The accurate study of cellular microenvironments is limited by the lack of technologies that can manipulate cells in 3D at a sufficiently small length scale. The ability to build and manipulate multicellular microscopic structures will facilitate a more detailed understanding of cellular function in fields such as developmental and stem cell biology. We present a holographic optical tweezers based technology to accurately generate bespoke cellular micro-architectures. Using embryonic stem cells, 3D structures of varying geometries were created and stabilized using hydrogels and cell-cell adhesion methods. Control of chemical microenvironments was achieved by the temporal release of specific factors from polymer microparticles positioned within these constructs. Complex co-culture micro-environmental analogues were also generated to reproduce structures found within adult stem cell niches. The application of holographic optical tweezers-based micromanipulation will enable novel insights into biological microenvironments by allowing researchers to form complex architectures with sub-micron precision of cells, matrices and molecules. The study of multicellular microenvironments is challenging due to the lack of effective technologies to recreate cellular architectures at the micron scale. The generation of more sophisticated tools to study these structures will have applications in numerous areas including basic biochemical and biomedical research, regenerative medicine, tissue engineering, biophysics and many others.The precise architectural position of cells within a particular microenvironment provides the basis for biological function. This is exemplified in early embryogenesis where the organization of less than 16 individual blastomeres controls subsequent development into the morula and blastulaThe structures present within adult organisms are also spatially organized at small length scales, for example within stem cell niches2Caenorhabditis elegans4Drosophila6910in vitro culture10In vitro screening techniques such as robotic spotting121413Early work to determine the structure and function of these interactions used genetic models such as Whilst these techniques have provided invaluable information on regulatory pathways they illustrate the current inability to determine the microscopic structure to function relationships between different cells as well as other micro-environmental components. Holographic optical tweezers (HOTs) are a micromanipulation tool of sufficient resolution to study these properties due to their capacity to position microscopic objects such as cells accurately and in three dimensions (3D)17181920212223Applications of optical tweezers technology have to date focused on determining the biophysical properties of cells2522241721We demonstrate the combination of a HOTs system with controllable and tailored structural elements including polymeric materials, ECM, controlled release microparticles and hydrogels. These elements were micro-manipulated into complex architectures precisely controlling physical and chemical factors to produce micro-environmental analogues. This powerful new tool will enable the study of regulatory mechanisms in diverse cellular microenvironments producing novel insights at a scale and level of micro-architectural complexity that previous techniques have been unable to provide.To enable the HOTs system  to positHaving established the capability of this technique to generate two dimensional structures we then moved on to investigate 3D patterning by positioning mES into simple, low cell number architectures . The posUsing this capacity we generated larger cellular structures by initially positioning a ring of eight cells around which we placed 14 mES cells forming a second ring that surrounded the initial structure with the cells on the outer and inner rings forming avidin-biotin adhesions. This unit was further expanded by the addition of two cellular layers in identical configurations making a multi-layer, integrated, stable, ring based structure of 66 cells . In thisTo retain architectures indefinitely within larger structures and cultures where elements were placed at defined distances away from other objects such as mES cells a physical, stabilizing medium was introduced in the form of hydrogels. The properties of this surrounding medium also introduced an additional level of biophysical control within the constructed microenvironment. This was achieved by use of a thermoresponsive gel system ULMP agarose and a polyethylene glycol (PEG) based modular system , 3a, 4.The PEG based modular gel matrix consisted of two multiarm PEG-peptide conjugates that were functionalized to enable cross-linking by the addition of the transglutaminase factor XIIIa31Having established the ability of this technique to generate architectures using a single cell type, we next built structures in which the location of multiple cell types could be independently controlled. We created patterns within co-cultures by positioning mES cells and mouse mesenchymal stem cells (mMSCs) into defined architectures  as well in vivo microenvironments. However, the technique can create more complex microenvironments by simultaneously patterning both cells and materials. To demonstrate this concept, polymer materials were positioned within cell cultures in the form of microparticles and electrospun fibers. We propose this as a means to control physical environments, tailoring materials and their surface properties to the required application. Poly(DL-lactic-co-glycolic acid) (PLGA) microparticles and electrospun fiber fragments were trapped and positioned within mES cell cultures forming predefined structures  and larger scale structures using this technology provides a tool for the study of many ructures . PLGA miructures . These mructures  and the ructures  or the aructures .To further expand the range of material structures that could be used in micro-environmental construction, PLGA microfibers were generated by electrospinning, cryogenically ground and fractioned according to size. The microfibers were then treated with fibronectin to facilitate cell adhesion; since neither the avidin-biotin nor hydrogel systems were used for the microfiber patterning. These cell adhesive fibers were suspended within mES cell cultures to generate cell and material co-patterns. Simple structures, for example consisting of one cell and two small PLGA fiber fragments were easily be constructed  as well This technology was also designed to introduce temporal control elements within the patterned structures that could release chemical factors which form part of specific cellular microenvironments. We utilized established polymer microparticle based controlled release technology with specific formulations tailored to produce desired release profiles 33. To viEach microparticle formulation was positioned on one side of a ring of mES cells as shown in the schematic . The posThe data thus far demonstrates the potential applications of this method of HOT manipulation in a wide range of applications. To further develop this technique we also investigated the capacity to construct more complex micro-environmental structures. We generated acid demineralized and enzymatically decellularized ECM fragments from mouse trabecular bone samples forming a bioactive scaffoldWe investigated the capacity of this construct to support the clonal expansion of individually seeded adult stem cells. To assist in the visualization of such colonies and to ensure continuous proliferation an immortalized cell line of human MSC (ihMSCs) origin was used with an integrated green fluorescent protein (GFP) reporter. Individual mPC cellularized ECM fragments were loaded into microwells for patterning on the tweezers micromanipulation system and a very dilute cell number suspension of ihMSCs was then added to each well. Single ihMSCs were placed in isolation within the porous structure of the re-cellularized ECM  and the The data discussed in this paper demonstrates the potential of this HOTs based biological manipulation technology but any detrimental effects are an important consideration. The damaging properties of infrared (IR) laser radiation are limited to heat induction35We also investigated if the mechanical effects of the optical trapping had negative effects on viability. To do this cells were positioned into six cell ring structures and exposed to 10, 20 or 30 minutes of optical trapping (a single trap per cell) . There win vitro has to date been challenging due to a lack of technologies that can position individual cells and matrices at a sufficiently small length-scale.The biological microenvironments within developing embryos and adult organisms consist of highly complex arrangements of cells, ECM and chemical factors910Techniques such as robotic spotting121413et al generated heterotypic microarrays in which mammalian and bacterial cells were positioned within co-culturesin vivo microenvironments.HOTs1718192021In order to realize the full potential of HOTs we positioned differing cell types, polymeric materials, ECM and controlled release technology into precisely defined 3D architectures ,2,3,4,5.The retention of micro-structural organization after HOTs patterning was achieved by use of either avidin-biotin chemical adhesion , 2 or hy18233031et al demonstrated biomolecule incorporation into defined channels (<200 μm diameter) within a bulk ULMP agarose structure which induced outgrowth of rat dorsal root gangliaDiverse hydrogel technologies are currently being developed, generating ever more sophisticated analogues of ECM function for application in the study of micro-cellular activitiesin vitro models mimicking, for example, the microenvironments present within developmental biology.While the precision control of cellular organization and hydrogel properties provides an invaluable micromanipulation tool-set, the control of the soluble factors is of equal importance. We used polymer microparticle based controlled release technologyThe complex patterning of cells in isolation will provide a tool within fields such as developmental biology , 2, 4 buin vitro microenvironments such as those found with stem cell niches, enabling new insights that other technologies are unable to provide.The application of our method of HOTs based micro-manipulation will enable micro-environmental characterization at a scale and resolution that to our knowledge has not previously been demonstrated. The key advantages of this technique are the ability to generate cellular constructs in 3D, introduce controllable and tailored structural elements at precise positions during construction and the temporal control of chemical factors. The systems we have demonstrated can be applied to a wide range of applications including developmental biology and the study of The HOTs instrument was similar to a system detailed previously42The mES cell line (denoted CGR8) was cultured on 0.1% (v/v) gelatin solution. Medium consisted of Dulbecco's modified Eagle's medium (DMEM) (Life Technologies) supplemented with 10% (v/v) fetal bovine serum (FBS) (Sigma-Aldrich), 2 mM L-glutamine (Life Technologies), 0.1 μM 2-mercaptoethanol (Sigma-Aldrich), 50 μg/ml penicillin/streptomycin (Life Technologies) and 5,000 U/ml leukemia inhibitory factor (LIF) . Cells were expanded, passaged and suspended in either avidin containing medium or pre-gel PEG solution ready for patterning on the HOTs system. Mouse MSCs (mMSCs) (Amsbio) were cultured according to manufacturer's guidelines and used for experimentation at passage four.Primary mPCs were extracted from CD-1 neonatal mouse pups by serial digestion with Trypsin II S and collagenase I (Sigma-Aldrich). Growth medium consisted of alpha modified Eagle's medium with 10% (v/v) FBS (Sigma-Aldrich), 2 mM L-glutamine (Life Technologies), 0.1 μM 2-mercaptoethanol (Sigma-Aldrich) and 50 μg/ml penicillin/streptomycin (Life Technologies). All mPCs were used at passage three.Immortalized human mesenchymal stem cells (ihMSCs) were generated by Lentiviral transfection of E6/E7 and hTERT genes as previously described442 and ground for approximately 10 minutes. The resulting ground fibers were then fractioned according to size by passing through a series of sieves (Fisher Scientific) with fragments of <50 μm were used for patterning experiments. The fragments were functionalized for cell adhesion (Sigma-Aldrich) by suspension in 100 μg/ml fibronectin solution containing 50 μg/ml penicillin/streptomycin for 30 minutes at 37°C under constant agitation. The fibronectin solution was removed by centrifugation and fibers re-suspended in complete mES medium for 24 hours prior to patterning.Polymer electropsun fibers were produced using poly (lactic-co-glycolic acid) (PLGA) (75:25)  as described previouslyMicroparticles were fabricated using the high loading efficiency water-in-oil-in-water (w/o/w) emulsion method as previously describedThe cellular organization during micromanipulation was in some cases stabilized by use of an avidin-biotin method as previously describedin situ gelation.The cellular organization during and after micromanipulation was in some cases stabilized by use of a