Fabrication and modeling of a continuous-flow microfluidic device for on-chip DNA amplification

This paper was presented at the 3rd Micro and Nano Flows Conference (MNF2011), which was held at the Makedonia Palace Hotel, Thessaloniki in Greece. The conference was organised by Brunel University and supported by the Italian Union of Thermofluiddynamics, Aristotle University of Thessaloniki, University of Thessaly, IPEM, the Process Intensification Network, the Institution of Mechanical Engineers, the Heat Transfer Society, HEXAG - the Heat Exchange Action Group, and the Energy Institute.The fabrication process and heat transfer computations for a continuous flow microfluidic device for DNA amplification by polymerase chain reaction (PCR) are described. The building blocks are thin polymeric materials aiming at a low cost and low power consumption device. The fabrication is performed by standard pattern transfer techniques (lithography and etching) used for microelectronics fabrication. The DNA sample flows in a meander shaped microchannel formed on a 100μm thick polyimide (PI) layer through three temperature regions defined by the integrated resistive heaters. The heat transfer computations are performed in a unit cell of the device. They show that, for the fabricated device, the variation of the temperature inside the channel zones where each step (denaturation, annealing, or extension) of PCR occur is less than 1.3K. This variation increases when the thickness of the PI layer increases. The computations also show that similar Silicon-based devices lead to lower temperature difference between the heaters and the DNA sample compared to the polymer-based fabricated device. However, the power consumption is estimated much greater for Silicon-based devices.This work was co-financed by Hellenic Funds and by the European Regional Development Fund (ERDF) under the Hellenic National Strategic Reference Framework (NSRF) 2007-2013, according to Contract no. MICRO2-45 of the Project “Microelectronic Components for Lab-on-chip molecular analysis instruments for genetic and environmental applications” within the Programme "Hellenic Technology Clusters in Microelectronics – Phase-2 Aid Measure"

Developing an infrastructure for secure patient summary exchange in the EU context: Lessons learned from the KONFIDO project

Background: The increase of healthcare digitalization comes along with potential information security risks. Thus, the EU H2020 KONFIDO project aimed to provide a toolkit supporting secure cross-border health data exchange. Methods: KONFIDO focused on the so-called “User Goals”, while also identifying barriers and facilitators regarding eHealth acceptance. Key user scenarios were elaborated both in terms of threat analysis and legal challenges. Moreover, KONFIDO developed a toolkit aiming to enhance the security of OpenNCP, the reference implementation framework. Results: The main project outcomes are highlighted and the “Lessons Learned,” the technical challenges and the EU context are detailed. Conclusions: The main “Lessons Learned” are summarized and a set of recommendations is provided, presenting the position of the KONFIDO consortium toward a robust EU-wide health data exchange infrastructure. To this end, the lack of infrastructure and technical capacity is highlighted, legal and policy challenges are identified and the need to focus on usability and semantic interoperability is emphasized. Regarding technical issues, an emphasis on transparent and standards-based development processes is recommended, especially for landmark software projects. Finally, promoting mentality change and knowledge dissemination is also identified as key step toward the development of secure cross-border health data exchange services

Strategies for Multiplexed Electrochemical Sensor Development

Detection of multiple biomarkers for disease diagnosis or treatment monitoring has received a lot of attention due to their potential impact on clinical decision making. Electrochemical biosensors have become one of the preferred detection approaches, due to the simplicity of the accompanying instrumentation. This chapter will explore how electrochemical sensors can be utilized for detection of multiple analytes by integration of sensors into microfluidic microsystems. Some key fabrication technologies for such devices will be presented utilizing polymer microfabrication, paper-based approaches, and the use of printed circuit boards. Next, the use of electrode arrays will be presented along with some commercial platforms, outlining plausible paths towards a successful electrochemical multiplexed sensor. Novel approaches based on microbeads and various labels will then be introduced along with various strategies and technologies utilized to achieve ultrasensitive multiplexed detection

