134 research outputs found
Capturing complex tumour biology in vitro: Histological and molecular characterisation of precision cut slices
Precision-cut slices of in vivo tumours permit interrogation in vitro of heterogeneous cells from solid tumours together with their native microenvironment. They offer a low throughput but high content in vitro experimental platform. Using mouse models as surrogates for three common human solid tumours, we describe a standardised workflow for systematic comparison of tumour slice cultivation methods and a tissue microarray-based method to archive them. Cultivated slices were compared to their in vivo source tissue using immunohistochemical and transcriptional biomarkers, particularly of cellular stress. Mechanical slicing induced minimal stress. Cultivation of tumour slices required organotypic support materials and atmospheric oxygen for maintenance of integrity and was associated with significant temporal and loco-regional changes in protein expression, for example HIF-1α. We recommend adherence to the robust workflow described, with recognition of temporal-spatial changes in protein expression before interrogation of tumour slices by pharmacological or other means
Orange jasmine as a trap crop to control Diaphorina citri
[EN] Novel, suitable and sustainable alternative control tactics that have the potential to reduce migration of Diaphorina citri into commercial citrus orchards are essential to improve management of huanglongbing (HLB). In this study, the effect of orange jasmine (Murraya paniculata) as a border trap crop on psyllid settlement and dispersal was assessed in citrus orchards. Furthermore, volatile emission profiles and relative attractiveness of both orange jasmine and sweet orange (Citrus¿×¿aurantium L., syn. Citrus sinensis (L.) Osbeck) nursery flushes to D. citri were investigated. In newly established citrus orchards, the trap crop reduced the capture of psyllids in yellow sticky traps and the number of psyllids that settled on citrus trees compared to fallow mowed grass fields by 40% and 83%, respectively. Psyllids were attracted and killed by thiamethoxam-treated orange jasmine suggesting that the trap crop could act as a `sink¿ for D. citri. Additionally, the presence of the trap crop reduced HLB incidence by 43%. Olfactometer experiments showed that orange jasmine plays an attractive role on psyllid behavior and that this attractiveness may be associated with differences in the volatile profiles emitted by orange jasmine in comparison with sweet orange. Results indicated that insecticide-treated M. paniculata may act as a trap crop to attract and kill D. citri before they settled on the edges of citrus orchards, which significantly contributes to the reduction of HLB primary spread.This work was supported by Fund for Citrus Protection (Fundecitrus) and by Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) (Proc. 2015/07011-3). We thank Moacir Celio Vizone, Felipe Marinho Martini and Joao Pedro Ancoma Lopes for technical support with experiments. Furthermore, we thank Cambuhy Agricola Ltda. and University of Araraquara (Uniara) for providing the areas in which the field experiments were performed. Second author received scholarship from National Council for Scientific and Technological Development (CNPq)/Brazil (Proc. 300153/2011-2).Tomaseto, AF.; Marques, RN.; Fereres, A.; Zanardi, OZ.; Volpe, HXL.; Alquézar-García, B.; Peña, L.... (2019). Orange jasmine as a trap crop to control Diaphorina citri. Scientific Reports. 9:1-11. https://doi.org/10.1038/s41598-019-38597-5S1119Bové, J. M. Huanglongbing: a destructive, newly-emerging, century-old disease of citrus. J Plant Pathol. 88, 7–37 (2006).Alvarez, S., Rohrig, E., Solís, D. & Thomas, M. H. Citrus greening disease (Huanglongbing) in Florida: economic impact, management and the potential for biological control. Agric. Res. 5, 109–118 (2016).Belasque, J. Jr. et al. Lessons from huanglongbing management in São Paulo state, Brazil. J. Plant Pathol. 92, 285–302 (2010).Boina, D. R., Meyer, W. L., Onagbola, E. O. & Stelinski, L. L. Quantifying dispersal of Diaphorina citri (Hemiptera: Psyllidae) by immunomarking and potential impact of unmanaged groves on commercial citrus management. Environ. Entomol. 38, 1250–8 (2009).Lewis-Rosenblum, H., Martini, X., Tiwari, S. & Stelinski, L. L. Seasonal movement patterns and long-range dispersal of Asian citrus psyllid in Florida citrus. J. Econ. Entomol. 108, 3–10 (2015).Hall, D. G. & Hentz, M. G. Seasonal flight activity by the Asian citrus psyllid in east central Florida. Entomol. Exp. Appl. 139, 75–85 (2011).Tomaseto, A. F., Krugner, R. & Lopes, J. R. S. Effect of plant barriers and citrus leaf age on dispersal of Diaphorina citri (Hemiptera: Liviidae). J. Appl. Entomol. 