (S473) phosphorylation in the epidermis after SSL, and show for the first time that this dysregulation can be inhibited in vivo by a selective PDK1/Akt inhibitor, PHT-427. Combining rapamycin with PHT-427 on tumor prone skin additively caused a significant reduction of tumor multiplicity compared with vehicle controls. Our findings indicate that patients taking rapamycin should avoid sun exposure, and that combining topical mTOR inhibitors and Akt inhibitors may be a viable chemoprevention option for individuals at high risk for cutaneous SCC.

Sleeping Beauty-Mediated Drug Resistance Gene Transfer in Human Hematopoietic Progenitor Cells.

The Sleeping Beauty (SB) transposon system can insert sequences into mammalian chromosomes, supporting long-term expression of both reporter and therapeutic genes. Hematopoietic progenitor cells (HPCs) are an ideal therapeutic gene transfer target as they are used in therapy for a variety of hematologic and metabolic conditions. As successful SB-mediated gene transfer into human CD34(+) HPCs has been reported by several laboratories, we sought to extend these studies to the introduction of a therapeutic gene conferring resistance to methotrexate (MTX), potentially providing a chemoprotective effect after engraftment. SB-mediated transposition of hematopoietic progenitors, using a transposon encoding an L22Y variant dihydrofolate reductase fused to green fluorescent protein, conferred resistance to methotrexate and dipyridamole, a nucleoside transport inhibitor that tightens MTX selection conditions, as assessed by in vitro hematopoietic colony formation. Transposition of individual transgenes was confirmed by sequence analysis of transposon-chromosome junctions recovered by linear amplification-mediated PCR. These studies demonstrate the potential of SB-mediated transposition of HPCs for expression of drug resistance genes for selective and chemoprotective applications.

Role of transgene regulation in ex vivo lentiviral correction of artemis deficiency.

Artemis is a single-stranded endonuclease, deficiency of which results in a radiation-sensitive form of severe combined immunodeficiency (SCID-A) most effectively treated by allogeneic hematopoietic stem cell (HSC) transplantation and potentially treatable by administration of genetically corrected autologous HSCs. We previously reported cytotoxicity associated with Artemis overexpression and subsequently characterized the human Artemis promoter with the intention to provide Artemis expression that is nontoxic yet sufficient to support immunodevelopment. Here we compare the human Artemis promoter (APro) with the moderate-strength human phosphoglycerate kinase (PGK) promoter and the strong human elongation factor-1α (EF1α) promoter to regulate expression of Artemis after ex vivo lentiviral transduction of HSCs in a murine model of SCID-A. Recipient animals treated with the PGK-Artemis vector exhibited moderate repopulation of their immune compartment, yet demonstrated a defective proliferative T lymphocyte response to in vitro antigen stimulation. Animals treated with the EF1α-Artemis vector displayed high levels of T lymphocytes but an absence of B lymphocytes and deficient lymphocyte function. In contrast, ex vivo transduction with the APro-Artemis vector supported effective immune reconstitution to wild-type levels, resulting in fully functional T and B lymphocyte responses. These results demonstrate the importance of regulated Artemis expression in immune reconstitution of Artemis-deficient SCID.

Simple and efficient methods for enrichment and isolation of endonuclease modified cells.

The advent of Transcription Activator-Like Effector Nucleases (TALENs), and similar technologies such as CRISPR, provide a straightforward and cost effective option for targeted gene knockout (KO). Yet, there is still a need for methods that allow for enrichment and isolation of modified cells for genetic studies and therapeutics based on gene modified human cells. We have developed and validated two methods for simple enrichment and isolation of single or multiplex gene KO's in transformed, immortalized, and human progenitor cells. These methods rely on selection of a phenotypic change such as resistance to a particular drug or ability to grow in a selective environment. The first method, termed co-transposition, utilizes integration of a piggyBac transposon vector encoding a drug resistance gene. The second method, termed co-targeting, utilizes TALENs to KO any gene that when lost induces a selectable phenotype. Using these methods we also show removal of entire genes and demonstrate that TALENs function in human CD34+ progenitor cells. Further, co-transposition can be used to generate conditional KO cell lines utilizing an inducible cDNA rescue transposon vector. These methods allow for robust enrichment and isolation of KO cells in a rapid and efficient manner.