Spodoptera frugiperda (FAW), first invaded Africa in 2016 and has since become established in many areas across the continent where it poses a serious threat to food and nutrition security. We re-parameterized the existing CLIMEX model to assess the FAW global invasion threat, emphasizing the risk of transient and permanent population establishment in Africa under current and projected future climates, considering irrigation patterns. FAW can establish itself in almost all countries in eastern and central Africa and a large part of western Africa under the current climate. Climatic barriers, such as heat and dry stresses, may limit the spread of FAW to North and South Africa. Future projections suggest that FAW invasive range will retract from both northern and southern regions towards the equator. However, a large area in eastern and central Africa is projected to have an optimal climate for FAW persistence. These areas will serve as FAW ‘hotspots’ from where it may migrate to the north and south during favorable seasons and then pose an economic threat. Our projections can be used to identify countries at risk for permanent and transient FAW-population establishment and inform timely integrated pest management interventions under present and future climate in Africa.The fall armyworm,  Spodoptera frugiperda J.E. Smith (Lepidoptera: Noctuidae), is an insect pest native to the Americas that has invaded and spread throughout sub-Saharan Africa within the last four years1. Since its introduction, FAW has emerged as a serious threat to cereal crops' productivity, such as maize and sorghum, two of the major staple food crops of smallholder farmers, thus jeopardizing food security throughout Africa3. In order to contain the FAW spread, many African countries have distributed and applied synthetic pesticides. In 2017, Zimbabwe distributed nearly 102,000 L of pesticide valued at 1.97 million US dollars to farmers4. Although subsidized by governments, the use of synthetic pesticides as the sole control measure is unsustainable due to their high cost, risk of increased pesticide resistance, pest resurgence, and risk to human health and the environment5. Hence, there is a need to develop new, more sustainable approaches to FAW management. Alternative control measures are being considered including the use of biopesticides6, biological control9 , and agroecological practices such as intercropping with legumes, push–pull system, and diversifying the farm environment12. The choice and scaling of these alternative solutions depend on accurate characterization of the potential permanent and temporal distribution of FAW across the continent as well as how FAW and the agriculture production system will respond to a changing climate. Further, such information will refine our understanding of the pest’s potential migratory behavior in Africa, and is essential for developing long-term strategies to mitigate FAW infestations in hotspots (regions where FAW is endemic) as well as transient areas where FAW may move from hotspots to occupy a region for a period of favorable climate conditions.The fall armyworm (FAW), 14. The most affected crops in the Americas, the native range of FAW, are maize and sorghum13. FAW larvae have voracious appetites and cause severe damage to plants15. The female FAW moth lays eggs on the distal part of the maize leaf. Upon hatching the first and second instar larvae feed on that leaf, but eventually they enter the leaf whorl and feed on the unfurling leaves causing extensive defoliation. As plants mature, FAW larvae often start feeding on the ear. Finally, the larvae pupate in the soil16. Pupation lasts 8–30 days until the adults emerge17. Under ideal conditions, adult moths live up to 14 days, capable of migrating to distant new areas. In warm climate, FAW completes its entire life cycle in 3–4 weeks, but in cold climate, it takes considerably longer17. Unlike other lepidopteran pests, FAW cannot survive in areas with extended freezing temperatures. It lacks any diapause mechanisms and overwinters only in warm and moist areas14. Although its year-round distribution is restricted to tropical and subtropical regions, adult moths can migrate substantial distances into temperate regions during warm summers14 and establish transient populations which can jeopardize harvests in these regions. If left unchecked, FAW in Africa has the potential to cause an economic loss of around 13 billion US dollars per year through damage to maize, sorghum, rice, and sugarcane, alone15.FAW is a polyphagous pest with a wide host range, feeding on 353 plant species, including many food crops, forages and turfgrass1, from where it is believed to have spread to other African countries, including Kenya, Uganda, Rwanda, Ethiopia, and Tanzania. By April 2018, FAW had invaded and spread throughout sub-Saharan Africa and Sudan18. Recently, FAW has been reported in Egypt19. The presence of FAW in North Africa substantially increases the risk of FAW invasion of Europe through migration. Beyond Africa, FAW has also invaded several countries in Asia23 and Australia24. The rapid spread of FAW across the world may be due to trade and weak phytosanitary regulations, but also due to migratory behavior of the pest itself28.Outside of the native range in North and South America, FAW was first reported in Nigeria, São Tomé and Príncipe in early 201631. If global temperatures increase as projected, multivoltine migratory insects such as FAW could increase their number of generations per year32 and expand their infestation range to higher latitudes and elevations. As FAW is rapidly spreading across Africa and beyond, we urgently need information about its potential invasion and establishment threat in the present and possible future climates.One of the major determining factors affecting pest populations and their distribution is climate, which may either directly affect the physiology of the pest or indirectly impact parasitoids or predators, competitors, and food sources41. Using the CLIMEX model, Ramirez-Cabral et al.38 assessed the possible FAW distribution changes in its native ranges under climate change scenarios. This model did not distinguish between permanent and transient FAW populations. It projected optimal climatic suitability in northeastern states of the USA. Later, Du Plessis et al.35 reparameterized the CLIMEX model and identified the FAW potential invasion and establishment areas under the historical climate conditions broadly. However, the pest's potential distribution in Africa, the most affected continent, under future climate conditions was not projected. Yet, the knowledge of areas of habitat suitability for transient, migrant and permanent FAW populations under climate change scenarios, including irrigation pattern is necessary to enable policymakers at the national and regional levels to develop sustainable management strategies and to identify research priorities under the present and future climates in Africa and beyond. This study aims to fill this knowledge gap.Species distribution