PEG hydrogel system as previously describedAll cell types (biotinylated and non-biotinylated) were suspended in avidin solution (10 μg/ml) PEG pre-gel solution, liquid agarose (<25°C) or an appropriate complete culture medium and placed into inbuilt microwells within polyHEMA coated glass bottomed culture dishes.mES cells were positioned according to predetermined architectural arrangements using the holographic optical tweezers system either in mono-cultures or in conjunction with mMSCs and mPCs. Stabilization of the resulting constructs was achieved using the appropriate method (gels or avidin-biotin). All cell co-culture experiments were performed by generating mixed suspensions in avidin containing medium at a ratio of five mES to one mPC or mMSC. A green fluorescent cell tracker probe  was used to label mPC cells and mMSCs for identification during co-culture experiments.Polymer microparticles were positioned within or near to cellular constructs using the same procedures as the cells but were not functionalized with biotin in any experiments and were stabilized using ULMP agarose. Fibronectin coated PLGA fragments were diluted at a ratio of 1:10 and suspended in 10 ml of 100 cell/ml mES solution.6 cells/ml and placed under continuous agitation for 12 hours. The remaining cell suspension was then removed and replaced with complete culture medium. The resulting re-cellularized fragments were left in culture for a further 24 hours before use with the HOTs patterning procedure. ihMSCs were passaged and suspended in a microwell containing complete medium and a single re-cellularized ECM fragment. HOTs were then used to position individual ihMSCs into specific locations within the re-cellularized ECM fragments and relative positions noted for later images. The fragments were then left at 37°C and 5% CO2 for 72 hours.Fragments of de-cellularized and demineralized trabecular bone were generated based on a previously described methodG.R.K. designed and performed experiments, analyzed data and wrote the manuscript and supplementary material. E.B., T.U. and J.W. designed and performed experiments and analyzed data. G.M.G. was the lead for the construction and design of optical tweezers instrument. Y.D. formulated and prepared hydrogel systems. M.E., M.P., S.A. and L.D.B. supplied supervisory input into experimental design and interpretation of data. K.S. was the project leader, supervised experimental design and interpretation of data and contributed to manuscript text. All authors discussed the results and implications of the project as well as commenting on the manuscript at all stages.Supplementary InformationSupplementary Movie 1Supplementary Movie 2


Intracerebral hemorrhage (ICH) is a devastating neurological disease with a grave prognosis. We evaluated microRNA (miRNA) expression after ICH and evaluated Let7c as a therapeutic target. We harvested hemorrhagic brain 24 hours after collagenase induced ICH in the rat. Microarray analysis was performed to compare the miRNAs expression pattern between hemorrhagic hemisphere and contralateral hemisphere. An in vitro thrombin toxicity model and blood injection ICH model were also used to evaluate miRNA expression. We selected miRNA for the therapeutic target study after reviewing target gene databases and their expression. The antagonistic sequence of the selected miRNA (antagomir) was used to evaluate its therapeutic potential in the in vitro thrombin toxicity and in vivo ICH models. Among 1,088 miRNAs analyzed, let7c was induced in the thrombin and ICH models. Let7c antagomir treatment increased cell survival in the in vitro thrombin injury model and improved neurological function at 4 weeks after ICH. Let7c antagomir decreased perihematoma edema, apoptotic cell death and inflammation around hematoma. Let7c antagomir also induced insulin like growth factor receptor 1 (IGF1R) protein and phosphorylated serine threonine kinase after ICH. This study shows a distinct miRNA expression pattern after ICH. The let7c antagomir reduced cell death and edema and enhanced neurological recovery at least in part by activating the IGF1R pro-survival pathway. This suggests blocking let7c might be a potential therapeutic target in ICH. Intracerebral hemorrhage (ICH) is one of the most devastating stroke subtypes with grave prognosis and has the highest mortality MicroRNA (miRNA) is a short sequence non-coding RNA with 20–25 base pairs which regulates gene expression in the post-transcription step by base-pairing with the target messenger RNA of the 3′ untranslated region In this study we tried to evaluate the miRNA expression pattern in a rat collagenase induced ICH model to understand ICH specific pathophysiology and to discover therapeutic targets by miRNA modulation. Using miRNA microarray and quantitative real-time polymerase chain reaction (qRT-PCR), we selected candidate miRNA with potential therapeutic effect. Whether its inhibition by counter sequenced miRNA, namely antagomir (AM), has a neuroprotective effect was studied using the in vitro thrombin toxicity model and in vivo ICH models.This study was approved by the Institutional Animal Care and Use Committee of Seoul National University Hospital, Korea. Male Sprague-Dawley rats  weighing 200 to 220 g were used in the experiment. Experimental ICH was induced by stereotactic administration of bacterial collagenase type VII, as described previously  24 hours after ICH induction, four rats were sacrificed and their brains were harvested. Total miRNA was extracted from each hemisphere with a miRNeasy Mini Kit , and the concentration and quality of each RNA sample was determined using the Agilent 2100 Bioanalyzer . The miRNAs from each hemisphere were labeled and hybridized by using the GenoExplorer microRNA array labeling kit and GenoExplorer microRNA chips including 1,088 miRNAs . The hybridized microRNA chips were scanned and analyzed using an Axon GenePix 4000B scanner and GenePix Pro software . The heat map pattern of miRNA expression was visualized according to Z-score, which is calculated by /(standard deviation of the miRNA expression), and analyzed by paired t-test.−ΔCt, and was normalized with the expression of control RNU6B  from each sample. The seed sequences of miRNAs in this study are as follows: let7c, 16-UGAGGUAGUAGGUUGUAUGGUU-37; miR-24-2, 25-GUGCCUACUGAGCUGAAA CAGU-46; miR-142-5p, 16-CAUAAAGUAGAAAGCACUACU-36.Selected miRNA from the microarray was validated by using the GenoExplorer miRNA qRT-PCR kit  and miRNA specific primer . Using the comparative threshold cycle (Ct), relative expression was calculated from the formula: relative expression  = 2www.targetscan.org), Pictar (http://.pictar.mdc-berlin.de/), microRNA.org (www.microrna.org), and miRecords (http://mirecords.biolead.org/). The miRNA which was up-regulated in the ICH hemisphere and was predicted to regulate genes of potential therapeutic effect was selected and its antagonistic sequences of single stranded RNAs with cholesterol residue at 3′ ending and phosphorothioate backbone was constructed to neutralize the targeted miRNAs .We reviewed four major miRNA target prediction sites to find potential target genes of elevated miRNAs; these prediction sites were TargetScan  to rat pheochromocytoma cell (PC12) line . The culture media consisted of RPMI 1640 , 10% horse serum , 5% fresh bovine serum , penicillin G  100 u/ml, and streptomycin  100 ug/ml which were placed in a chamber with 5% CO2 and 100% humidity. Thrombin was applied at constant concentration and the cell viability assay was measured by the WST-1 assay kit , according to the manufacturer's instructions. After applying various concentrations of thrombin, we found that 200 U/ml thrombin administration resulted in 50% cell death and 500 U/ml thrombin administration resulted in 90% cell death after 24 hours. The therapeutic potential of AM was studied from the in vitro thrombin injury model. The cells were transfected by selected AM with Lipofectamine 2000 .In a previous experiment we showed that intranasal delivery of AM is a convenient and efficient way of modulating miRNA expression in a neurological disease model For immunochemistry, seven rats from each group were anesthetized and perfusion of the heart was maintained with 50 mL cold saline and 50 ml of 4% paraformaldehyde, in 0.1 mol/l of phosphate buffered saline. The brain sections were stained with antibodies against myeloperoxidase  and Ox-42  as described previously Modified limb placing test every week until the fifth week was performed by two investigators unaware of the experimental groups (n = 5 per group) to evaluate functional improvement as described previously with slight modification Four anesthetized animals from each group were decapitated after perfusion of the heart with 10 mL cold saline and homogenates of each brain were serially processed for western blotting. For in vitro study we transfected 70 nmol/L of the candidate mature double stranded miRNA with neuroprotective potential or scrambled miRNA control  to the PC12 cell line using lipofectamine 2000  and evaluated the potential target protein after 24 hours, as described previously Continuous values were expressed as mean value ± standard deviation. The Mann-Whitney U test was used to compare values between the two groups. Analysis of variance for repeated measures followed by the Bonferroni method for post-hoc inter-group comparison was performed to evaluate recovery of neurological function. Two tailed p-value less than 0.05 was considered as statistically significant.The microarray result showed several miRNAs were up-regulated in the hemorrhagic hemisphere compared to the opposite hemisphere . Those mNext the miRNA expression level was evaluated from two additional ICH models and in vitro thrombin injury model. The level of let7c increased significantly in blood injection model, but not in saline injected model . The levThe immediate intranasal application of AM let7c was not associated with any noticeable adverse event throughout experiment, and abrogated the brain water content  . InflammThe transfection of let7c in PC12 cells decreased IGF1R protein expression significantly compared to either the negative control or transfection of scrambled miRNA sequence, which suggests that let7c determines IGF1R expression . The in This study shows the miRNA expression pattern after ICH induction, and the results indicate that up-regulated miRNAs could be a potential therapeutic target in ICH. When let7c was abrogated by its antagonist application, neuronal survival was increased in the in vitro thrombin toxicity model and functional improvement was facilitated after ICH, with reduced apoptotic cell death. The IGF1R expression was influenced by let7c modulation, and the activated cell survival pathway after IGF1R signaling is proposed as a plausible therapeutic mechanism of AM let7c.This study shows the characteristic miRNA expression profile in the ICH model with region-specific elevation. The expression pattern of miRNA had been previously reported from three different brain hemorrhage models produced by intraventricular autologous blood, lysed blood and thrombin injection The role of let7 has been widely studied in cancer and in stem cell biology, and its potential targets include multiple anti-apoptotic and cell proliferation pathways The possible anti-inflammatory effect of AM let7c treatment is another interesting finding. There exist several lines of evidence suggesting IGF1 signaling relates to inflammation Intranasal delivery has been suggested as a convenient and effective transmission modality for central nervous system acting medications 2 It is impossible to simulate perihematomal inflammation or tissue hypoperfusion by an in vitro thrombin injury model. Considering that the PC12 cell line is from pheochromocytoma tissue, the miRNA expression level and protective effect of let7c derived from in vitro thrombin injury may be different from the brain tissue response after ICH. However, the neuroprotective effect of AM let7c in the in vitro thrombin injury model was validated in the vivo ICH model, in which apoptotic cell death was reduced by promoting IGF1R signaling. Only the intranasal delivery of AM let7c was studied in this study, although there is a chance that other administration routes such as intracerebral injection could be more efficient. Future studies regarding dose response, time window, as well as the route of administration will help to strengthen the feasibility of miRNA modulation strategy in ICH.There are several weak points of this study. First of all, thrombin was applied on the PC12 cell line to imitate an in vitro ICH condition, but it is evident that more extensive mechanisms are related to post-hemorrhagic neuronal damage in the real clinical situation.In conclusion, we demonstrated a distinct miRNA expression pattern after ICH and its modulation can have therapeutic potential. The let7c antagomir reduced cell death and inflammation, and enhanced neurological recovery by activating the IGF1R pro-survival pathway. This suggests blocking let7c might be a potential therapeutic target in ICH.Checklist S1ARRIVE Checklist.(DOC)Click here for additional data file.