Disposable sensors in diagnostics, food and environmental monitoring

Disposable sensors are low‐cost and easy‐to‐use sensing devices intended for short‐term or rapid single‐point measurements. The growing demand for fast, accessible, and reliable information in a vastly connected world makes disposable sensors increasingly important. The areas of application for such devices are numerous, ranging from pharmaceutical, agricultural, environmental, forensic, and food sciences to wearables and clinical diagnostics, especially in resource‐limited settings. The capabilities of disposable sensors can extend beyond measuring traditional physical quantities (for example, temperature or pressure); they can provide critical chemical and biological information (chemo‐ and biosensors) that can be digitized and made available to users and centralized/decentralized facilities for data storage, remotely. These features could pave the way for new classes of low‐cost systems for health, food, and environmental monitoring that can democratize sensing across the globe. Here, a brief insight into the materials and basics of sensors (methods of transduction, molecular recognition, and amplification) is provided followed by a comprehensive and critical overview of the disposable sensors currently used for medical diagnostics, food, and environmental analysis. Finally, views on how the field of disposable sensing devices will continue its evolution are discussed, including the future trends, challenges, and opportunities

Correction for Johansson et al., An open challenge to advance probabilistic forecasting for dengue epidemics.

Correction for “An open challenge to advance probabilistic forecasting for dengue epidemics,” by Michael A. Johansson, Karyn M. Apfeldorf, Scott Dobson, Jason Devita, Anna L. Buczak, Benjamin Baugher, Linda J. Moniz, Thomas Bagley, Steven M. Babin, Erhan Guven, Teresa K. Yamana, Jeffrey Shaman, Terry Moschou, Nick Lothian, Aaron Lane, Grant Osborne, Gao Jiang, Logan C. Brooks, David C. Farrow, Sangwon Hyun, Ryan J. Tibshirani, Roni Rosenfeld, Justin Lessler, Nicholas G. Reich, Derek A. T. Cummings, Stephen A. Lauer, Sean M. Moore, Hannah E. Clapham, Rachel Lowe, Trevor C. Bailey, Markel García-Díez, Marilia Sá Carvalho, Xavier Rodó, Tridip Sardar, Richard Paul, Evan L. Ray, Krzysztof Sakrejda, Alexandria C. Brown, Xi Meng, Osonde Osoba, Raffaele Vardavas, David Manheim, Melinda Moore, Dhananjai M. Rao, Travis C. Porco, Sarah Ackley, Fengchen Liu, Lee Worden, Matteo Convertino, Yang Liu, Abraham Reddy, Eloy Ortiz, Jorge Rivero, Humberto Brito, Alicia Juarrero, Leah R. Johnson, Robert B. Gramacy, Jeremy M. Cohen, Erin A. Mordecai, Courtney C. Murdock, Jason R. Rohr, Sadie J. Ryan, Anna M. Stewart-Ibarra, Daniel P. Weikel, Antarpreet Jutla, Rakibul Khan, Marissa Poultney, Rita R. Colwell, Brenda Rivera-García, Christopher M. Barker, Jesse E. Bell, Matthew Biggerstaff, David Swerdlow, Luis Mier-y-Teran-Romero, Brett M. Forshey, Juli Trtanj, Jason Asher, Matt Clay, Harold S. Margolis, Andrew M. Hebbeler, Dylan George, and Jean-Paul Chretien, which was first published November 11, 2019; 10.1073/pnas.1909865116. The authors note that the affiliation for Xavier Rodó should instead appear as Catalan Institution for Research and Advanced Studies (ICREA) and Climate and Health Program, Barcelona Institute for Global Health (ISGlobal). The corrected author and affiliation lines appear below. The online version has been corrected

Thermospermine catabolism increases Arabidopsis thaliana resistance to Pseudomonas viridiflava