modeling is an effective method of predicting pest invasion risk and establishment potential in the region of focus. The correlative model MaxEnt and the process-based semi-mechanistic model CLIMEX are the two most popular models used to assess the risk of FAW invasion and establishment42, combined with eco-physiological tolerances and global occurrence records, to assess and predict the FAW invasion and establishment risk under both historical and future climate conditions, taking also the effect of irrigation into account. In addition, we performed the sensitivity analysis to test the effect of parameter changes on the modeling outcomes and identify the parameters significantly influencing the CLIMEX results for the distribution of FAW. By mapping where transient and permanent FAW populations might potentially become established, the results from this paper can support strategic decision-making.We used CLIMEX 4.017. The current study used CLIMEX (version 4)42, a semi-mechanistic niche modeling platform, to project FAW distribution in relation to climate. The model parameters that describe the species’ response to climate were overlaid onto FAW occurrence data and climate data to project the species’ potential global distribution. Briefly, the annual growth index (GI) was used to describe the potential for FAW population growth during favorable climatic conditions, while stress indices  and interaction stresses   was used to visualize the result obtained from the CLIMEX analysis and calculate the areas under various EI categories for the species.The ArcGIS software 10.8 , PestWatch (www.pestwatch.psu.edu), Butterflies and Moths of North America , and literature resources  global platform45.FAW occurrence observations (n = 304) within its native range were obtained from the Global Biodiversity Information Facility  or field scouting (with confirmed FAW larvae) data were collected. To make data visualization and manipulation easier, FAW occurrence records were spatially filtered to retain a single record in each 10-arc minute (~ 18 km) grid. This resulted in 2968 records  used for further analysis. The distribution records used in this study are shown in Fig. 1https://www.climond.org)46. This dataset consisted of long-term averages centered at the year 1975 for maximum and minimum temperatures, precipitation and relative humidity at 09:00 and 15:00 h. Future climatic suitability for FAW was projected using the 10-arc minutes gridded spatial resolution climate data for 2030, 2050, and 2080 retrieved from the CliMond (Version 2) in CLIMEX format46. The future climate projections used in this study were based on two global climate models (GCM), CSIRO-Mk3.0 GCM developed by CSIRO Atmospheric Research, Australia47 and MIROC-H GCM developed by Center for Climate Research, Japan. These were run with the A1B SRES  emission scenarios. The SRES A1B was chosen with the assumption that, in the future, the use of fossil intensive and non-fossil energy sources will be balanced. For an A1B emission scenario, the CSIRO-Mk3.0 and MIROCH-H GCMs predict a rise in temperature of 2.11 °C and 4.31 °C, respectively, by the end of twenty-first century48. Similarly, these two GCMs predict different rainfall patterns49.The current suitability of FAW was modeled with the CliMond historical dataset interpolated at 10-arc minute 50.The Intergovernmental Panel on Climate Change (IPCC) Fifth Assessment Report (AR5) released a new family of emission scenario called Representative Concentration Pathway (RCP), to replace the SRES family. This consists of four-climate change scenarios- RCP2.6, RCP4.5, RCP6 and RCP8.5. The closest similar RCP scenario to SRES A1B is RCP 6.0, which represents an intermediate emission scenariohttp://www.fao.org/aquastat/en/geospatial-information/global-maps-irrigated-areas/).The information on global irrigation areas was derived from the Food and Agriculture Organization of the United Nations (http://www.earthstat.org/) created by Monfreda et al.52.Geographic distribution of maize and sorghum, two major host crops of FAW, were obtained from the EarthStat database  by fitting the projected distributions to the occurrence records of FAW in its native range and the recently invaded range in East Africa. We initially adopted parameter values from a previously conducted CLIMEX study on FAW38. Then, parameter values were determined through an iterative process to fit the simulated CLIMEX results to the known distribution of FAW in the world in 2020. FAW occurrence records in North America include reports of transient or migrant populations , Asia, and Australia. The process of parameter fitting was repeated until the reliability and consistency of the model projections were established. The parameter values used in this study are listed in Table Using the “Compare Locations” modules in CLIMEX, the values of FAW parameters Table  were detons Fig. . Therefo35. These values are supported by multiple publications56. FAW reared at 25 °C constant temperature are less likely to emerge deformed55, and the adult moths have the highest adult longevity and fecundity53. A recent study by Du Plessis et al.17 identified 30 °C as the upper optimal temperature for FAW growth and development. The development rate of FAW increases linearly with increasing temperature from 18 to 30 °C, but declines when temperature increases above 30 °C17.The lower (DV1) and upper (DV2) optimal temperatures for FAW population growth were left unchanged at 25 °C and 30 °C, respectively38. FAW lacks any diapause mechanisms and overwinters only in warm and humid areas; hence, FAW cannot tolerate freezing temperature14. Wood et al.59 reported that a temperature above 10 °C is required for pupal eclosion. The pupae held at 10 °C live for 50–62 days but do not eclose59.The limiting low temperature (DV0) was also kept the same at 12 °C55.The limiting high temperature (DV3) was set at 36 °C because more than 50% of FAW reared at 35 °C or above exhibit physical deformity and die within 24 h of emergence38, with some assumptions. The lower soil moisture threshold (SM0) was kept the same at 0.15 to allow FAW invasion in semi-arid areas in Africa. Silvain & Ti-A-Hing60 reported higher FAW populations (both adult moths and larvae) during rainy seasons than in the dry seasons. At any time, the larval population is affected by the amount of rainfall that was experienced three weeks earlier60. Although a heavy downpour reduces adult emergence by trapping moths in their pupation tunnel61, FAW larvae can tolerate substantial waterlogging conditions35. Therefore, the upper soil moisture threshold (SM3) was set to 2. Reducing the value of SM3 from 2.5 to 2 had no effect on defining the potential range of FAW. The lower (SM1) and upper (SM2) limits for optimal growth were left unchanged at 0.8 and 1.5, respectively35. The upper optimal soil moisture (SM2) value allows the persistence of FAW in tropical areas that experience high rainfall, such as Central America. Here, the SM value 0 indicates no soil moisture; SM 0.5 indicates soil