Anthonomus grandis). Transcriptome analysis was performed to understand the molecular interactions between Gossypium hirsutum L. and cotton boll weevil larvae. The Illumina HiSeq 2000 platform was used to sequence the transcriptome of cotton flower buds infested with boll weevil larvae.Cotton is a major fibre crop grown worldwide that suffers extensive damage from chewing insects, including the cotton boll weevil larvae  in cotton flower buds infected with boll weevil larvae. Among them, 402 (90.7%) were up-regulated, 41 (9.3%) were down-regulated and 432 (97.5%) were identified as orthologues of A. thaliana genes using Blastx. Mapman analysis of DEG indicated that many genes were involved in the biotic stress response spanning a range of functions, from a gene encoding a receptor-like kinase to genes involved in triggering defensive responses such as MAPK, transcription factors (WRKY and ERF) and signalling by ethylene (ET) and jasmonic acid (JA) hormones. Furthermore, the spatial expression pattern of 32 of the genes responsive to boll weevil larvae feeding was determined by “in situ” qPCR analysis from RNA isolated from two flower structures, the stamen and the carpel, by laser microdissection (LMD).The analysis generated a total of 327,489,418 sequence reads that were aligned to the 2+ influxes, and reactive oxygen species; and the modulation of transcripts encoding enzymes from phytohormone signalling pathways. These data will aid in the selection of target genes to genetically engineer cotton to control the cotton boll weevil.A large number of cotton transcripts were significantly altered upon infestation by larvae. Among the changes in gene expression, we highlighted the transcription of receptors/sensors that recognise chitin or insect oral secretions; the altered regulation of transcripts encoding enzymes related to kinase cascades, transcription factors, CaThe online version of this article (doi:10.1186/1471-2164-15-854) contains supplementary material, which is available to authorized users. Gossypium hirsutum L. contributes the most to the total lint cotton production worldwide ..CCR4, RIMAPK, MAPKK, MAPKKK) cascade [MAPK3 (At3g45640) and MKK9 (At1g73500) genes with the GO term “response to chitin”. The in situ qPCR analysis showed that these genes were highly expressed in the stamen and carpel from cotton flower buds infested by boll weevil larvae , suggesting that ERF-5 might play an important role in plant defence by positively regulating ethylene (ET) signalling [GhERF-4 and GhERF-5 genes were more highly expressed in the stamen than in the carpel, and the GhERF-98 gene was slightly more highly expressed in the carpel  as well as in response to chitin, a main elicitor of the plant defence response against insects [The recognition of conserved HAMP, such as chitin or fatty acid-amino acid conjugates  by PRR triggers intracellular signalling via a mitogen-activated protein kinase  and infection by the biotrophic pathogen P. syringae, working redundantly with the structurally related genes WRKY70 and WRKY53 to positively regulate basal resistance to P. syringae[Arabidopsis WRKY genes are rapidly and strongly induced by chitin [WRKYs are target genes of the upstream MAPK3 (At3g45640), such as WRKY33. Studies in Arabidopsis have shown that in response to pathogen invasion, MAPK3 (At3g45640) phosphorylates WRKY33, which directly binds to the W-boxes in the promoter of the ACS6 gene in vivo, suggesting that WRKY33 is directly involved in the activation of ACS6 expression downstream of the MAPK3 cascade [AtWRKY33 is required to activate the synthesis of antimicrobial substances such as camalexin [WRKY genes  showed a higher expression in the carpel than in the stamen. In an elegant series of experiments, Asai et al. [AtWRKY22 functions downstream of the flagellin receptor, a leucine-rich repeat (LRR) receptor kinase, and that a mitogen-activated protein kinase (MAPK) cascade conferred resistance to bacterial (P. syringae) and fungal (Botrytis cinerea) pathogens. These results strongly indicate that signalling events initiated by boll weevil larvae feeding might converge on a conserved system of pathogen-associated molecular pattern recognition, a MAPK cascade and transcription factors.RNAseq analysis identified transcripts for 88 TFs that were differentially expressed in 6 mm cotton flower buds damaged by boll weevil larvae. In the present study, we selected the 21 cotton genes identified as WRKY for a phylogenetic analysis in combination with the full set of syringae. Previouy chitin . Some WR cascade . This siamalexin . Interesi et al.  showed t2+) have been implicated as a second messenger in many plant signalling pathways including responses to herbivory [2+ levels [Spodoptera littoralis) caused a transient increase in cytosolic Ca2+ in Phaseolus lunatus cells adjacent to the insect damage [EDA39, CML38 and CML24) showed a higher expression close to the damage (stamen tissues) than in carpels, suggesting that these proteins might be a component of Ca2+ signalling that modulate plant defence responses against herbivory attack.Relatively little is known about the signal transduction pathways triggered by insect damage. Calcium ions (Caerbivory . It has + levels . For inst damage . Cotton 2−), hydrogen peroxide (H2O2), singlet oxygen (1O2), and hydroxyl radical (·OH) are collectively called ROS; they are produced in mitochondria, chloroplasts, and peroxisomes, as well as on the external surfaces of plasma membranes. Feeding of Helicoverpa zea on soybean, as well as S. littoralis and Tetranychus urticae feeding on Medicago truncatula plants, increase ROS levels [In situ qPCR analysis showed that transcripts encoding an NADPH oxidase (RbohD) were expressed at a higher level in carpel tissues than in stamen tissues compared to the control samples  used for plant defence. Jasmonate plays a central role in regulating the defence responses to herbivores that inflict various types of tissue damage. Jasmonate mutants are affected in their resistance to a wide range of arthropod herbivores, including caterpillars (Lepidoptera), beetles (Coleoptera), thrips (Thysanoptera) and leafhoppers (Homoptera) [ACS and ACO, were also up-regulated by boll weevil larvae damage. The primary function of ET in plant resistance to herbivores is the fine-tuning of JA-induced responses [Arabidopsis plants with ethephon, a synthetic ET donor, transiently elevates the levels of JA and AOS transcripts [The boll weevil larval damage in flower buds also induced the expression of genes associated with phytohormone biosynthetic processes and signalling. Among the gene transcripts associated with JA, ET and ABA biosynthesis selected for analysis by qPCR were moptera) , 59, 62.moptera) . The genesponses . In tomaesponses . The trenscripts . Blockinnscripts . By usinnscripts  showed tArabidopsis PUB17 is required for the initiation of the hypersensitive response (HR). Therefore, two predicted members of this family, PUB23 and CMPG1, which are among the DEGs in our experiment and were annotated in response to chitin and/or death, were also analysed by in situ qPCR. Spatial analysis of gene expression revealed that the transcripts of both genes were strongly up-regulated at the site close to the damage (stamen) compared to carpel tissues. These genes are among the 30 ubiquitin-ligase genes that up-regulated either 15 or 30 min after chitooctaose treatment in Arabidopsis[PUB23 gene in Arabidopsis was induced by PAMPs and infection by pathogens. Moreover, the pub22/pub23/pub24 triple mutant displayed negative regulation of PAMP-triggered immunity [The ubiquitin-ligase genes are also known to play a role in the plant defence response . Yang etbidopsis. Previouimmunity .GER3 transcript was strongly down-regulated in the stamen and in the carpel  production, a type of ROS that is implicated in the herbivory-induced response in plants, and was also repressed by P. brassicae eggs and P. rapae herbivory in a study in A. thaliana[HSP20, HSP90.1 and HSP90.2 was observed in both stamen and carpel tissues  and many genes encoding heat shock proteins (Hsp). qPCR analysis revealed that the l Figure . Interesy Figure . GER3 lethaliana. The tras Figure . In manyectively –72. HSP9g AvrPto, 74. In G. hirsutum flower buds in response to feeding by cotton boll weevil (A. grandis) larvae. A large number of cotton transcripts and biological processes were significantly altered upon larvae infestation. Among the changes observed, we highlighted the induction of transcripts for the receptors/sensors that recognise chitin, insect oral secretions and signals from injured plant cells; differential modulation of transcripts encoding enzymes related to kinase cascades; transcription factors (such as WRKY and ERF); Ca2+ influx; reactive oxygen species; and modulation of transcripts encoding enzymes from phytohormone signalling pathways, mainly the JA and ET pathways. Cotton boll weevil larvae feeding affected many genes that have also been shown to be regulated in response to microbial or fungal infection, indicating the existence of complex crosstalk in the response to these different pathogens. The qPCR analysis associated with microdissection showed that almost all of the genes were more highly expressed in the stamen, the region close to the damage caused by larvae feeding, than in the carpel, farther away from the injured area. However, herbivory-induced defence responses in cotton flower buds were observed not only in the wounded regions but also in undamaged regions of the damaged flower buds. It is possible that a signal travels to other parts of the flower bud that transmit an herbivory alert. Although the identity of the mobile signal responsible for this systemic response remains unknown in many plants, studies performed in solanaceous plants demonstrated that herbivory rapidly induces a short-distance mobile signal that travels and activates MAPKs and triggers JA accumulation. Deciphering the signals that regulate herbivore-responsive gene expression in G. hirsutum flower buds might provide new strategies to manipulate this response for the production of insect-resistant transgenic plants.Alternative strategies for protecting crops from insect pests