This work investigated the roles of the tetraamine thermospermine (TSpm) by analysing its contribution to Arabidopsis basal defence against the biotrophic bacterium Pseudomonas viridiflava. The participation of polyamine oxidases (PAOs) in TSpm homeostasis and TSpm-mediated defence was also investigated. Exogenous supply of TSpm, as well as ectopic expression of the TSpm biosynthetic gene ACL5, increased Arabidopsis Col-0 resistance to P. viridiflava, while null acl5 mutants were less resistant than Col-0 plants. The above-mentioned increase in resistance was blocked by the PAO inhibitor SL-11061, thus demonstrating the participation of TSpm oxidation. Analysis of PAO genes expression in transgenic 35S::ACL5 and Col-0 plants supplied with TSpm suggests that PAO 1, 3, and 5 are the main PAOs involved in TSpm catabolism. In summary, TSpm exhibited the potential to perform defensive functions previously reported for its structural isomer Spm, and the relevance of these findings is discussed in the context of ACL5 expression and TSpm concentration in planta. Moreover, this work demonstrates that manipulation of TSpm metabolism modifies plant resistance to pathogens.The gift of SL-11061 by Dr Frydman (SLIL Biomedical Corporation, Madison, WI) is greatly appreciated. This work was supported by Consejo Nacional de Investigaciones Cientificas y Tecnicas (CONICET), Argentina (PIP 5740, PIP 0395), Agencia Nacional de Promocion Cientifica y Tecnologica, Argentina (PICT 1119, ANPCYT), Universidad Nacional de General San Martin, Argentina (SJ10/30), Ministerio de Economia y Competitividad, Spain (BIO2011-23828), and Fundacion Carolina (postdoctoral fellowship to MM). MM, FLP, and OAR are members of the Research Career of CONICET.Marina, M.; Vera Sirera, FJ.; Rambla Nebot, JL.; Gonzalez, ME.; Blazquez Rodriguez, MA.; Carbonell Gisbert, J.; Pieckenstain, FL.... (2013). Thermospermine catabolism increases Arabidopsis thaliana resistance to Pseudomonas viridiflava. Journal of Experimental Botany. 64(5):1393-1402. https://doi.org/10.1093/jxb/ert012S13931402645Alonso, J. M. (2003). Genome-Wide Insertional Mutagenesis of Arabidopsis thaliana. Science, 301(5633), 653-657. doi:10.1126/science.1086391Alippi, A. M., Dal Bo, E., Ronco, L. B., Lopez, M. V., Lopez, A. C., & Aguilar, O. M. (2003). Pseudomonas populations causing pith necrosis of tomato and pepper in Argentina are highly diverse. Plant Pathology, 52(3), 287-302. doi:10.1046/j.1365-3059.2003.00850.xAngelini, R., Bragaloni, M., Federico, R., Infantino, A., & Porta-Pugua, A. (1993). Involvement of Polyamines, Diamine Oxidase and Peroxidase in Resistance of Chickpea to Ascochyta rabiei. Journal of Plant Physiology, 142(6), 704-709. doi:10.1016/s0176-1617(11)80906-5Clough, S. J., & Bent, A. F. (1998). Floral dip: a simplified method forAgrobacterium-mediated transformation ofArabidopsis thaliana. The Plant Journal, 16(6), 735-743. doi:10.1046/j.1365-313x.1998.00343.xCona, A., Rea, G., Angelini, R., Federico, R., & Tavladoraki, P. (2006). Functions of amine oxidases in plant development and defence. Trends in Plant Science, 11(2), 80-88. doi:10.1016/j.tplants.2005.12.009Fincato, P., Moschou, P. N., Spedaletti, V., Tavazza, R., Angelini, R., Federico, R., … Tavladoraki, P. (2010). Functional diversity inside the Arabidopsis polyamine oxidase gene family. Journal of Experimental Botany, 62(3), 1155-1168. doi:10.1093/jxb/erq341Gonzalez, M. E., Marco, F., Minguet, E. G., Carrasco-Sorli, P., Blázquez, M. A., Carbonell, J., … Pieckenstain, F. L. (2011). Perturbation of spermine synthase Gene Expression and Transcript Profiling Provide New Insights on the Role of the Tetraamine Spermine in Arabidopsis Defense against Pseudomonas viridiflava. Plant Physiology, 156(4), 2266-2277. doi:10.1104/pp.110.171413Hanzawa, Y., Imai, A., Michael, A. J., Komeda, Y., & Takahashi, T. (2002). Characterization of the spermidine synthase-related gene family inArabidopsis thaliana. FEBS Letters, 527(1-3), 176-180. doi:10.1016/s0014-5793(02)03217-9Hanzawa, Y. (2000). ACAULIS5, an Arabidopsis gene required for stem elongation, encodes a spermine synthase. The EMBO Journal, 19(16), 4248-4256. doi:10.1093/emboj/19.16.4248Igarashi, K., & Kashiwagi, K. (2000). Polyamines: Mysterious Modulators of Cellular Functions. Biochemical and Biophysical Research Communications, 271(3), 559-564. doi:10.1006/bbrc.2000.2601Imai, A., Akiyama, T., Kato, T., Sato, S., Tabata, S., Yamamoto, K. T., & Takahashi, T. (2003). Spermine is not essential for survival of Arabidopsis. FEBS Letters, 556(1-3), 148-152. doi:10.1016/s0014-5793(03)01395-4Imai, A. (2006). The dwarf phenotype of the Arabidopsis acl5 mutant is suppressed by a mutation in an upstream ORF of a bHLH gene. Development, 133(18), 3575-3585. doi:10.1242/dev.02535Jakob, K., Goss, E. M., Araki, H., Van, T., Kreitman, M., & Bergelson, J. (2002). Pseudomonas viridiflavaandP. syringae—Natural Pathogens ofArabidopsis thaliana. Molecular Plant-Microbe Interactions, 15(12), 1195-1203. doi:10.1094/mpmi.2002.15.12.1195Kakehi, J. -i., Kuwashiro, Y., Niitsu, M., & Takahashi, T. (2008). Thermospermine is Required for Stem Elongation in Arabidopsis thaliana. Plant and Cell Physiology, 49(9), 1342-1349. doi:10.1093/pcp/pcn109Kamada-Nobusada, T., Hayashi, M., Fukazawa, M., Sakakibara, H., & Nishimura, M. (2008). A Putative Peroxisomal Polyamine Oxidase, AtPAO4, is Involved in Polyamine Catabolism in Arabidopsis thaliana. Plant and Cell Physiology, 49(9), 1272-1282. doi:10.1093/pcp/pcn114Knott, J. M., Römer, P., & Sumper, M. (2007). Putative spermine synthases fromThalassiosira pseudonanaandArabidopsis thalianasynthesize thermospermine rather than spermine. FEBS Letters, 581(16), 3081-3086. doi:10.1016/j.febslet.2007.05.074Maiale, S. J., Marina, M., Sánchez, D. H., Pieckenstain, F. L., & Ruiz, O. A. (2008). In vitro and in vivo inhibition of plant polyamine oxidase activity by polyamine analogues. Phytochemistry, 69(14), 2552-2558. doi:10.1016/j.phytochem.2008.07.003Marina, M., Maiale, S. J., Rossi, F. R., Romero, M. F., Rivas, E. I., Gárriz, A., … Pieckenstain, F. L. (2008). Apoplastic Polyamine Oxidation Plays Different Roles in Local Responses of Tobacco to Infection by the Necrotrophic Fungus Sclerotinia sclerotiorum and the Biotrophic Bacterium Pseudomonas viridiflava. Plant Physiology, 147(4), 2164-2178. doi:10.1104/pp.108.122614Marini, F., Betti, L., Scaramagli, S., Biondi, S., & Torrigiani, P. (2001). Polyamine metabolism is upregulated in response to tobacco mosaic virus in hypersensitive, but not in susceptible, tobacco. New Phytologist, 149(2), 