moisture is 50% of soil water holding capacity, and SM > 1 indicates a run-off situation.Not much is known about the relationship between soil moisture and the FAW lifecycle, so we adopted the species parameters from previous studiesCLIMEX mainly uses four stress indices  to determine the species' geographical distribution. Species population growth occurs between the temperature parameters DV0 and DV3, and moisture parameters SM0 and SM3 Table . Values The cold stress temperature threshold (TTCS) and cold stress accumulation rate (THCS) was decreased to 8 °C and − 0.005, respectively, to fit the FAW distribution in the Rio Grande valley Texas (overwintering site in North America), Mediterranean coast in North Africa and the Yunnan province (first FAW-invaded province) in China.35. Heat stress accumulation rate (THHS) was set to 0.0025 week−1 to allow the pest development in Nile River basins in Egypt and irrigated areas in Yemen and Pakistan.The heat stress temperature threshold (TTHS) for HS was kept the same at 39ºC to allow pest development in western African countries35. Soil moisture dry stress threshold (SMDS) was set to the same value as SM0, i.e., 0.15, and dry stress rate (HDS) was set to 0.005.Dry stress indices were the same as those of the existing model of Du Plessis et al. −1 to exclude extremely wet areas from being suitable. This change does not limit the FAW persistence in areas with known FAW distribution but reduces the modeled risk in extremely wet areas.Soil moisture threshold for wet stress (SMWS) was set at the same value as SM3 in our model. The wet stress accumulation rate (HWS) was increased to 0.01 week.62 estimated PDD for FAW at 346.2 degree-days with base temperature of 13.8 °C while Du Plessis et al.17 calculated PDD value of 390 degree-days with base temperature 12.57 °C. In the current study, the base temperature was set at 12 °C. To get the same number of generations per year, PDD value was increased to 400-degree days.In CLIMEX, the PDD parameter indicates the degree-day above the minimum base temperature (DV0) necessary for species to complete one generation. Hogg et al−1 as top-up irrigation throughout the year (Irrigation scenario I) to capture the risk posed by FAW in areas where cropping should be sustained by irrigation57. This top-up irrigation was added only when the weekly rainfall was less than 25 mm. Second, a composite FAW-risk map (Irrigation scenario II) was developed by combining the rainfed and irrigation scenario I results; EI from irrigation scenario I was mapped in areas under irrigation reported by Siebert et al.63 and EI for rainfed scenario was mapped elsewhere.FAW was reported in dry areas in North Africa, Pakistan and Yemen. These areas did not fall within climatically suitable areas projected under rainfed conditions. These dry area records might reflect FAW populations able to persist only when irrigation is applied to sustain the crop. To simulate the effect of irrigation in FAW distribution, two irrigation scenarios were taken into account. First, the CLIMEX model for FAW was run using 2.5 mm dayFAW occurrence records were overlaid on the projected layer surfaces to evaluate the model performance. EI values of the pixel where each FAW occurrence records lie were extracted from the projected raster layers. A histogram and a normal distribution curve were fitted on the projected EI values of each dataset under the current and projected future climates. Descriptive statistics were also generated from the extracted EI values to recognize the projected models that captured the presence records better. This analysis was used to measure and confirm the ability of the developed model to predict the FAW habitat areas successfully.CLIMEX model is a robust tool to assess the risk of pest invasion and establishment, but it includes several sources of uncertainty that need to be communicated to risk assessors and decision makers. The CLIMEX Version 4 has the parameter sensitivity and model uncertainty analysis tool available to evaluate the model. The sensitivity analysis identifies the degree to which each species parameter affects the projected areas of climatic suitability, whereas uncertainty analysis reflects the ability of the model to accurately predict the climatic suitability for a species. For the parameter sensitivity and uncertainty analysis, the default model parameters were run using the historic climate (CM10 1975H V1.2) under the rainfed scenario. Both parameter sensitivity and model uncertainty analysis were run for the entire world..43  Fig. a. This aThe model projected a notable difference in the potential distribution of FAW between irrigation scenario-II and the rainfed scenario, especially in North Africa, the Middle East and Australia Fig. c,d. ManyPotential FAW distribution in the Americas, its native range, is consistent with its known geographical range. The model confirmed that FAW over-wintering locations in the Americas are climatically suitable (with EI > 30) for the persistence of the FAW populations Fig. a,c under63  for FAW population persistence under rainfed conditions Fig. a. A few Although Europe has an unsuitable climate for FAW year-round establishment Fig. a,c, a fe23. There is a considerable chance that FAW will extend beyond its current range in Asia. Based on historical climate data, South East Asian and South Asian countries have optimal climate conditions for FAW population establishment  than projections under the MIROC-H model (3.7%). While all the currently suitable countries  lose climatic suitability, the reduction is significant in Eastern and West African countries  of the land area in Africa has suitable to optimal climate for both maize and FAW  and limiting low moisture (SM0), with 1 and 0.8% impact, respectively  than colder areas  Fig. .Figure 6The high proportion (0.98) of the occurrence records in the validation area falls within the area of projected suitable to optimal categories for FAW establishment  is currently suitable for FAW population establishment. In the future, areas of climatic suitability for FAW establishment are expected to gradually decrease over time mainly due to heat and dry stress. However, FAW may still survive and become established in significant areas in Africa  by the end of 2080. If certain conditions are met, these established persistent populations, could, in turn, serve as a source of seasonal invasions and migrate into less favorable climatic regions. Therefore, it is likely that FAW will establish permanent populations and cause substantial damage and economic losses to certain crops production every year in Africa unless FAW populations can be effectively managed.24. While FAW has not yet invaded Europe, our model identifies several pockets in Europe that are climatically suitable for FAW invasion, suggesting that FAW has not reached its full potential range and its range is still expanding.Our CLIMEX model projects that, under the current climate, there is an increased risk of global FAW invasion and establishment. Outside of