are exploring the endogenous resistance mechanisms exhibited by plants to most herbivore insects through a greater understanding of induced defences in plants. This study aimed to unravel the changes in the transcriptome of Gossypium hirsutum L. variety “BRS Cedro”) plants were grown under a controlled temperature (27 ± 2°C) and natural photoperiod at Embrapa Genetic Resources and Biotechnology in Brasilia . Three-month old plants containing 6 mm flower buds were selected for the experiments. A population of A. grandis (Coleoptera: Curculionidae) was maintained at 27 ± 2°C, 70 ± 10% relative humidity, and a photoperiod of 14 h. Insects were routinely maintained on a standard rearing diet [A. grandis egg containing an active embryo. The orifice resulting from drilling was sealed with Vaseline to prevent dehydration of the egg. The hatching of larvae occurred approximately 2 h after egg inoculation. The larvae were removed with tweezers from the flower buds 48 h after inoculation, and four flower buds from different plants were immediately frozen in liquid nitrogen for the isolation of total RNA to perform RNAseq experiments. Similarly, a set of twelve flower buds infected with larvae were collected and fixed in cold ethanol:acetic acid (3:1) to subsequently isolate the selected tissue through laser microdissection. The insect infection experiments were performed in two biological replicates. The control samples were drilled cotton flower buds into which no eggs were introduced.Cotton  according to the manufacturer’s protocol. RNA quality and quantity were determined using a Nanodrop 2000 (Thermo Scientific) and a Bioanalyser Chip RNA 7500 series II (Agilent), respectively. The analysis of RNA integrity was judged by the RNA Integrity Number (RIN), which was calculated with 2100 Expert Software. The software algorithm, which was developed by Schroeder et al. , categorG. hirsutum expressed sequence tag (EST) assembly  as the transcriptome reference  [2 (FC) ≥ 2 were determined to be significantly differentially expressed genes (DEG) and were selected.Raw reads were filtered to obtain high-quality reads by removing low-quality reads containing more than 30% bases with Q < 20. After trimming low-quality bases from the 5′ and 3′ ends of the remaining reads, the resulting high-quality reads were aligned against a xie.com)  using thxie.com) . Differexie.com) . The couhttp://www.blast2go.com/b2ghome) [The DEG were subjected to blast using the program Blastx with the TAIR 9 protein database and further classified into categories according to the GO classification system   was alsoA. thaliana, that share the same ontology based on the GO database. The GSEA method identified biological processes (BP), molecular functions (MF) and cellular components (CC) that were overrepresented among the list of DEG  was performed . A gene G Figure . The oveArabidopsis thaliana leaves that were infested by diamond back moth (Plutella xylostella) larvae 24 h after the onset of herbivory (GEO:GSE10681) ; two proteins from the mitogen-activated kinase signalling cascade ; three calmodulin-like proteins ; two genes from the oxidative burst process ; twelve TFs, including nine WRKY transcription factors  and three AP2/ERE transcription factors ; four genes involved in phytohormone biosynthesis ; and two ubiquitin ligase proteins involved in targeted protein degradation (PUB23 and CMPG1). Among the down-regulated DEGs, we evaluated the expression pattern of a germin-like protein (GER3) and three members of the heat shock protein family . Primers were designed using Primer 3 software tools (http://frodo.wi.mit.edu/primer3/) with a melting temperature of 60°C, an amplicon length of 150 to 200 bp, and a GC content of 50 to 60% [Real time PCR (qPCR) assays were performed on 32 differentially expressed genes identified by RNAseq analysis . These areas Figure  included260. Equal amounts of aRNA (5.0 μg) of the two samples in biological replicates were reverse transcribed using 0.5 μl of Random Primers  and Superscript III reverse transcriptase (Invitrogen) according to the manufacturer’s instructions. Polymerase chain reactions were performed in the 7500 Fast Real-Time PCR detection system (Applied Biosystem) and SYBR® Green was used to monitor dsDNA synthesis. PCR efficiencies and the optimal quantification cycle threshold or Cq values were estimated using the online Real time PCR Miner[GhACT4 and GhFBX6 were used to normalise the qPCR data and have been discussed previously [©), which compares two treatment groups with multiple data points in the sample compared to the control groups and calculates the relative expression ratio between them. The mathematical model used is based on the PCR efficiencies and the mean CP deviation between the sample and control group of target genes and is normalised to the mean CP deviation of the reference genes [P values by a Pair-Wise Fixed Reallocation Randomization Test[Isolated areas of carpel and stamen with biological replicates of infested and control samples  were collected in microfuge tubes containing 100 μl RNALater solution (Ambion) and were used for RNA isolation Figure . RNA wasCR Miner. Two indeviously   from 72 AtWRKY protein sequences from Arabidopsis (downloaded from AtTFDB (http://arabidopsis.med.ohio-state.edu/AtTFDB/) and 21 cotton WRKY genes. Phylogenetic trees were constructed after using a model test to pick the most appropriate evolutionary model for our analysis in the Mega 5.2 program [http://www.atgc-montpellier.fr/phyml/) [To identify the twenty-one cotton WRKY genes that were differentially expressed in our RNAseq experiment, a phylogenetic tree based on the WRKY domain of  program . The bes program  with an /phyml/)  and asseJPEG file showing contig size distribution from the aligned reads of transcriptome sequencing of cotton flower buds.(TIFF 11 MB)Additional file 1:Full list of the up-regulated and down-regulated cotton genes found in the RNA-seq analysis. Only those differentially expressed genes with significant expression changes  are shown. Columns A-G are the results from Blastx with Arabidopsis, and columns H-P are the results from the Blast2GO program. (XLS 780 KB)Additional file 2:Molecular function (A) and Cellular component (B) ontologies overrepresented by the Gene Set Enrichment Analysis (GSEA).(TIFF 521 KB)Additional file 3:Subset of differentially expressed genes (DEG) in cotton flower buds in response to cotton boll weevil larvae feeding that were annotated with the GO term “plasma membrane”.(XLS 44 KB)Additional file 4:Subset of differentially expressed genes (DEG) in cotton flower buds in response to cotton boll weevil larvae feeding that were annotated with the GO term “cell wall”.(XLS 38 KB)Additional file 5:Biological processes (BP) overrepresented by the Gene Set Enrichment Analysis (GSEA).(XLS 58 KB)Additional file 6:The twenty-one cotton WRKY genes that were differentially expressed in our RNAseq experiment, the domain sequence used in the phylogenetic tree, the protein sequence and the nucleotide sequence.(XLS 77 KB)Additional file 7:Comparison of qPCR and RNA sequencing expression data. Thirty-two differentially expressed genes responding to cotton boll weevil larvae feeding were analysed for RNA abundance using qPCR. The log fold-change in RNA sequencing was estimated and tested by DEGseq software, in which the count data were normalised to the total number of counts, taking the variance and the mean of the biological replicates into account for each library with reference to the control (blue bars). qPCR results (red bars) were calculated with the relative REST software using a mathematical model based on the PCR efficiencies and the mean CP deviation between the sample and control group of target genes and were normalised to the mean CP deviation of reference genes. We calculated the Pearson correlation coefficient (r) between the different methods for all transcripts. The correlation coefficient was 0.9406. (TIFF 8 MB)Additional file 8:Primers used for the qPCR expression analysis of DEG in tissues isolated from cotton flower buds infested by cotton boll weevil larvae.(XLS 52 KB)Additional file 9:


MLL gene and two different known translocation partners (AF4 and AF9), which resulted in specific chromosomal translocations in K562 cells as well as primary hematopoietic stem and progenitor cells (HSPCs). The initiation of a specific MLL translocation in a small number of HSPCs likely mimics the leukemia-initiating event that occurs in patients. In our studies, the creation of specific MLL translocations in CD34+ cells was not sufficient to transform cells in vitro. Rather, a variety of fates was observed for translocation positive cells including cell loss over time, a transient proliferative advantage followed by loss of the clone, or a persistent proliferative advantage. These studies highlight the application of genome engineering tools in primary human HSPCs to induce and prospectively study the consequences of initiating translocation events in leukemia pathogenesis.One of the challenging questions in cancer biology is how a normal cell transforms into a cancer cell. There is strong evidence that specific chromosomal translocations are a key element in this transformation process. Our studies focus on understanding the developmental mechanism by which a normal stem or progenitor cell transforms into leukemia. Here we used engineered nucleases to induce simultaneous specific double strand breaks in the  Mixed Lineage Leukemia (MLL) gene  for 38–40 cycles depending on primer set, and final extension at 72°C for 1 min. PCR products were visualized on a 1% agarose gel with ethidium bromide. PCR products were extracted from the gel (Qiagen), cloned into the pCRII-Blunt-TOPO vector (Invitrogen), transformed into competent cells. After incubation at 37°C overnight colonies were isolated and enriched in liquid culture. Plasmid DNA of the transformed competent cells was purified (Qiagen Miniprep) and submitted for Sanger Sequencing using the M13 forward and M13 reverse primers.5’CAGGAGGATTGTGAAGCAGAAAA3’; AF4 primer: 5’TAGGGAAAGGAAACTTGGATGG3’] using the following cycling parameters: denature at 95°C for 5 min,  for 36 cycles, and final extension at 72°C for 1 min. PCR products were visualized, gel extracted, and prepared for Sanger sequencing as noted above. Primers used for real-time analysis of MLL-AF4 fusion transcript were previously described by Siraj et al. [RNA was isolated from CD34+ cell cultures using the RNeasy Mini Kit (Qiagen). 