140, 91–102 (2016).Gottwald, T. R. Current epidemiological understanding of citrus huanglongbing. Annu. Rev. Phytopathol. 48, 119–139 (2010).Bassanezi, R. B. et al. Efficacy of area-wide inoculum reduction and vector control on temporal progress of huanglongbing in young sweet orange plantings. Plant Dis. 97, 789–796 (2013).Sétamou, M. & Bartels, D. W. Living on the edges: spatial niche occupation of Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae), in citrus groves. PLoS One 10, 1–21 (2015).Gottwald, T., Irey, M., Gast, T. & Parnell, S. Spatio-temporal analysis of an HLB epidemic in Florida and implications for spread. In Proceedings of the 17 th Conference of International Organization of Citrus Virologists, IOCV, University of California, Riverside, CA, 84–97 (2010).Shelton, A. M. & Badenes-Perez, F. R. Concepts and applications of trap cropping in pest management. Annu. Rev. Entomol 51, 285–308 (2006).Hokkanen, H. M. T. Trap cropping in pest management. Annu. Rev. Entomol. 36, 119–138 (1991).Stern, V. M., Mueller, A., Sevacherian, V. & Way, M. Lygus bug control in cotton through alfalfa interplanting. Calif. Agric. 8–10 (1969).Godfrey, L. D. & Leigh, T. F. Alfalfa harvest strategy effect on lygus bug (Hemiptera: Miridae) and insect predator population density: Implications for use as trap crop in cotton. Environ. Entomol. 23, 1106–1118 (1994).Gonsalves, D. & Ferreira, S. Transgenic papaya: a case for managing risks of Papaya ringspot virus in Hawaii. Plant Heal. Prog. 1–6, https://doi.org/10.1094/PHP-2003-1113-03-RV (2003)Aubert, B. Trioza erytheae del Guercio and Diaphorina citri Kuwayama (Homoptera: Psylloidea), the two vectors of citrus greening disease: biological aspects and possible control strategies. Fruits 42, 149–162 (1987).Leong, S. C. T., Fatimah, A., Beattie, A., Heng, R. K. J. & King, W. S. Influence of host plant species and flush growth stage on the Asian citrus psyllid, Diaphorina citri Kuwayama. Am. J. Agric. Biol. Sci. 6, 536–543 (2011).Patt, J. M. & Sétamou, M. Responses of the Asian citrus psyllid to volatiles emitted by the flushing shoots of its rutaceous host plants. Environ. Entomol. 39, 618–24 (2010).Damsteegt, V. D. et al. Murraya paniculata and related species as potential hosts and inoculum reservoirs of ‘Candidatus Liberibacter asiaticus’, causal agent of huanglongbing. Plant Dis. 94, 528–533 (2010).Lopes, S. A. et al. Liberibacters associated with orange jasmine in Brazil: Incidence in urban areas and relatedness to citrus liberibacters. Plant Pathol. 59, 1044–1053 (2010).Cifuentes-Arenas, J. C. Huanglongbing e Diaphorina citri: Estudos das relações patógeno-vetor-hospedeiro. Ph.D. Thesis. Faculdade de Ciências Agrárias e Veterinárias/Universidade Estadual Paulista (UNESP), Jaboticabal, SP, Brazil. 1–133 (2017).Morilla, G. et al. Pepper (Capsicum annuum) is a dead-end host for Tomato yellow leaf curl virus. Phytopathology 95, 1089–1097 (2005).Midega, C. A. O., Pittchar, J. O., Pickett, J. A., Hailu, G. W. & Khan, Z. R. A climate-adapted push-pull system effectively controls fall armyworm, Spodoptera frugiperda (J. E. Smith), in maize in East Africa. Crop Prot. 105, 10–15 (2018).Miranda, M. P. et al. Processed kaolin affects the probing and settling behavior of Diaphorina citri (Hemiptera: Liviidae). Pest Manag. Sci. 74, 1964–1972 (2018).Kobori, Y., Nakata, T., Ohto, Y. & Takasu, F. Dispersal of adult Asian citrus psyllid, Diaphorina citri Kuwayama (Homoptera: Psyllidae), the vector of citrus greening disease, in artificial release experiments. Appl. Entomol. Zool. 46, 27–30 (2011).Sétamou, M. et al. Diurnal patterns of flight activity and effects of light on host finding behavior of the Asian citrus psyllid. J. Insect Behav. 25, 264–276 (2012).Wenninger, E. J., Stelinski, L. L. & Hall, D. G. Roles of olfactory cues, visual cues, and mating status in orientation of Diaphorina citri Kuwayama (Hemiptera: Psyllidae) to four different host plants. Environ. Entomol. 38, 225–234 (2009).Miranda, M. P., Dos Santos, F. L., Felippe, M. R., Moreno, A. & Fereres, A. Effect of UV-blocking plastic films on take-off and host plant finding ability of Diaphorina citri (Hemiptera: Liviidae). J. Econ. Entomol. 108, 245–251 (2015).Visser, J. H. Host odor perception in phytophagous insects. Annu. Rev. Entomol. 31, 121–144 (1986).Robbins, P. S., Alessandro, R. T., Stelinski, L. L. & Lapointe, S. L. Volatile profiles of young leaves of Rutaceae spp. varying in susceptibility to the Asian citrus psyllid (Hemiptera: Psyllidae). Florida Entomol. 