its native range in North and South America, the current climatic conditions in many parts of Africa, South and South-East Asia, southeastern parts of China, the north coast of Australia, and a few pockets in Europe are favorable for FAW invasion and establishment. Until 2016, FAW was confined to areas with a subtropical wet and dry climate in the Americas. After invading Africa in 2016, FAW has spread rapidly throughout vast regions of Africa that have a climate similar to its native range. By 2020, FAW has further spread to the Middle East, South Asia, South East Asia, and Australia14. However, if global temperature increases as projected, some areas currently classified as climatically unsuitable may become suitable for FAW invasion and establishment in the future. Expansion towards the north has already been reported for several invasive insect species under climate change65.The model suggests that extreme temperatures, cold winters and hot summers, as well as limited soil moisture, are the most critical factors constraining the survival of FAW. Currently, cold stress limits FAW’s potential to permanently establish in northern North America, southern parts of South America, the northern part of Asia, and Europe. Presently, the permanent establishment of FAW in these regions is not possible because FAW does not diapauseSimilarly, dry stress limits FAW’s establishment in North African countries, the Middle East, and a large part of Australia. In the future, these areas are projected to experience a reduction in total annual rainfall and would not be expected to support FAW populations. However, irrigated crop fields in these areas could support seasonal FAW populations during warm winters.28. The projected FAW overwintering areas in the present study are consistent with those in previous studies35. Further, the projected climatic suitability for FAW also closely matches the projected distribution of maize, its preferred host under current and future climates , which further enhances the probability of FAW to invade and even establish persistent populations in the areas projected by our model.In Africa, central, eastern, and western African countries with sub-tropical wet and dry climates offer optimal conditions for FAW population establishment and are under high risk. These areas could serve as overwintering areas and sources of seasonal invasions to other parts of Africa, in the same way, that southern Florida, Texas, and Mexico serve in North America55. However, microclimates near maize growing areas in the Nile River basins and irrigated zones could provide channels of suitable habitat66 and support the establishment of FAW permanent populations. In addition, a small portion of the Mediterranean coast in North Africa is suitable for FAW persistence but has not yet been invaded. The arrival of FAW in those areas by its own means is unlikely because FAW has to migrate across the Sahara and face these harsh desert conditions. Yet, since FAW has already invaded the humid irrigated zones of Aswan Governorate in South Egypt19, we should not rule out the possibility of seasonal invasion of the north through natural migration. Migrant FAW moths could use maize fields along the Nile River basins as ‘stepping-stones’ before finding a suitable location along the North African coast. The flight biology of FAW in North America suggests that it can complete such a long-distance migration28. The establishment of significant populations along the North African coast and the Nile River basin will increase the threat to North Africa and Europe through seasonal migration67. The risk posed by migrant FAW populations north of the Sahara should not be ignored, since a similar scenario occurs in North America where the climate also does not support FAW establishment, yet crops are still devastated by seasonal invasions of FAW28.In Africa, the northern part of FAW's current distribution is currently limited by extreme heat and dry stress. The permanent establishment of FAW in this region is unlikely, as FAW cannot tolerate extreme heat27. Rwomushana et al.4 reported several interceptions of FAW in consignments from Africa. Therefore, to prevent travel-assisted introduction of the FAW in these places, close monitoring of the FAW invasion and activity in North Africa as well as adopting strict phytosanitary measures are and will continue to be necessary. In addition, pest species, including FAW, can move long distances aided by meteorological phenomena such as storm fronts68. While the present study did not consider it, an analysis of synoptic and mesoscale wind and storm patterns may be useful, especially since these patterns are projected to change over time70.Furthermore, several pest species have crossed unfavorable geographic barriers by ‘piggybacking’ on human transit routes. Therefore, intra-continental travel and trade increase the risk of FAW introduction to the North African coast and Europe38. These range contraction projections are similar to those made for its preferred host, Z. mays43  would expand the projected range of distributionThe large area overlap between the projected geographical distributions of FAW and its major host maize also indicates the substantial ecological and economical risk posed by this species. Under current climate, 33% of the total land area in Africa has suitable to optimal climate for FAW persistence and maize cultivation. Since this species feeds on over 350 plant species, the area overlap between FAW and it’s host crops could be greater than 33%. This FAW-maize overlap area is expected to decrease over the next 50–60 years, mainly due to loss of climatic suitability for maize cultivation. FAW could still persist in areas outside the FAW-maize overlap areas if other host crops are present. Our analysis only shows the potential area overlap between FAW and host crop maize, assuming that maize is grown in all the areas climatically suitable for maize cultivation. Therefore, actual areas of maize and other host crops cultivation should be considered in the future studies.74. This study focuses on abiotic factors for temperature-dependent life history events to map climatically suitable areas for FAW because abiotic factors such as climatic variables are considered prime factors affecting species distribution at the continental and global scales75. Therefore, we examined the influence of climate change only on FAW's range of invasion and establishment. At these large scales, the results from the bioclimatic models alone are sufficient to suggest which regions are at high risk for pest invasion and establishment under future climates75.The geographic distribution of FAW is determined by biotic interactions, abiotic factors, and its active or passive movement74. FAW requires a suitable place to live and complete its lifecycle over several generations. FAW also requires an adequate supply of host plants in synchrony with its lifecycle, without which it cannot persist in that environment. Furthermore, FAW's interactions with other species, such as natural