500 ng of RNA from each sample was used to generate cDNA using the RevertAid First Strand DNA Synthesis Kit (Thermo Scientific). PCR was subsequently performed for the MLL-AF4 transcript [MLL primer: j et al.  qPCR usiMLL Dual Color Break Apart Rearrangement Probe . Two hundred cells were analyzed per sample for the presence or absence of an MLL rearrangement. A representative image was obtained for each sample.Live cultures of the indicated populations were submitted for FISH analysis to the Cytogenetics Laboratory of Stanford Hospital and Clinics on a fee for service basis. Cells were fixed and hybridized with the Vysis t test. P<0.05 was considered statistically significant.CD34+ cells were nucleofected with MLL and AF4 or AF9 TALENs or control (GFP) and were seeded in triplicate  at days 25 and 39 of extended liquid culture in Methocult H4230 methylcellulose medium  supplemented with the same cytokines used in liquid cultures. Cultures were incubated at 37°C, 5% CO2, and colonies were scored after 12–14 days. Secondary CFC assays were performed by harvesting all cells from the primary cultures after colony enumeration, and replating them into new assays under identical conditions. Morphology of the colonies was determined as previously described . All staMLL, AF4, and AF9 genes, known as breakpoint cluster regions, that are frequently the sites of chromosomal translocations in patients with leukemia [MLL and AF4 genes, we chose a specific patient translocation sequence (UPN010) that is available through the GenBank database (AJ408902.1) and is representative of the most common site of translocations in patients with infant leukemia [MLL and AF4 corresponding to the UPN010 translocation  and the reciprocal translocation  . These studies demonstrate that TALENs specifically targeted to the respective breakpoint cluster regions are capable of generating MLL chromosomal translocations.To test whether the simultaneous induction of two double strand breaks is sufficient to induce a specific chromosomal translocation, we nucleofected K562 cells with either the AF9-MLL) . Transloquencing . A sub-cMLL translocation. After 1–2 weeks following nucleofection, MLL-AF4 or MLL-AF9 translocations and the respective reciprocal translocations  were detected . Parallel cultures were maintained over time and monitored by PCR for the presence of the MLL-AF4 as well as the reciprocal AF4-MLL translocation. The estimated MLL-AF4 translocation frequency following nucleofection with TALENs was 2.6–4.0 x 10−5. The reciprocal AF4-MLL translocation was found to have a lower frequency of 1.3–2 x 10−5. Taken together, these data demonstrate the induction of MLL chromosomal translocations by TALEN-mediated genome engineering of primary human HSPCs.Since K562 cells have significant chromosomal abnormalities at baseline and likely do not recapitulate what would be observed in primary cells, we explored the feasibility of inducing translocations in human primary HSPCs. After isolation of human primary CD34+ cells from fresh umbilical cord blood, the indicated TALENs were introduced by nucleofection. Cells were maintained in culture and monitored over time for the appearance of an detected . The resdetected . In ordede novo MLL translocations result in a survival advantage in primary cells, human CD34+ cells were nucleofected with MLL and AF4 or AF9 TALENs or GFP and maintained in culture. Cell viability was determined by flow cytometry over time  of cells in subculture 9, 17.5% (35/200) of cells in subculture 20, and 0% (0/200) of cells in the GFP control culture. Interestingly, the four subcultures that demonstrated the most significant expansion and prolonged survival in extended culture also had a detectable reciprocal AF4-MLL translocation by PCR  or control samples each on day 25 and 39 of the extended culture in semisolid medium supplemented with the same cytokines that were used during the extended cultures. All three cultures generated colonies after 12–14 days, but the MLL-AF4 and MLL-AF9 samples displayed significantly higher clonogenic potential after the second round of replating as compared to the control samples , and prospectively investigated what happened to the cells as a result of the initiating event. We believe that this is the first example in which TALEN nucleases have induced the relevant translocation in primary human HSPCs, a critically important milestone in using engineered nucleases to study the ontogeny of translocation associated leukemia.Using tools developed for genome editing/engineering, we induced patient specific MLL-rearranged leukemia presents a unique opportunity to explore leukemia pathogenesis in a prospective manner. We know from infant studies that MLL rearrangements are an initiating event that occurs in utero in infants who develop MLL-rearranged leukemia [TEL-AML1 and BCR-ABL have been shown to occur in utero, but demonstrate more twin discordance, longer latency, and are thought to require subsequent mutations for leukemia development [MLL translocation alone may be sufficient for leukemogenesis given the high penetrance and short latency seen in twin studies [MLL translocations also occur in therapy related leukemias in which a secondary leukemia usually occurs 1–3 years after exposure to topoisomerase inhibitors such as etoposide and teniposide [leukemia , 23. Othelopment , 25. Epi studies . MLL traniposide –29. ThusMLL translocations in the pathogenesis of acute leukemia is still not complete. Many animal models have been developed and helped to define MLL fusion proteins as key transcriptional and epigenetic regulators that lead to leukemia pathogenesis [MLL fusion proteins, especially in the case of the MLL-AF4 translocation. The MLL-AF4 translocation has been difficult to model in mice due both to the size of the MLL-AF4 translocation product and its chromosomal orientation [MLL-AF4 fusion product have not fully recapitulated the clinical phenotype seen in infants and are complicated by potential effects related to retroviral insertion. There is ongoing debate as to whether the MLL-AF4 fusion protein alone is sufficient to induce leukemogenesis or whether subsequent mutations are required [MLL-AF4 leukemias to have fewer additional mutations as compared to other leukemias, but have identified N-Ras, K-Ras, and Flt-3 as a source of potential cooperating mutations [AF4-MLL translocation product, as only a fraction of patients have a detectable reciprocal translocation; however one report has demonstrated leukemia development in a mouse model by enforced expression of the reciprocal AF4-MLL fusion product alone [Despite extensive investigations over the past 20 years, our understanding of the role of ogenesis , 30–34. entation . Additiorequired . Previouutations –40. Addict alone . The genIn vitro, we observed that many cells with an induced MLL translocation do not persist in culture. This is likely a result of an intact apoptotic response in primary cells. However, a subset of cells that acquired an MLL translocation persisted over time, appeared to have a selective advantage in extended culture, and demonstrated a higher clonogenic potential in colony forming assays. While this effect was not as robust as that seen in previous studies when the fusion product has been introduced with retroviral vectors, this is likely related to lower expression of the fusion product under the control of the endogenous promoter. Interestingly, the clones that demonstrated the most robust survival advantage either in subculture analysis or colony forming assay contained the reciprocal translocation product. This further suggests that the reciprocal translocation may play an active role in leukemia pathogenesis. While the MLL-rearranged cells demonstrated a selective advantage, which may represent a pre-leukemic clone, our studies would suggest that they were not fully transformed as these cells eventually died out in culture as well as with serial replating in colony forming assays. This is consistent with other studies showing that enforced expression of an MLL-AF4 transgene in primary HSPCs conveys a proliferative advantage but is insufficient to generate leukemia [in vitro and in vivo to explore the immunophenotypic, gene expression and epigenetic changes that occur in cells over time as a result of the MLL translocation. Additionally, we are using this system to generate MLL translocations in CD34+ cells freshly isolated from umbilical cord blood followed by transplantation into immunocompromised (NSG) mice in order to develop a novel mouse model of MLL-rearranged leukemia. We believe this system will allow us to more fully and accurately answer whether or not MLL translocations are sufficient for leukemogenesis or whether they require cooperating events. It will also allow exploration of the relationship between the MLL translocation partner as well as cell of origin in relation to disease phenotype.leukemia , 38. An S1 FigMLL and AF4 or AF9 TALENs or GFP (control) and maintained in culture in vitro. Cell viability was monitored over time by flow cytometry.CD34+ cells were nucleofected with (TIF)Click here for additional data file.S2 FigMLL and AF4 TALENs to induce MLL translocations. Subpopulations were sampled over time for the presence of the MLL-AF4 and AF4-MLL translocations. PCR products were isolated and sequenced to evaluate evolution of sequence specific clones.Human CD34+ cells were nucleofected with (TIF)Click here for additional data file.S3 FigMLL-AF4 translocation as well as the negative control cultures. RT-PCR detected the MLL-AF4 transcript expressed in subcultures 9 and 20. The PCR products were isolated and sequenced for confirmation. (B) Real-time RT-PCR confirmed MLL-AF4 transcript expression in subcultures 3, 9, 16, and 20 above the threshold limit of detection.(A) RNA was isolated at day 42 for each subculture showing a prominent (TIF)Click here for additional data file.