95, 774–776 (2012).Fancelli, M. et al. Attractiveness of host plant volatile extracts to the Asian citrus psyllid, Diaphorina citri, is reduced by terpenoids from the non-host cashew. J. Chem. Ecol. 44, 397–405 (2018).Alquézar, B. et al. β-caryophyllene emitted from a transgenic Arabidopsis or chemical dispenser repels Diaphorina citri, vector of Candidatus Liberibacters. Sci. Rep. 7, 5639 (2017).Jones, R. A. C. Effects of cereal borders, admixture with cereals and plant density on the spread of bean yellow mosaic potyvirus into narrow‐leafed lupins (Lupinus angustifolius). Ann. Appl. Biol. 122, 501–518 (1993).Beloti, V. H., Alves, G. R., Coletta-Filho, H. D. & Yamamoto, P. T. The Asian citrus psyllid host Murraya koenigii is immune to citrus huanglongbing pathogen ‘Candidatus Liberibacter asiaticus’. Phytopathology 108, 1089–1094 (2018).Walter, A. J., Duan, Y. & Hall, D. G. Titers of ‘Ca. Liberibacter asiaticus’ in Murraya paniculata and Murraya-reared Diaphorina citri are much lower than in Citrus and Citrus-reared psyllids. HortScience 47, 1449–1452 (2012).Walter, A. J., Hall, D. G. & Duan, Y. P. Low incidence of ‘Candidatus Liberibacter asiaticus’ in Murraya paniculata and associated Diaphorina citri. Plant Dis. 96, 827–832 (2012).Ammar, E.-D. D., Ramos, J. E., Hall, D. G., Dawson, W. O. & Shatters, R. G. Acquisition, replication and inoculation of Candidatus Liberibacter asiaticus following various acquisition periods on huanglongbing-infected citrus by nymphs and adults of the Asian citrus psyllid. PLoS One 11, e0159594 (2016).Inoue, H. et al. Enhanced proliferation and efficient transmission of Candidatus Liberibacter asiaticus by adult Diaphorina citri after acquisition feeding in the nymphal stage. Ann. Appl. Biol. 155, 29–36 (2009).Pelz-Stelinski, K. S., Brlansky, R. H., Ebert, T. A. & Rogers, M. E. Transmission parameters for Candidatus Liberibacter asiaticus by Asian citrus psyllid (Hemiptera: Psyllidae). J. Econ. Entomol. 103, 1531–1541 (2010).Canale, M. C. et al. Latency and persistence of ‘Candidatus Liberibacter asiaticus’ in its psyllid vector, Diaphorina citri (Hemiptera: Liviidae). Phytopathology 107, 264–272 (2017).Li, W., Hartung, J. S. & Levy, L. Quantitative real-time PCR for detection and identification of Candidatus Liberibacter species associated with citrus huanglongbing. J. Microbiol. Methods 66, 104–115 (2006).Nakata, T. Effectiveness of micronized fluorescent powder for marking citrus psyllid. Diaphorina citri. Appl. Entomol. Zool. 43, 33–36 (2008).Tomaseto, A. F. et al. Environmental conditions for Diaphorina citri Kuwayama (Hemiptera: Liviidae) take-off. J. Appl. Entomol. 142, 104–113 (2018).Paris, T. M., Croxton, S. D., Stansly, P. A. & Allan, S. A. Temporal response and attraction of Diaphorina citri to visual stimuli. Entomol. Exp. Appl. 155, 137–147 (2015).Zanardi, O. Z. et al. Putative sex pheromone of the Asian citrus psyllid, Diaphorina citri, breaks down into an attractant. Sci. Rep. 8, 455 (2018).Metsalu, T. & Vilo, J. ClustVis: A web tool for visualizing clustering of multivariate data using Principal Component Analysis and heatmap. Nucleic Acids Res. 43, W566–W570 (2015).Fournier, D. A. et al. AD Model Builder: using automatic differentiation for statistical inference of highly parameterized complex nonlinear models. Optim. Methods Softw. 27, 233–249 (2012).Bates, D., Mächler, M., Bolker, B. & Walker, S. Fitting linear mixed-effects models using lme4. J. Stat. Softw. 67, 1–48 (2015).Nelder, J. A. & Wedderburn, R. W. M. Generalized linear models. J. R. Stat. Soc. 135, 370–384 (1972).Demétrio, C. G. B., Hinde, J. & Moral, R. A. In Ecological Modelling Applied to Entomology (eds Ferreira, C. P. & Godoy, W. A. C.) 219–259 (Springer, 2014).Lenth, R. V. Least-Squares Means: the R package lsmeans. J. Stat. Softw. 69, (2016).R Core Team R: A language and environment for statistical computing. 2015. R Foundation for Statistical Computing, Vienna, Austria (2015). Available at, http://www.r-project.org/ . (Accessed: 20th July 2017)
In vitro fertilization does not increase the incidence of de novo copy number alterations in fetal and placental lineages