enemies and competitors in a particular region, affect species distribution. Unfortunately, at this point combining all these factors in the bioclimatic model would increase the complexity and is not currently possible75.Biotic factors should be incorporated in the bioclimatic model results to provide a more refined understanding of the species distribution under changing climates76. In this case, modeling habitat suitability for the pest under current and future climatic conditions provides robust tools and recommendations across multiple stakeholder levels and geographical scales. The current study will guide farmers, extension agents, researchers, policymakers, and public and private sectors to develop risk assessment protocols and climate-smart pest management strategies to prevent or reduce the economic loss due to FAW.The projected distribution of the FAW in Africa has indicated that FAW will severely impact agriculture over the next several decades. Insect pest management will be more challenging in a changing climate since climatic factors affect the timing of pest infestation, host preference, the efficacy of chemical and biological measures of control, and their utilization within integrated pest management strategiesThe FAW poses a considerable threat to farmers worldwide. Projection of the pest distribution and its potential to establish in a targeted geographical location is crucial in enhancing preparedness, particularly selecting appropriate pest management control. Since FAW performance and survival is limited by temperature and humidity, we constructed a model that predicts areas suitable for FAW occurrence under current and future climate conditions. Here we considered two fundamentally different scenarios: areas where FAW persist year-round with multiple generations per year, and areas where FAW invade seasonally and each invasion starts from persistent populations. Under current climatic conditions, FAW has not yet reached all areas where it could potentially establish year-round populations, and therefore also not all areas that it can occasionally or seasonally invade. Under projected global temperature increases, the optimal areas for FAW persistence will shrink and, with them, the areas of seasonal invasions. Our model does not consider the multitudes of biotic interactions. However, it appears that at least the distribution of maize, FAW’s preferred host plant, follows a similar pattern, which means that under future climatic conditions successful cultivation of maize will require successful management of FAW populations.Supplementary Information 1.Supplementary Information 2.


Solanum tuberosum (Stub) and S. bulbocastanum (Sblb) for breeding purposes. In 2007, 23 somatic hybrids were produced using an electrofusion of mesophyll protoplasts of diploid (2n = 2x = 24) potato line StubDH165 and S. bulbocastanum PI24351 (Sblb66). Phenotype of somatic hybrids in field conditions were evaluated, together with constitution and stability of 30 nuclear (ncSSR) and 27 cytoplasmic (cpSSR) microsatellite markers and content of main glycoalkaloids. All somatic hybrids had very high field resistance against late blight, but the plants were infertile: the viability of pollen grains insignificantly varied between 0.58 and 8.97%. A significant somaclonal variation was observed in terms of the morphology of plants, the date of emergence, the quantity of harvested tubers, the content of glycoalkaloids in foliage, and nuclear microsatellite markers (ncSSR). The analysis of ncSSR identified five distinct genotypes of hybrids partly associated with phenotype variations. The process of somatic hybridization with regeneration of shoots was identified as the most likely source of somaclonal variation because the ncSSR genotypes of hybrids, which were maintained in vitro, remained stable for more than 10 years. The infertile somatic hybrids have no practical breeding potential, but they are considered very suitable for advanced studies of the differential expression of genes in the pathways linked to dormancy of tubers and synthesis of glycoalkaloids.Somatic hybridization has been frequently used to overcome sexual incompatibility between potato and its secondary germplasm. The primary objective of this study was to produce and evaluate somatic hybrids of  Solanum tuberosum ssp. tuberosum L.), the third most important food crop worldwide, following rice and wheat1, belongs to the genus Solanum section Petota., that includes four cultivated species2 and around 100 wild tuber-bearing species3. The latest complex analyses of DNA sorted the species into four clades4, which have helped in understanding phylogenetic, geographical, and genetic relationships potentially influencing the utilization of germplasm in breeding. In particular, the wild species have been frequently used in potato breeding programs, as they are genetic resources for many highly valued resistance genes. Interspecific hybridization, used for introgression of the genes to the potato germplasm, is frequently disturbed or prevented by different prezygotic and postzygotic barriers5, which can be overcome through somatic hybridization8. In the last decade, S. bulbocastanum Dun. , which is sexually incompatible with cultivated potato9 and has a very high resistance to late blight caused by Phytophthora infestans (Mont.) de Bary10, has been frequently used for somatic hybridization. In this regard, the genomes of potato and S. bulbocastanum, respectively reported previously as A and B genomes12, have been studied. Significant structural differences in Stub + Sblb somatic hybrids were reported7 and documented by typical meiotic anomalies, such as loops in paired “A and B” chromosomes at pachytene, dicentric chromatid bridges at M-I anaphase, various meiotic spindle orientations and various numbers of resulting meiotic cells. Subsequently, the 2S and 8S chromosomal regions in the B genome distinct from that found in the A genome were described using a DArT (Diversity Arrays Technology) marker-based linkage map9.Potato  markers detecting abundant polymorphisms of microsatellitesS. tuberosum ssp. tuberosum (Stub) and S. bulbocastanum (Sblb) PI24351 resistant to late blight and evaluate their potential for breeding. Being based on the hypothesis that phenotype differences should associate with changes of microsatellite loci, the article informs about findings of our long-term study of somaclonal variation observed within the somatic hybrids.The primary objective of this research was to produce somatic hybrids of 2n = 2x = 24 StubDH165  was induced by inter-specific hybridization of 2n = 4x = 48 potato variety ‘Apta’ with 2n = 2x = 24 inducer of diploidy S. phureja Juz. & Bukasov (Sphu) 85/16. The parental genotype Sblb66  was chosen from population PI243510 as a donor of the Rpi-blb1 gene.The plants obtained and analyzed in the presented study are introduced in Table .25 and fused by Electro Cell Manipulator BTX 2001  in a glass microslide with rectangular electrodes with a 3.2 mm gap. The protoplasts were arranged in