Desmodus rotundus). RABV was also isolated from oral swabs, lungs and kidneys of both bats, whereas viral RNA was detected in all of the tissues examined by hemi-nested RT-PCR except for the liver of one bat. Sequencing of the nucleoprotein gene showed that both viruses were 100% identical, whereas sequencing of the glycoprotein gene revealed one non-synonymous substitution . The two vampire bat RABV isolates in this study were phylogenetically related to viruses associated with vampire bats in the eastern states of Mexico and El Salvador. Additionally, 7% of sera collected from 398 bats demonstrated RABV neutralizing antibody. The proportion of seropositive bats varied significantly across trophic guilds, suggestive of complex intraspecific compartmentalization of RABV perpetuation.Rabies in bats is considered enzootic throughout the New World, but few comparative data are available for most countries in the region. As part of a larger pathogen detection program, enhanced bat rabies surveillance was conducted in Guatemala, between 2009 and 2011. A total of 672 bats of 31 species were sampled and tested for rabies. The prevalence of rabies virus (RABV) detection among all collected bats was low (0.3%). Viral antigens were detected and infectious virus was isolated from the brains of two common vampire bats ( Desmodus rotundus) collected in two different locations in Guatemala. Sequencing of the isolates revealed a closer relationship to Mexican and Central American vampire bat isolates than to South American isolates. The detection of RABV neutralizing antibodies in 11 species, including insectivorous, frugivorous, and sanguivorous bats, demonstrates viral circulation in both hematophagous and non-hematophagous bat species in Guatemala. The presence of bat RABV in rural communities requires new strategies for public health education regarding contact with bats, improved laboratory-based surveillance of animals associated with human exposures, and novel techniques for modern rabies prevention and control. Additionally, healthcare practitioners should emphasize the collection of a detailed medical history, including questions regarding bat exposure, for patients presenting with clinical syndromes compatible with rabies or any clinically diagnosed progressive encephalitis.In this study we provide results of the first active and extensive surveillance effort for rabies virus (RABV) circulation among bats in Guatemala. The survey included multiple geographic areas and multiple species of bats, to assess the broader public and veterinary health risks associated with rabies in bats in Guatemala. RABV was isolated from vampire bats ( Rabies virus (RABV), the only Lyssavirus documented in the New World Bats (Order: Chiroptera) have been implicated as hosts and reservoirs for numerous emerging infectious diseases, and are considered one of the most relevant groups of mammals in the study of disease ecology Rabies epizootiology is well appreciated in countries with an established laboratory-based surveillance network. Combined with molecular epidemiology, enhanced and passive surveillance are used to define the geographic distribution of RABV variants, infer the temporal and spatial spread of infections associated with diverse reservoir hosts, identify spillover infections into humans, and to devise relevant prevention and control based upon such information Desmodus rotundus) represents the most apparent economic and public health threat associated with bats RABV transmitted by vampire bats  Guatemala was selected as one major comparative New World study location as part of the U.S. Centers for Disease Control and Prevention's (CDC) Global Disease Detection (GDD) program among ten international locations. The objective of the CDC GDD program is to develop and strengthen global capacity to rapidly detect, accurately identify, and promptly contain emerging infectious threats. Nineteen field sites for sampling bats in Guatemala were selected on the basis of historical outbreaks of rabies, contemporary national surveillance data, known or suspected vampire bat depredation upon human populations, or neurological illness reported in livestock. Brain impressions prepared from frozen bat tissues were fixed in acetone at −20°C, and RABV antigens were detected by the DFA test, using fluorescein isothiocyanate (FITC)-labeled monoclonal antibody (mAb) conjugate , as described The presence of RABV neutralizing antibodies (rVNA) was determined by the rapid fluorescent focus inhibition test (RFFIT) or a modified micro-RFFIT test on sera collected from ten field sites Antigenic characterization was performed by indirect immunofluorescence using eight mAbs directed against the RABV nucleoprotein (N) antigens , supplied by EMD Millipore Corporation , as previously described 6 mouse neuroblastoma cells (MNA) in a T-25 tissue culture flask . Tissue culture flasks were incubated at 0.5% CO2 at 37°C for 72 hours. All cultures were sub-passaged a minimum of four times. For infectivity assessments, Teflon-coated four well slides were seeded with 30 uL of MEM-10 containing 0.5×106 cells per mL, and incubated in a humid chamber at 0.5% CO2 at 37°C for 24 hours. The slides were then rinsed with phosphate-buffered saline (PBS 4550), and fixed in cold acetone at −20°C for one hour. RABV antigens were visualized by use of the DFA test, using optimal working dilutions of FITC-labeled anti-RABV mAb conjugate  after each passage.Virus isolation was attempted on oral and fecal swabs stored in growth medium (MEM-10), and organs, obtained from DFA-positive bats. Homogenates (10% w/v) were prepared in MagnaNA lyser tissue homogenizer tubes containing 1.4-mm (diameter) ceramic beads , using 1.0 mL of MEM-10 as a diluent. The entire solution from the oral swab, and 0.5 ml from the fecal swab was used to inoculate cells for isolation. For virus recovery, 100 uL of test inoculum was added to 1 mL of MEM-10 containing 5×10TTGACGAAGATCTTGCTCAT (positions 1514–1533). The hemi-nested reaction was performed with sense primer 1087F, GAGAARGAACTTCARGA (positions 1157–1173). The glycoprotein (G) gene was amplified as two overlapping fragments using primer combinations umf2/994b and 760f/308b, as previously described Total RNA was extracted from fecal swabs and organ tissues of RABV-infected bats using TRIZol reagent . Fecal swabs were stored in 1 ml of MEM-10 in the field, and 200 µl of the swab suspension was mixed with 1 ml of TRIzol for RNA extraction. Primers were selected within the coding region of the N gene and the initial reaction was performed with sense primer 1066F, GARAGAAGATTCTTCAGRGA (positions 1157–1173), which was also used for reverse transcription and antisense primer 304B, The complete N and G sequences were assembled and translated to amino acid sequences using the Bio Edit program 2 tests, and the level of significance was evaluated at α = 0.05.All statistical analyses were performed using JMP version 9.0.2 or SAS version 9.2 . The 95% confidence intervals (CI) were calculated for proportions of seropositive bats by species and location of capture. A nested mixed logistic model was used to test the effect of trophic guild on seroprevalence. Species nested within trophic guild was treated as a random effect and trophic guild was treated as a fixed effect. As only a single sanguivorous species was tested, we also compared the model outputs with a data set excluding vampire bats. The antibody prevalence between sex were also compared using χD. rotundus , followed by the Jamaican fruit bat, Artibeus jamaicensis   .3,20 = 4.65, p = 0.04; −21Species (Diet) = 3.4×10) and a data set without vampire bats  = 4.4×10). In both models, pairwise contrasts revealed a significant difference between frugivorous and insectivorous bats. Insectivorous bats were 8.5 times as likely to be seropositive compared to frugivorous bats. No other pairwise contrasts in rVNA seroprevalence between trophic guild levels were significant. Among capture locations, the proportion of seropositive bats was highest for Naranjo (21%), followed by El Jobo (19%), and El Penate (11%). P = 0.77). Species composition of bats captured varied across sites (From sera available for testing (n = 398), 28 bats demonstrated detectable rVNA for an antibody prevalence of 7% (95% CI 5–10%). Seroprevalence was highest for insectivorous species (21%), followed by omnivorous (8%) and sanguivorous (9%) taxa. The proportion of rVNA seropositive bats varied significantly across trophic guilds in a complete data set .T,S). The viruses were most similar to one of the lineages of vampire bat rabies viruses circulating in Mexico, and were relatively distant from the lineages circulating in South America, with the exception of Columbian lineages. The most phylogenetically related viruses were described previously from Chiapas, Tabasco and Veracruz states of eastern Mexico The N gene sequences of both viruses were 100% identical, whereas the G sequences differed in one non-synonymous substitution , even among vampire bats (1%). The two vampire bat RABV isolated were related phylogenetically to viruses associated with vampire bats in the eastern states of Mexico and El Salvador, which is not unexpected given their geographic proximity. Rabies epizootics and phylogenetic clusters of RABV circulating in vampire bat populations are relatively constrained in space In experimentally infected bats, rVNA are typically generated only among survivors, and bats that succumb to infection may only seroconvert during late stages of clinical disease Strategies to control RABV transmitted by vampire bats in Latin America have relied historically on population reduction methods, either by non-specific destruction of roosts or by anti-coagulants applied to cattle or individual bats. When the anti-coagulant paste is applied to vampire bats, it is suspected that the treated bats then return to a roost where is spread to other conspecifics through allogrooming. Culling of vampire bats is thought to benefit agriculture and public health in the short-term by alleviating bat bites on livestock and humans respectively. However, there is little evidence that this method actually targets rabid bats, and the apparent positive effect of culling on seroprevalence, combined with demographic and behavioral responses, may actually increase the proportion of susceptible bats D. rotundus, though this strategy has not been tested in the field Rabies surveillance at the national level in Guatemala falls under the jurisdiction of the Ministry of Public Health and Social Welfare Unfortunately, despite significant progress in the prevention and control of rabies, once clinical signs manifest in humans the case fatality rate approaches 100%, even with intensive supportive care As the first focused rabies study in Guatemala, gaps were evident in our study. Limitations to the current study included a lack of more focal seasonal sampling, a broader spatial scale, and a need for greater engagement of the Ministries of Health and Agriculture in a One Health context to improve routine laboratory based surveillance. The presence of bat RABV in rural communities likely requires new strategies for joint public health and veterinary education and outreach, increased availability for diagnostic laboratory testing in remote areas, and continued enhanced surveillance for rabies prevention and control, as suggested for other developing countries Table S1Bats collected for rabies testing from eight field sites in Guatemala, 2009.(DOCX)Click here for additional data file.Table S2Bats collected for rabies testing from two field sites in Guatemala, 2010.(DOCX)Click here for additional data file.Table S3Bats collected for rabies testing from nine field sites in Guatemala, 2011.(DOCX)Click here for additional data file.Table S4List of sequences included in the data set from Central and South American vampire bat RABV.(DOCX)Click here for additional data file.