301-309. doi:10.1046/j.1469-8137.2001.00017.xMinguet, E. G., Vera-Sirera, F., Marina, A., Carbonell, J., & Blazquez, M. A. (2008). Evolutionary Diversification in Polyamine Biosynthesis. Molecular Biology and Evolution, 25(10), 2119-2128. doi:10.1093/molbev/msn161Mitsuya, Y., Takahashi, Y., Berberich, T., Miyazaki, A., Matsumura, H., Takahashi, H., … Kusano, T. (2009). Spermine signaling plays a significant role in the defense response of Arabidopsis thaliana to cucumber mosaic virus. Journal of Plant Physiology, 166(6), 626-643. doi:10.1016/j.jplph.2008.08.006Mitsuya, Y., Takahashi, Y., Uehara, Y., Berberich, T., Miyazaki, A., Takahashi, H., & Kusano, T. (2007). Identification of a novel Cys2/His2-type zinc-finger protein as a component of a spermine-signaling pathway in tobacco. Journal of Plant Physiology, 164(6), 785-793. doi:10.1016/j.jplph.2006.05.011Møller, S. G., & McPherson, M. J. (1998). Developmental expression and biochemical analysis of the Arabidopsis atao1 gene encoding an H 2 O 2 ‐generating diamine oxidase. The Plant Journal, 13(6), 781-791. doi:10.1046/j.1365-313x.1998.00080.xMoschou, P. N., Sarris, P. F., Skandalis, N., Andriopoulou, A. H., Paschalidis, K. A., Panopoulos, N. J., & Roubelakis-Angelakis, K. A. (2009). Engineered Polyamine Catabolism Preinduces Tolerance of Tobacco to Bacteria and Oomycetes. Plant Physiology, 149(4), 1970-1981. doi:10.1104/pp.108.134932Muniz, L., Minguet, E. G., Singh, S. K., Pesquet, E., Vera-Sirera, F., Moreau-Courtois, C. L., … Tuominen, H. (2008). ACAULIS5 controls Arabidopsis xylem specification through the prevention of premature cell death. Development, 135(15), 2573-2582. doi:10.1242/dev.019349Murashige, T., & Skoog, F. (1962). A Revised Medium for Rapid Growth and Bio Assays with Tobacco Tissue Cultures. Physiologia Plantarum, 15(3), 473-497. doi:10.1111/j.1399-3054.1962.tb08052.xOber, D., Gibas, L., Witte, L., & Hartmann, T. (2003). Evidence for general occurrence of homospermidine in plants and its supposed origin as by-product of deoxyhypusine synthase. Phytochemistry, 62(3), 339-344. doi:10.1016/s0031-9422(02)00553-8Oshima, T. (2007). Unique polyamines produced by an extreme thermophile, Thermus thermophilus. Amino Acids, 33(2), 367-372. doi:10.1007/s00726-007-0526-zPanicot, M., Minguet, E. G., Ferrando, A., Alcázar, R., Blázquez, M. A., Carbonell, J., … Tiburcio, A. F. (2002). A Polyamine Metabolon Involving Aminopropyl Transferase Complexes in Arabidopsis. The Plant Cell, 14(10), 2539-2551. doi:10.1105/tpc.004077Pfaffl, M. W. (2002). Relative expression software tool (REST(C)) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Research, 30(9), 36e-36. doi:10.1093/nar/30.9.e36Rambla, J. L., Vera-Sirera, F., Blázquez, M. A., Carbonell, J., & Granell, A. (2010). Quantitation of biogenic tetraamines in Arabidopsis thaliana. Analytical Biochemistry, 397(2), 208-211. doi:10.1016/j.ab.2009.10.013Rea, G., Metoui, O., Infantino, A., Federico, R., & Angelini, R. (2002). Copper Amine Oxidase Expression in Defense Responses to Wounding and Ascochyta rabiei Invasion. Plant Physiology, 128(3), 865-875. doi:10.1104/pp.010646Sagor, G. H. M., Takahashi, H., Niitsu, M., Takahashi, Y., Berberich, T., & Kusano, T. (2012). Exogenous thermospermine has an activity to induce a subset of the defense genes and restrict cucumber mosaic virus multiplication in Arabidopsis thaliana. Plant Cell Reports, 31(7), 1227-1232. doi:10.1007/s00299-012-1243-yShah, N., Thomas, T., Shirahata, A., Sigal, L. H., & Thomas, T. J. (1999). Activation of Nuclear Factor κB by Polyamines in Breast Cancer Cells†. Biochemistry, 38(45), 14763-14774. doi:10.1021/bi991291vTakahashi, Y., Berberich, T., Miyazaki, A., Seo, S., Ohashi, Y., & Kusano, T. (2003). Spermine signalling in tobacco: activation of mitogen-activated protein kinases by spermine is mediated through mitochondrial dysfunction. The Plant Journal, 36(6), 820-829. doi:10.1046/j.1365-313x.2003.01923.xTakahashi, Y., Berberich, T., Yamashita, K., Uehara, Y., Miyazaki, A., & Kusano, T. (2004). Identification of Tobacco HIN1 and Two Closely Related Genes as Spermine-Responsive Genes and their Differential Expression During the Tobacco Mosaic Virus-Induced Hypersensitive Response and During Leaf- and Flower-Senescence. Plant Molecular Biology, 54(4), 613-622. doi:10.1023/b:plan.0000038276.95539.39Takahashi, Y., Cong, R., Sagor, G. H. M., Niitsu, M., Berberich, T., & Kusano, T. (2010). Characterization of five polyamine oxidase isoforms in Arabidopsis thaliana. Plant Cell Reports, 29(9), 955-965. doi:10.1007/s00299-010-0881-1Takahashi, Y., Uehara, Y., Berberich, T., Ito, A., Saitoh, H., Miyazaki, A., … Kusano, T. (2004). A subset of hypersensitive response marker genes, including HSR203J, is the downstream target of a spermine signal transduction pathway in tobacco. The Plant Journal, 40(4), 586-595. doi:10.1111/j.1365-313x.2004.02234.xTakano, A., Kakehi, J.-I., & Takahashi, T. (2012). Thermospermine is Not a Minor Polyamine in the Plant Kingdom. Plant and Cell Physiology, 53(4), 606-616. doi:10.1093/pcp/pcs019Thomas, T., Shah, N., Klinge, C., Faaland, C., Adihkarakunnathu, S., Gallo, M., & Thomas, T. (1999). Polyamine biosynthesis inhibitors alter protein-protein interactions involving estrogen receptor in MCF-7 breast cancer cells. Journal of Molecular Endocrinology, 131-139. doi:10.1677/jme.0.0220131Tiburcio, A. F., Altabella, T., Borrell, A., & Masgrau, C. (1997). Polyamine metabolism and its regulation. Physiologia Plantarum, 100(3), 664-674. doi:10.1111/j.1399-3054.1997.tb03073.xUehara, Y., Takahashi, Y., Berberich, T., Miyazaki, A., Takahashi, H., Matsui, K., … Kusano, T. (2005). Tobacco ZFT1, a Transcriptional Repressor with a Cys2/His2 Type Zinc Finger Motif that Functions in Spermine-Signaling Pathway. Plant Molecular Biology, 59(3), 435-448. doi:10.1007/s11103-005-0272-0URANO, K., YOSHIBA, Y., NANJO, T., IGARASHI, Y., SEKI, M., SEKIGUCHI, F., … SHINOZAKI, K. (2003). Characterization of Arabidopsis genes involved in biosynthesis of polyamines in abiotic stress responses and developmental stages. Plant, Cell and Environment, 26(11), 1917-1926. doi:10.1046/j.1365-3040.2003.01108.xVera-Sirera, F., Minguet, E. G., Singh, S. K., Ljung, K., Tuominen, H., Blázquez, M. A., & Carbonell, J. (2010). Role of polyamines in plant vascular development. Plant Physiology and Biochemistry, 48(7), 534-539. doi:10.1016/j.plaphy.2010.01.011Yamakawa, H., Kamada, H., Satoh, M., & Ohashi, Y. (1998). 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An open challenge to advance probabilistic forecasting for dengue epidemics