chains using an impulse of AC , fused by an impulse of DC , and recovered post-fusion . Media for electrofusion, cultivation, and regeneration of calli and shoots and the remaining cultivation conditions were as reported by Cheng and Saunders26. After rooting, the maintained plants were sub-cultivated monthly on original Murashige and Skoog27 medium containing vitamins , supplemented with 6.5% agar and 30 g of sucrose. A representative selection of the hybrids is available in the Potato Gene Bank in vitro of the Potato Research Institute Havlíčkův Brod Ltd.The protoplasts were isolated based on methods by Carlberg et al2 of leaf tissue was cut with a razor blade in 0.5 mL of Otto I buffer (0.1 M citric acid monohydrate and 0.5% (v/v) Tween 20), and the suspension was filtered through a 42 μm nylon mesh. The filtrate was incubated for 10 min at room temperature and 1 mL of Otto II buffer (0.4 M Na2HPO4.12H2O), 4,6-diamidino-2-phenylindole (4 μg mL−1) and β-mercaptoethanol (2 μL mL−1) were added. The fluorescence intensity was measured using a Partec PAS analyzer (Germany).The ploidy of regenerated plants was detected using flow cytometry. Approximately 1 cm28, pollen viability as in Wang et al.29, and resistance against late blight of somatic hybrids was evaluated in field and laboratory30 experiments. Also, the content of glycoalkaloids (α-chaconine and α-solanine) in mg kg−1 of dry matter was quantified in both foliage and tubers. Detailed conditions of the field, laboratory and biochemical evaluations are presented in Supplementary file The morphological characteristics of foliage and tubers were evaluated according to Vidner et al.31. The final set of markers comprised 23 plastid SSR markers by Bryan et al.32, 4 mitochondrial SSR markers by Hosaka and Sanetomo33, 24 ncSSR from the potato genetic identity kit by Ghislain et al.21 and 6 ncSSR markers related to the tuber starch content22. The obtained PCR products were analyzed by capillary electrophoresis ABI PRISM 310 . Length polymorphisms were detected using size standard GeneScan LIZ600  by GeneMapper v 4.1 software . In addition to cpSSR markers, the type of cytoplasm was identified for all genotypes using the original methodology by Hosaka and Sanetomo34. All methodological details of DNA analyses and needed references are presented in Supplementary Tables DNA of all samples were isolated twice, in 2009 and 2020, using the DNeasy Plant Mini Kit  and gradually analyzed using 57 SSR markers; 27 cytoplasmic microsatellites (cpSSR) and 30 nuclear microsatellites (ncSSR) were divided into 13 multiplexes. The positioning of ncSSR loci is presented in Supplementary Figure Quantitative data collected during field evaluations and glycoalkaloids content were statistically evaluated using ANOVA and Tukey’s post-hoc analysis. The variability of morphological descriptors was analyzed by Kruskal–Wallis ANOVA. Statistical evaluations were performed on the standard level of significance (α = 0.05) using Dell Statistica Software .35. The dendrogram was then calculated using the method of neighbor joining with 30,000 bootstraps and designed within the same software.Allelic data from SSR analysis were processed and sorted in MS Excel 2016. The matrix of dissimilarities was calculated directly from allelic data based on simple matching using the Darwin 6 softwareThe study complied with relevant institutional, national, and international guidelines and legislation.The methods of isolation, fusion and regeneration of protoplasts resulted in 3042 pieces of microcallus (approximately 0.5 mm) which were individually plated. Subsequently, 595 calli were grown, but only three of them  regenerated into 40 independent shoots. Once the shoots were rooted, 23 green, well-growing plants were obtained. Except REG52E, all adapted well ex vitro. All plants were identified as tetraploid (2n = 4x = 48) somatic hybrids using flow cytometry and subsequent analyses of DNA. Another 17 shoots were obtained, 11 being green and 6 exhibiting albinism, which gradually died being unable to form roots.Stub/Sblb-specific alleles). In somatic hybrids generally, the appearance of a new 161 bp allele in locus STI028 on chromosome 11 was detected as a result of deletion in Sblb specific 164 bp allele. Also, a loss of 3–5 parental alleles to benefit the null alleles was detected in somatic hybrids. Comparing the ncSSR allelic patterns in somatic hybrids, 5 different ncSSR genotypes (A–E) were identified  as with Sblb. Flowering started in mid-June and persisted to the first frosts in October.Compared to parental species, field tests showed that all somatic hybrids had a more intensive growth and sturdy habitus with a generally intermediate appearance in morphology, particularly regarding characteristics of stems, leaves, and flowers Fig. . For exaSblb66, since StubDH165 failed to grow effectively in field conditions and did not provide reliable data.Somaclonal variation was observed in the phenological development of plants, when the individuals with the ncSSR genotype A, tended to be earlier in terms of the date of emergence and flowering start . Moreover, the tubers were not suitable for consumption based on the subsequent strong bitter taste. The genotype REG35E (12D) showed the significantly highest contents of glycoalkaloids in foliage, which was associated with the loss of Stub 126 bp allele of the marker STI0014 positioned on the chromosome 09.The values of α-chaconine and α-solanine in tubers and foliage of Sblb66. In the field experiments, very high resistance was detected without any symptoms of disease in either leaves and stems. In laboratory experiments, however, the majority of somatic hybrids showed medium to high resistance accompanied by the saprophytic growth and occasionally very weak sporulation of the pathogen in necrotic tissues of leaves. This behavior of pathogen was linked to the environment of the Petri dish. In contrast, the susceptible progenitor StubDH165 allowed the pathogen to produce spores in the whole leaf area and then destructions of leaf tissues were observed.All somatic hybrids showed a high late blight resistance in both laboratory and field experiments, although it was not as strong as in Stub varieties did not result in any berry or seed, and all concerned flowers of somatic hybrids fell within the three days following pollination. The viability of the pollen tested using the methodology by Wang et al.29 varied insignificantly within somatic hybrids in a range from 0.58 to 8.97%. In contrast, the standards (variety ‘Apta’ and Sblb66) showed 74% viability which differed significantly from the somatic hybrids (P < 0.01). These genotypes were able to produce vigorous seeds from open pollination.All the somatic hybrids were infertile, even though they produced a considerable amount of pollen grains and visible eggs in ovaries for the entirety of the growing season. Self-pollinations and artificial crossing between somatic hybrids, backcrosses, or crosses with StubDH165 was successfully used in somatic hybridization with Sblb66. The