Glycine max (L.) Merr.], (2) discuss the relationship between prediction accuracy and numbers of markers, and (3) evaluate the effect of marker preselection based on different methods on the prediction accuracy. Our study is based on a population of 235 soybean varieties which were evaluated for plant height and yield per plant at multiple locations and genotyped by 5361 single nucleotide polymorphism markers. We applied ridge regression best linear unbiased prediction coupled with fivefold cross-validations and evaluated three strategies of marker preselection. For plant height, marker density and marker preselection procedure impacted prediction accuracy only marginally. In contrast, for grain yield, prediction accuracy based on markers selected with a haplotype block analyses-based approach increased by approximately 4 % compared with random or equidistant marker sampling. Thus, applying marker preselection based on haplotype blocks is an interesting option for a cost-efficient implementation of genomic selection for grain yield in soybean breeding.Genomic selection is a promising molecular breeding strategy enhancing genetic gain per unit time. The objectives of our study were to (1) explore the prediction accuracy of genomic selection for plant height and yield per plant in soybean [The online version of this article (doi:10.1007/s11032-016-0504-9) contains supplementary material, which is available to authorized users. Glycine max (L.) Merr.] is one of the most important sources of oil and plant protein  explore the genomic prediction accuracy for plant height and yield per plant, (2) discuss the relationship between prediction accuracy and numbers of markers, and (3) evaluate the effect of marker preselection based on different methods on the prediction accuracy.Our study comprised phenotypic data of 235 soybean varieties provided by the National Key Facility for Crop Gene Resources and Genetic Improvement (NFCIR), Institute of Crop Science, Chinese Academy of Agricultural Science. Out of the 235 varieties, 185 were North Spring soybean (NSs) and 50 HuangHuai summer soybean (HHSs) lines. The 235 varieties were evaluated in replicated field trials in 23 locations in Northeast China and in the HuangHuai region in the year 2011 (Supplementary Table S1). The experimental designs were randomized complete block designs with two replications. Plots consisted of three rows with 3 m in length and 0.2 m apart. Fertility and pest management were performed following standard management recommendations. Plant height (cm) and yield per plant (g) were determined in each location following standard protocols  of each line was determined using the same model mentioned above by assuming fixed genotypic effect and random location effects. The difference of target traits average between NSs subsets and HHSs subsets was evaluated applying a t test using PASW statistics.Variance components and heritability of plant height and yield per plant were estimated using the lme4 package implemented in the software package R . Linkage disequilibrium parameter (r2) between SNP pairs was estimated using the statistical software R (Team https://www.r-project.org/). Decay of linkage disequilibrium was explored based on the data of estimated r2 against genetic distance for all SNP pairs, by fitting a curve with the locally weighted polynomial regression method (Cleveland http://www.maizegenetics.net/). The first two principal components were used to examine the presence of subpopulation structure.The 235 soybean lines were genotyped with Illumina SoySNP 6 k iSelect BeadChip which comprised 5361 SNPs. These SNPs were chosen from the Illumina SoySNP 50 k iSelect BeadChip   is the n × p matrix of markers with xij being the number of a chosen allele at the jth locus for the ith genotype, and e is the vector of residual terms. In the model, we assumed that marker and residual effects are randomly distributed with Ip and In denote identity matrices with respective dimensions, l refers to the number of locations.The potential of genomic selection was examined focusing on ridge regression best linear unbiased prediction (RR-BLUP) implemented in the statistical package “rrBLUP” Endelman . Let n brMP) was estimated, and prediction accuracy (rGS) was calculated by standardizing rMP by the square root of the broad-sense heritability. We repeated the procedure 500 times to reduce the sampling error. In addition, we examined the prediction accuracy also within the North Spring soybean (NSs) subpopulation contrasting it with a random subset of the total population with the same sample size.We evaluated the prediction accuracy of genomic selection applying fivefold cross-validations. Marker effects were estimated in the training population and the effects were used to predict the genotypic values in the test population. The Pearson product-moment correlation coefficient between the predicted and observed phenotype  and broad genetic variation for the assayed 235 soybean varieties. Lines belonging to the HuangHuai summer group (HHSs) displayed significantly (P < 0.01) higher plant height and larger grain yield per plant as compared to North Spring (NSs) lines  compared to yield per plant (rGS = 0.47)  Fig. .Fig. 5BoWe studied the effects of different marker sampling strategies on genomic prediction accuracy for a broad range of marker densities. The marker sampling strategies were a random sampling method (RSM), a haplotype block analysis-based sampling (HBA), and evenly sampling method (ESM). Using a step of 250 SNPs, 265 to 5015 SNPs were randomly selected for RSM in order to estimate the prediction accuracies (Supplementary Table S2). In contrast, for HBA we selected one SNP for each of the 357 identified haplotype blocks. These SNPs were combined with the remaining 3197 “SNPs”. From this data set, we randomly selected 172 to 3554 SNPs with a step of 178 SNPs and examined the prediction accuracy for the target traits (Supplementary Table S2). We also selected from 172 to 2664 SNPs evenly around genome with a step of 178 SNPs for ESM strategy and evaluated the prediction accuracies (Supplementary Table S2). Generally, prediction accuracies for both plant height and yield per plant increased with increasing number of SNPs for both sampling strategies Below is the link to the electronic supplementary material.


Caretta caretta) make up one of the smallest subpopulations of this threatened species and have declining nest numbers. We used satellite telemetry and a switching state-space model to identify distinct foraging areas used by 59 NGoM loggerheads tagged during 2010–2013. We tagged turtles after nesting at three sites, 1 in Alabama  and 2 in Florida . Peak migration time was 22 July to 9 August during which >40% of turtles were in migration mode; the mean post-nesting migration period was 23.0 d (±13.8 d SD). After displacement from nesting beaches, 44 turtles traveled to foraging sites where they remained resident throughout tracking durations. Selected foraging locations were variable distances from tagging sites, and in 5 geographic regions; no turtles selected foraging sites outside the Gulf of Mexico (GoM). Foraging sites delineated using 50% kernel density estimation were located a mean distance of 47.6 km from land and in water with mean depth of −32.5 m; other foraging sites, delineated using minimum convex polygons, were located a mean distance of 43.0 km from land and in water with a mean depth of −24.9 m. Foraging sites overlapped with known trawling activities, oil and gas extraction activities, and the footprint of surface oiling during the 2010 Deepwater Horizon oil spill (n = 10). Our results highlight the year-round use of habitats in the GoM by loggerheads that nest in the NGoM. Our findings indicate that protection of females in this subpopulation requires both international collaborations and management of threats that spatially overlap with distinct foraging habitats.Northern Gulf of Mexico (NGoM) loggerheads ( Many highly mobile marine vertebrates, including turtles, make long distance migrations to breeding areas. The locations of marine turtle breeding and nesting sites are often up to hundreds to thousands of kilometers from foraging areas in which animals are resident for the majority of their lives Lepidochelys kempii) in the Gulf of Mexico (GoM), Shaver et al. Caretta caretta) in particular, combined data sets from multiple subpopulations in the Northwest Atlantic have produced a more comprehensive understanding of movement patterns and foraging sites used by breeding individuals Satellite tracking has emerged as a strong tool for uncovering oceanic routes taken during marine animal migrations, as well as areas of residence at-sea. Recently, analyses of multiple long-term tracking datasets have contributed to a broad understanding of marine animal habitat use across taxa at regional scales Loggerhead marine turtles in the Northwest Atlantic are listed as threatened under the U.S. Endangered Species Act. The species exists as five subpopulations Declines in nest abundance for the NGoM subpopulation Previous loggerhead tracking studies where turtles were tagged on beaches in the NGoM This work was conducted under the authority of research permits from the Florida Fish and Wildlife Conservation Commission Marine Turtle Permit #094, Bon Secour National Wildlife Refuge Special Use Permit #12-006S (issued to K. Hart), Federal U.S. Fish and Wildlife Permit #TE206903-1 (issued to J. Phillips), as well as the U.S Geological Survey Animal Care and Use permit #SESC-2011-05.Turtle tagging occurred at three study sites in the NGoM . The GulIn GS, nightly surveys were conducted from 9 pm to 6 am every day from 1 June to ∼30 June. On SJP, nightly surveys were conducted from 9 pm to 6 am every day from 15 May to 15 Aug. On EAFB, nightly surveys were conducted from 9 pm to 6 am for one week in July 2012 (10 to 17 July). We used 59 satellite transmitters to monitor the movements of 59 post-nesting loggerhead turtles in the GoM over a 4 year period between 2010 and 2013; details on those tagged in 2010 were previously published in Hart et al. 1 from June–November, then every 3rd day from November–May. We compared mean turtle size (SCL-tip) in two tagging locations, Alabama and Florida, with t-test.Turtles were documented and outfitted with transmitters using established protocols www.seaturtle.org. Location classes (LC) 3, 2, 1, 0, A, and B were used to reconstruct routes and calculate straight-line and total distances that the turtles traveled. Locations were rejected if they were LC Z (for which no error estimation was available). Argos assigns accuracy estimates of <250 m for LC 3, 250 to <500 m for LC 2, 500 to <1500 m for LC 1, and >1500 m for LC 0 Location data were filtered using Satellite Tracking and Analysis Tool STAT;  availablBalaenoptera musculus), and several species of turtles Location data obtained through satellite transmitters are often received at irregular time intervals, and sometimes involve large gaps and positional errors. Ad hoc filtering of location data based on location quality is not sufficient to remove erroneous locations and also results in loss of information We used a switching SSM approach to determine the beginning and end date of migration and foraging periods for each turtle following Hart et al. After fitting the switching SSM to individual loggerhead tracks, we identified locations where turtles were in migration mode. For this analysis, we summarized migration periods after inter-nesting periods and before arrival at foraging grounds. To do this, we used the migration period directly before the foraging period . We then visually confirmed these migration periods with location data and summarized migration both temporally (number of days) and spatially.From the foraging periods determined with SSM, we filtered out locations requiring speeds >5 kph, along with any other obviously erroneous locations . We also removed locations deeper than −200 m (neritic zone cutoff); Hawkes et al. cv) for each KDE 2) within each kernel density contour (50% and 95%) and to plot