This is the final version. Available on open access from the National Academy of Sciences via the DOI in this recordData Availability: Data deposition: The data are available at https://github.com/cdcepi/dengue-forecasting-project-2015 (DOI: https://doi.org/10.5281/zenodo.3519270).A wide range of research has promised new tools for forecasting infectious disease dynamics, but little of that research is currently being applied in practice, because tools do not address key public health needs, do not produce probabilistic forecasts, have not been evaluated on external data, or do not provide sufficient forecast skill to be useful. We developed an open collaborative forecasting challenge to assess probabilistic forecasts for seasonal epidemics of dengue, a major global public health problem. Sixteen teams used a variety of methods and data to generate forecasts for 3 epidemiological targets (peak incidence, the week of the peak, and total incidence) over 8 dengue seasons in Iquitos, Peru and San Juan, Puerto Rico. Forecast skill was highly variable across teams and targets. While numerous forecasts showed high skill for midseason situational awareness, early season skill was low, and skill was generally lowest for high incidence seasons, those for which forecasts would be most valuable. A comparison of modeling approaches revealed that average forecast skill was lower for models including biologically meaningful data and mechanisms and that both multimodel and multiteam ensemble forecasts consistently outperformed individual model forecasts. Leveraging these insights, data, and the forecasting framework will be critical to improve forecast skill and the application of forecasts in real time for epidemic preparedness and response. Moreover, key components of this project-integration with public health needs, a common forecasting framework, shared and standardized data, and open participation-can help advance infectious disease forecasting beyond dengue