infertility of somatic hybrids is the most challenging problem and is explainable by considering the evolutionary distinctness of Stub and Sblb genomes4. This distinctness was previously experimentally shown9 and is documented herein by ncSSR analysis. The SSR panel designed for the Stub genome was able to detect significant differences in studied loci, especially in telomeric parts. Another factor is the general chromosomal instability of dihaploids36. Since the dihaploid potato used for fusion was induced by S. phureja, a risk of occurrence of frequent Stub/Sphu DNA rearrangements37 affecting meiotic division can be expected. It is important to consider whether fertility could be improved using dihaploid potato derived from microspores. This is difficult to assess, because the latest published experiments with somatic hybrids7 also worked with Sphu-induced potato dihaploids and the experience with fertility was similar. Successful and fertile somatic hybrids were most likely obtained by the fusion of 4n Stub and 2n Sblb only38, when the predominating genome A likely enabled the hybrids to be fertile. The results obtained have supported the general recognition that with tighter ratios of incompatible genomes in somatic hybrids, problems arise with meiotic division as well as with the development of functional gametes.Yielding 23 vital somatic hybrids, the 15. Naturally, an extension of variability in relatively uniform progenies can be detected in qualitative, quantitative, and molecular characteristics of tightly related somatic hybrids. The initiation of substantial genetic changes was related to the first three months, up until the regeneration of shoots from the callus. During long-term maintenance of plants from meristematic tissues (approximately 100 subcultures), the genotypes of somatic hybrids did not change. In the regeneration period, cells were exposed to the cytokinin zeatin, which can weaken the activity of enzymes responsible for repairing DNA16. This is supported by ncSSR analysis, since microsatellites alone are very susceptible to disfunctions of repairing enzymes23, in potato represented by MSH2 gene. Mutations of the gene induces microsatellite instability39 but we probably can exclude this in relation to experience from long time maintenance. Detected genetic variations further agree with results of previous SSR40 and DArT24 analyses. The ncSSR panel presented here is informative in terms of reduced repair activity, but not as informative when regarding the identification of loci associated with phenotype differentiation. The phenotype variations supported by frequent molecular changes in DNA were the most substantial result of the study, with some complexes of mutations associated with specific phenotypes . Although genetic variations occurred in nuclear microsatellites, they were not observed in the cytoplasmic DNA. Plasmatic DNA was completely inherited from the Stub progenitor, which agrees with previous studies42 where the preferential inheritance of the Stub plastome was observed in several interspecific somatic hybrids, however Chandel et al.43 found significant differences just in cytoplasmic DNA. Considering the epigenetic background, molecular results indicated that the somaclonal variation in the progeny is well explainable by structural changes in DNA. However, epigenetic changes cannot be excluded completely because they have not been objectively studied here. Recent studies of somaclonal variation confirmed genetic changes in potato protoplast cultures44 and epigenetic (methylation) changes45 in clonally propagated potato. In contrast, study on epigenetics in coffee excluded these influences, and all somaclonal variation was explained by structural changes in DNA46. Another phenomenon to consider in our case is a somatic incompatibility47, which seems to be analogous to sexual incompatibility and incongruity. It is linked to chromosomal incompatibility, which reflects in many genetic rearrangements , and also to genome-plasmon interactions. Both usually prevent regeneration of plants because lead to discoordination of life cell cycle and metabolism. To synchronize all aspects to reach successful somatic hybrids is challenging, which is here documented by low ratio of regenerated plants to the current number of initiated microcalli.Somaclonal variation is a phenomenon regarding breeding potential in potato and has been widely studied50. Additionally, the flower traits reported here, including shape arrangement and color of the flower corolla, are assumed to be controlled by several interacting genes, some of which have an additional pleiotropic effect on the color of stems, petioles, peels, or pulp of tubers. The adaptation of tubers induction on the short day and very late maturity agreed with the behavior of S. bulbocastanum51. The resistance against P. infestans was in accordance with previous observations of interspecific hybrids of S. tuberosum and S. pinnatisectum53 and interspecific hybrids of S. tuberosum and S. cardiophyllum43. Even though the expression of resistance was not as strong as in Sblb66, this study presents a high efficiency of the Rpi-Blb-1 gene10 in the agri-environmental conditions of the Czech Republic.The intermediate appearance of somatic hybrids in the majority of aboveground morphological traits and more intensive growth agreed with previous studiesStub varieties. The low concentration of alkaloids found in Sblb was consistent with previous studies54. However, this contradicted the results by Savarese et al.55, who did not detect chaconine and solanine in S. bulbocastanum. Newly detected unknown glycoalkaloids in the hybrids presented here likely belong to the Sblb family of solanidine and solanidine-like alkaloids56. Their identification requires other analyses; a study of their ecological effects in interaction with the Colorado potato beetle could have potential. We observed that ncSSR genotype D significantly differed in the content of glycoalkaloids and that the characteristics associated with the loss of Stub allele 126 bp of the marker STI0014 was positioned on chromosome 09. This can indicate a hypothetical presence of some regulatory factors influencing the expression of glycoalkaloids on chromosome 09, which were neutralized by linked rearrangements near locus STI0014. However, an array of 10 genes that participate in glycoalkaloids biosynthesis were positioned57; six of them create a cluster on chromosome 07, and two are present in a duplicated region in chromosome 12. No other pathways linked to the glycoalkaloid biosynthesis have been reported.The total content of glycoalkaloids in somatic hybrids was more comparable to 15. The material presented here can be used in comparative studies of differences in transcriptomes and the expressional value of genes related to the synthesis of glycoalkaloids58 and dormancy, which can be influenced by the differential activity of specific cyclins with cyclin-dependent kinases59. Similarly, the activity of genes responsible for DNA repair, likely associated