the data; we used 95% KDEs to represent the overall home foraging range, and the 50% KDEs to represent core area of activity at foraging sites If an individual foraging period was at least 20 days in length, we generated mean daily locations using the filtered locations to minimize autocorrelation; the resulting coordinates provided raw data for kernel density estimation (KDE). Kernel density is a non-parametric method used to identify one or more areas of disproportionately heavy use  within a home-range boundary For foraging periods that did not qualify for KDE analysis(those without 20 mean daily locations) but at least six days long, we performed minimum convex polygon (MCP) analysis , we tested tracks during the foraging period for spatial randomness against randomly generated walks http://www.ngdc.noaa.gov/m,gg/geodas/geodas.html; accessed 26 January 2012). We also plotted 2 KDE foraging centroids for loggerheads tagged in the NGoM and previously published in Hart et al. We calculated the centroid of each MCP and 50% KDE; if a 50% KDE included multiple activity centers, we calculated the centroid of the largest activity center. We extracted depths for all centroids and the distance from each to the nearest land. For bathymetry, we used the NOAA National Geophysical Data Center (GEODAS) ETOPO1, 1 arc-minute global relief model of Earth’s surface , and those that failed to run successfully in SSM , we plotted the last transmitted, filtered location. We also plotted last filtered locations for turtles that had KDEs or MCPs but for which site-fidelity tests failed. We plotted these “last points” for a visual representation of general areas these turtles occupied either during migration or foraging. We conducted simple regression analysis to examine association between turtle size and each of size of 50% KDE and bathymetry of foraging site. We also used simple regression analysis to examine whether size of 50% KDE is associated with number of tracking days.We assigned foraging sites into 5 geographic regions, following H. Vander Zanden (pers. comm.), and similar to VanderZanden et al. To depict all foraging locations used by turtles over time, we calculated the number of turtle-foraging days in grid cells (10×10 km); the grid extended across the extent of the GoM within the −200 m isobath. For each turtle track with days in foraging mode, we counted number of days each turtle was observed (turtle days) in each grid cell during foraging periods using all satellite locations except for LC Z.http://www.data.boem.gov/homepg/data_center/mapping/geographic_mapping.asp, accessed on 4 December 2013. Platforms in this layer included all platforms existing in the U.S. Bureau of Ocean and Energy Management (BOEM) database. To relate centroids to the DWH oil spill, we counted the number of foraging centroids located within the surface oiling layer from . In addition, we determined how many centroids were within state, U.S. federal or international waters. We obtained layers on 16 December 2013 for the Exclusive Economic Zone  and State Submerged Lands .Finally, to help provide guidance for conservation and management actions with foraging habitat, we also mapped the overlap of commercial shrimp trawling during 2011–2012 (sum of days fished over the two years), the locations of active oil and gas platforms, and the oiling extent from the DWH oil spill. We created a layer in ArcGIS 9.3 using shrimp trawling data and statistical zone cutoffs provided by NOAA . We determined the number of shrimp trawling days in each area as it related to the centroids. The layer for oil and gas platforms was obtained from 30.3 = 1.066, p = 0.295). In a total of 9229 tracking days, individual tracking durations for all 59 turtles ranged from 24 to 582 d . In 2010, we tracked 4 turtles and those 4 tracks were analyzed and published in Hart et al. We attempted to run each of 59 turtle tracks through SSM; however, we dropped 13 tracks due to short tracking durations or large transmission gaps. Four of the turtles were originally analyzed in Hart et al. We considered the migration period directly before the foraging period to be the main migration period; this classification was appropriate for all but 3 turtles . Turtle size and size of 50% KDE were not significantly associated  and the size of 50% KDEs were not influenced by tracking duration .As stated above, SSM was successful for 46 turtles see  and 2 pr2 .We calculated centroids for the 38 KDEs and 10 MCPs  for turt2,41 = 0.071, p-value = 0.931; means: NGoM = 93.9 cm SCL-tip); SGulf (SGoM+SNWA)  = 94.6; WFL = 94.4). The results of the Chi-square test did not indicate that the proportion of turtles in each foraging site region was significantly different between turtles tagged in Florida and Alabama ; however, this analysis was based on small number of turtles in each region.In this study, loggerheads selected foraging sites in each of the 5 regions defined earlier . TurtlesHigh numbers of turtle-days per grid cell occurred during foraging at locations throughout the Gulf . The disThe spatial information for oil and gas platforms only extended into U.S. waters so we could only compare the locations of n = 37/45 centroids 82%, 36 turtles). These platforms currently do not spread into waters off the Florida coast and therefore many of these turtle centroids were greater than 10 km from a platform. Only 4 centroids were within 10 km of platforms and these centroids were located off the coasts of Alabama (n = 1), Mississippi/Louisiana (n = 2) and Texas n = 1; . Shrimp  turtles.For many marine species, locations of key foraging areas are beginning to emerge through the combined use of satellite tracking technologies and advanced spatial modeling approaches. Loggerheads in the NGoM subpopulation may be in decline Using the SSM technique, we were able to quantitatively identify the initiation and termination of the migratory period, as well as date of arrival of loggerheads at foraging grounds. Previous studies have defined departure from nesting beaches or arrival at foraging grounds through visual inspection of location data Eubalaena glacialis) along the U.S. Atlantic coast For loggerheads in the Northern Gulf, timing of peak post-nesting migration clearly varied by individual and year, supporting previous findings of Rees et al. One other study see  tracked 2, similar to findings presented in Hart et al. 2. This selection of almost the exact same ‘patch’ of foraging habitat by a loggerhead is similar to findings presented in Marcovaldi et al. 2 and home ranges of approximately 500 km2 , followed by NGoM (32%), SGoM (18%), SNWA (11%), and WGoM (2%). Of the turtles selecting foraging sites in the NGoM, 35% were tagged in Alabama and 14% were tagged in Florida. These rankings mirror those found in Foley et al. Phalacrocorax aristotelis) Even though we tracked a large number of turtles in this study, the map of foraging site locations for this subpopulation may still be incomplete. In a two decade-long study on European shags , and other threats. Although foraging habitat characteristics and suitability for marine turtles in this region are poorly understood, locations of core-use foraging habitats identified here  indicate that important habitat exists for loggerheads at these same potentially affected sites Acipenser oxyrinchus desoto) Trichechus manatus) Tursiops truncatus) With the help of satellite telemetry and other techniques see , the locMany turtle home ranges overlapped with areas heavily used by commercial trawlers see . In theiFewer home ranges overlapped with areas with active oil and gas platforms, as the majority of the turtles we tracked foraged off WFL and the NGoM, where few oil and gas platforms are located. However, our SSM results indicated that neritic habitat off Texas, Louisiana, Mississippi, and Alabama, where oil and gas platforms are prevalent, appears to serve both as important movement corridors and foraging sites for turtles nesting throughout the NGoM see also . Oil andDue to the varied anthropogenic threats in the area, a cumulative impact assessment in the GoM could inform management efforts. A 2008 global impact assessment on marine ecosystems found the GoM to be an area with a medium-high impact from human activities Our results highlight the importance of habitats within the GoM for sustaining nesting females within the NGoM loggerhead subpopulation, and they clearly demonstrate the potential for interactions with anthropogenic activities that may threaten population persistence and recovery. Because adult female survival rates have an especially strong effect on population recovery The DWH oil spill is a key concern for exposure of adult females in this declining subpopulation to oil and oil clean-up activities. Potential impacts of oil and dispersants from the DWH oil spill on Gulf sea turtles may range from mortality to sublethal stress and chronic impairment, including potential deleterious effects on reproduction and recruitment Figure S1Predicted and actual tracks for Turtles 119941, 129506 and 129515 to show examples of exceptions for migration days  in between migration (red points), and both the migration before and after were used. Turtle 129506 had two foraging periods as defined by our date cut-off . The first was in the inter-nesting area near land and this period did not pass site fidelity. The visual inspection confirmed a ‘main’ migration after this first foraging period (red points). Turtle 129515 had a ‘main’ migration (red points) before an early small foraging period that was not the main foraging period used for analysis .(TIF)Click here for additional data file.Figure S2Migration tracks for 46 adult female loggerheads (Caretta caretta) after nesting in the Northern Gulf of Mexico 2010–2013. Tagging locations (black stars) from left to right are Gulf Shores, AL, Eglin Air Force Base, FL and St. Joseph Peninsula, FL. Lines created by connecting main migration locations (see ions see  filtered(TIF)Click here for additional data file.Figure S3Example switching state-space model (SSM) prediction (red and blue points) over raw unfiltered locations (open grey circles) for Turtle119946 tagged in Gulf Shores, Alabama in 2012.(TIF)Click here for additional data file.Figure S4Foraging site kernel density estimate  for 2 adult female loggerheads (Caretta caretta) tagged in 2010 and previously summarized in Hart et al. (2012), shown in dark blue, and with an additional 489 days (turtle 52968) and 62 days (turtle 47755) of tracking data added during ‘foraging’ mode (light blue).(TIF)Click here for additional data file.Figure S5Foraging site kernel density estimates  for adult female loggerheads (Caretta caretta) that nested in the Northern Gulf at study sites in Alabama and Florida between 2010–2013. Red asterisks denote KDEs for 2 turtles from previous study .(TIF)Click here for additional data file.Figure S6Ten minimum convex polygon (MCP) areas for 7 adult female loggerheads (Caretta caretta) satellite-tagged in the Northern Gulf (Turtle 129496 had 3 MCPs and Turtle 120439 had 2). MCPs are colored by tagging location.(TIF)Click here for additional data file.Figure S7Kernel Density Estimation (KDE) for 38 adult female loggerheads  satellite-tagged in the Northern Gulf of Mexico (NGoM); contours are colored by original tagging location . One turtle was tracked in both 2011 and 2012, and turtle 120439 had two foraging KDEs. Two turtles from Hart et al. (2012) are included. Gulf regions are denoted by red lines and are as follows: WGoM, Western Gulf of Mexico; NGoM; WFL, Western Florida; SNWA, Subtropical Northwest Atlantic; and SGoM, Southern Gulf of Mexico.(TIF)Click here for additional data file.Table S1Turtle info.(DOCX)Click here for additional data file.Table S2Tags used by site and year.(DOCX)Click here for additional data file.Table S3SSM prediction and model parameters for turtles 119946, 129515, 119941, and 129506.(DOC)Click here for additional data file.Methods S1Detailed methods for why we selected or ruled out turtles for specific analyses and any exceptions made.(DOCX)Click here for additional data file.