An open challenge to advance probabilistic forecasting for dengue epidemics.

A wide range of research has promised new tools for forecasting infectious disease dynamics, but little of that research is currently being applied in practice, because tools do not address key public health needs, do not produce probabilistic forecasts, have not been evaluated on external data, or do not provide sufficient forecast skill to be useful. We developed an open collaborative forecasting challenge to assess probabilistic forecasts for seasonal epidemics of dengue, a major global public health problem. Sixteen teams used a variety of methods and data to generate forecasts for 3 epidemiological targets (peak incidence, the week of the peak, and total incidence) over 8 dengue seasons in Iquitos, Peru and San Juan, Puerto Rico. Forecast skill was highly variable across teams and targets. While numerous forecasts showed high skill for midseason situational awareness, early season skill was low, and skill was generally lowest for high incidence seasons, those for which forecasts would be most valuable. A comparison of modeling approaches revealed that average forecast skill was lower for models including biologically meaningful data and mechanisms and that both multimodel and multiteam ensemble forecasts consistently outperformed individual model forecasts. Leveraging these insights, data, and the forecasting framework will be critical to improve forecast skill and the application of forecasts in real time for epidemic preparedness and response. Moreover, key components of this project-integration with public health needs, a common forecasting framework, shared and standardized data, and open participation-can help advance infectious disease forecasting beyond dengue