Although chromosomal instability (CIN) is a common phenomenon in cleavage-stage embryogenesis following in vitro fertilization (IVF)1,2,3, its rate in naturally conceived human embryos is unknown. CIN leads to mosaic embryos that contain a combination of genetically normal and abnormal cells, and is significantly higher in in vitro-produced preimplantation embryos as compared to in vivo-conceived preimplantation embryos4. Even though embryos with CIN-derived complex aneuploidies may arrest between the cleavage and blastocyst stages of embryogenesis5,6, a high number of embryos containing abnormal cells can pass this strong selection barrier7,8. However, neither the prevalence nor extent of CIN during prenatal development and at birth, following IVF treatment, is well understood. Here we profiled the genomic landscape of fetal and placental tissues postpartum from both IVF and naturally conceived children, to investigate the prevalence and persistence of large genetic aberrations that probably arose from IVF-related CIN. We demonstrate that CIN is not preserved at later stages of prenatal development, and that de novo numerical aberrations or large structural DNA imbalances occur at similar rates in IVF and naturally conceived live-born neonates. Our findings affirm that human IVF treatment has no detrimental effect on the chromosomal constitution of fetal and placental lineages
Altered miRNA expression network in locus coeruleus of depressed suicide subjects
Norepinephrine (NE) is produced primarily by neurons in the locus coeruleus (LC). Retrograde and ultrastructural examinations reveal that the core of the LC and its surrounding region receives afferent projections from several brain areas which provide multiple neurochemical inputs to the LC with changes in LC neuronal firing, making it a highly coordinated event. Although NE and mediated signaling systems have been studied in relation to suicide and psychiatric disorders that increase the risk of suicide including depression, less is known about the corresponding changes in molecular network within LC. In this study, we examined miRNA networks in the LC of depressed suicide completers and healthy controls. Expression array revealed differential regulation of 13 miRNAs. Interaction between altered miRNAs and target genes showed dense interconnected molecular network. Functional clustering of predicated target genes yielded stress induced disorders that collectively showed the complex nature of suicidal behavior. In addition, 25 miRNAs were pairwise correlated specifically in the depressed suicide group, but not in the control group. Altogether, our study revealed for the first time the involvement of LC based dysregulated miRNA network in disrupting cellular pathways associated with suicidal behavior
MicroScope: ChIP-seq and RNA-seq software analysis suite for gene expression heatmaps
BACKGROUND: Heatmaps are an indispensible visualization tool for examining large-scale snapshots of genomic activity across various types of next-generation sequencing datasets. However, traditional heatmap software do not typically offer multi-scale insight across multiple layers of genomic analysis (e.g., differential expression analysis, principal component analysis, gene ontology analysis, and network analysis) or multiple types of next-generation sequencing datasets (e.g., ChIP-seq and RNA-seq). As such, it is natural to want to interact with a heatmap’s contents using an extensive set of integrated analysis tools applicable to a broad array of genomic data types. RESULTS: We propose a user-friendly ChIP-seq and RNA-seq software suite for the interactive visualization and analysis of genomic data, including integrated features to support differential expression analysis, interactive heatmap production, principal component analysis, gene ontology analysis, and dynamic network analysis. CONCLUSIONS: MicroScope is hosted online as an R Shiny web application based on the D3 JavaScript library: http://microscopebioinformatics.org/. The methods are implemented in R, and are available as part of the MicroScope project at: https://github.com/Bohdan-Khomtchouk/Microscope
Capturing complex tumour biology in vitro : histological and molecular characterisation of precision cut slices
Precision-cut slices of in vivo tumours permit interrogation in vitro of heterogeneous cells from solid tumours together with their native microenvironment. They offer a low throughput but high content in vitro experimental platform. Using mouse models as surrogates for three common human solid tumours, we describe a standardised workflow for systematic comparison of tumour slice cultivation methods and a tissue microarray-based method to archive them. Cultivated slices were compared to their in vivo source tissue using immunohistochemical and transcriptional biomarkers, particularly of cellular stress. Mechanical slicing induced minimal stress. Cultivation of tumour slices required organotypic support materials and atmospheric oxygen for maintenance of integrity and was associated with significant temporal and loco-regional changes in protein expression, for example HIF-1 alpha. We recommend adherence to the robust workflow described, with recognition of temporal-spatial changes in protein expression before interrogation of tumour slices by pharmacological or other means.Peer reviewe
SARS-CoV-2 susceptibility and COVID-19 disease severity are associated with genetic variants affecting gene expression in a variety of tissues
Variability in SARS-CoV-2 susceptibility and COVID-19 disease severity between individuals is partly due to genetic factors. Here, we identify 4 genomic loci with suggestive associations for SARS-CoV-2 susceptibility and 19 for COVID-19 disease severity. Four of these 23 loci likely have an ethnicity-specific component. Genome-wide association study (GWAS) signals in 11 loci colocalize with expression quantitative trait loci (eQTLs) associated with the expression of 20 genes in 62 tissues/cell types (range: 1:43 tissues/gene), including lung, brain, heart, muscle, and skin as well as the digestive system and immune system. We perform genetic fine mapping to compute 99% credible SNP sets, which identify 10 GWAS loci that have eight or fewer SNPs in the credible set, including three loci with one single likely causal SNP. Our study suggests that the diverse symptoms and disease severity of COVID-19 observed between individuals is associated with variants across the genome, affecting gene expression levels in a wide variety of tissue types
Whole-genome sequencing reveals host factors underlying critical COVID-19
Critical COVID-19 is caused by immune-mediated inflammatory lung injury. Host genetic variation influences the development of illness requiring critical care1 or hospitalization2–4 after infection with SARS-CoV-2. The GenOMICC (Genetics of Mortality in Critical Care) study enables the comparison of genomes from individuals who are critically ill with those of population controls to find underlying disease mechanisms. Here we use whole-genome sequencing in 7,491 critically ill individuals compared with 48,400 controls to discover and replicate 23 independent variants that significantly predispose to critical COVID-19. We identify 16 new independent associations, including variants within genes that are involved in interferon signalling (IL10RB and PLSCR1), leucocyte differentiation (BCL11A) and blood-type antigen secretor status (FUT2). Using transcriptome-wide association and colocalization to infer the effect of gene expression on disease severity, we find evidence that implicates multiple genes—including reduced expression of a membrane flippase (ATP11A), and increased expression of a mucin (MUC1)—in critical disease. Mendelian randomization provides evidence in support of causal roles for myeloid cell adhesion molecules (SELE, ICAM5 and CD209) and the coagulation factor F8, all of which are potentially druggable targets. Our results are broadly consistent with a multi-component model of COVID-19 pathophysiology, in which at least two distinct mechanisms can predispose to life-threatening disease: failure to control viral replication; or an enhanced tendency towards pulmonary inflammation and intravascular coagulation. We show that comparison between cases of critical illness and population controls is highly efficient for the detection of therapeutically relevant mechanisms of disease
SARS-CoV-2 susceptibility and COVID-19 disease severity are associated with genetic variants affecting gene expression in a variety of tissues
Variability in SARS-CoV-2 susceptibility and COVID-19 disease severity between individuals is partly due to
genetic factors. Here, we identify 4 genomic loci with suggestive associations for SARS-CoV-2 susceptibility
and 19 for COVID-19 disease severity. Four of these 23 loci likely have an ethnicity-specific component.
Genome-wide association study (GWAS) signals in 11 loci colocalize with expression quantitative trait loci
(eQTLs) associated with the expression of 20 genes in 62 tissues/cell types (range: 1:43 tissues/gene),
including lung, brain, heart, muscle, and skin as well as the digestive system and immune system. We perform
genetic fine mapping to compute 99% credible SNP sets, which identify 10 GWAS loci that have eight or fewer
SNPs in the credible set, including three loci with one single likely causal SNP. Our study suggests that the
diverse symptoms and disease severity of COVID-19 observed between individuals is associated with variants across the genome, affecting gene expression levels in a wide variety of tissue types
Genome-wide DNA methylation analysis of transient neonatal diabetes type 1 patients with mutations in ZFP57
- …