with homoeologous recombination23 and mismatch repair39, should be studied.Somaclonal variability is suitable for experimental research on genetic control of metabolic pathwaysThe experimental conditions of protoplasts regeneration into plants were suitable for both production of somatic hybrids and a broadening of their genetic diversity. Detection of five different ncSSR genotype groups in hybrids proved a range of genetic changes which were responsible especially for significant differences in the total content of glycoalkaloids in foliage and in the date of emergence. Other less significant changes were observed in habitus and production characteristics of the plants. Although the somatic hybrids were highly resistant against late blight, they are out of any breeding potential being infertile. Instead, they will be used as “isogenic lines” for comparative analyses of transcriptomes in metabolic pathways linked to dormancy of tubers and biosynthesis of glycoalkaloids.Supplementary Figure 1.Supplementary Figure 2.Supplementary Tables 1-4.Supplementary Tables 5-6.Supplementary Table 7.Supplementary Information 8.


MYC, FXN) and ferroptosis genes  are significantly altered with pancreatic tumor grades, which may contribute to differential expression of ferritin related to pancreatic cancer prognosis.Pancreatic cancer is one of the most lethal diseases which lack an early diagnostic marker. We investigated whether serum ferritin (SF) reflects risk for pancreatic cancer and potential genes that may contribute ferritin and pancreatic cancer risks. We performed a meta-analysis of relevant studies on SF and pancreatic cancer risk by searching articles in PUBMED and EMBASE published up to 1 March 2020. We also collected serum samples from Taipei Medical University Joint Biobank and compared SF levels in 34 healthy controls and 34 pancreatic cancer patients. An Oncomine database was applied as a platform to explore a series of genes that exhibited strong associations between ferritin and pancreatic cancer. Herein, we show that high levels of SF can indicate risk of pancreatic cancer, suggesting SF as the new tumor marker that may be used to help pancreatic cancer diagnosis. We also found that expressions of iron homeostasis genes ( The age-standardized mortality rate for pancreatic cancer in Taiwan is 5.6 per 100,000 people, and it was ranked the seventh leading cause of mortality in 20183. It is reported that approximately 80% of pancreatic cancer patients present an unresectable tumor with distant metastasis at the time of diagnosis, due in part to the absence of an early diagnostic test4. As a result, the 5-year survival rate of pancreatic cancer was 9% in 2020, in which the pattern of cancer deaths closely paralleled that of the incidence5. Considering the poor prognoses and limited therapeutic options, there is an urgent need to develop potential biomarkers for the early diagnosis of pancreatic cancer.Pancreatic cancer is the fourth leading cause of cancer-related deaths in the US, and the number of deaths also continues to increase in Taiwan9. Ferritin is the primary iron storage protein, and its serum level reflects iron stores in the body. It was reported that 1 ng/mL serum ferritin (SF) reflects approximately 8 mg of stored iron12. As high levels of iron are potentially toxic, several epidemiological studies also found associations between high levels of SF and increased risks for various cancers, suggesting that SF could be used as an iron index and also a tumor marker16. Degradation of ferritin, on the other hand, can elevate the level of cellular labile iron, which increases ROS levels and ultimately induces an iron-dependent form of non-apoptotic cell death, called ferroptosis17. Ferritin, thus, is an important mediator that regulates cellular iron metabolism and carcinogenesis, which may ultimately provide new insights into cancer research and therapeutic strategies.Iron is an essential element for most forms of life due to its roles in synthesizing oxygen transport proteins such as hemoglobin, myoglobin, and other iron-containing proteins. It is widely acknowledged that an excess accumulation of iron can be harmful by increasing production of reactive oxygen species (ROS), which ultimately cause DNA damage and genetic alterations, leading to tumor growthCorrespondingly, SF was suggested to be potential biomarker for diagnosing several types of cancer or monitoring cancer progression. Recent studies also suggested a plausible link between SF and pancreatic cancer, but those findings are not yet definitive. The present study was designed to explore if SF is associated with pancreatic cancer. We specifically assessed associations between SF and pancreatic cancer risk in Taiwan, by analyzing ferritin assays in 34 Taiwanese patients with histologically proven pancreatic carcinoma and in 34 healthy Taiwanese controls. We also performed a pooled analysis of six studies for a comprehensive quantitative assessment of the association of SF levels with pancreatic cancer risks. Lastly, an Oncomine database analysis of patient-derived gene expressions was used to assess potential genes that might mediate the association between ferritin and pancreatic cancer.Combined together, in response to pancreatic cancer is being considered one of the most lethal malignancies with an absence of an early diagnostic test, our study investigated the significance of SF as a prognostic marker for pancreatic cancer and offers new possibilities for pancreatic cancer research to improve overall prognoses. SF levels could serve as a potential marker to predict individuals at increased risk during pancreatic cancer screening, along with being a novel therapeutic strategy for pancreatic cancer.To summarize previous studies investigating the association between SF and pancreatic cancer risks, we conducted a meta-analysis of individual datasets. In total, 144 articles were searched from PUBMED and EMBASE, among all articles published up to 1 March 2020. After screening the title and abstract, 51 articles were retrieved and reviewed, of which five articles met the inclusion criteria. An additional article was included from a hand search  with extreme heterogeneity (I2 = 90.4%) and publication bias  and patients  were the possible sources of heterogeneity (Supplementary Table p for the Z test < 0.0001), and no heterogeneity was found (I2 = 0.0%). Indeed, only the study from Fabris et al.22 reported the stage of pancreatic cancer patients, but no subgroup analysis of SF and cancer stage was performed (Table p = 0.112), the results of which indicated that no publication bias existed , and the same pattern existed when we sub-grouped subjects by gender  but no significant difference was found in SF level of patients at stage III–IV  PDAC patients aged 41–82 years and 34 (50%) healthy controls aged 57–74 years, to investigate the relationship between SF and pancreatic cancer RNA expressions of FTH1 and FTL in pancreatic cancer patients using Oncomine datasets. We analyzed a dataset from Collisson et al.29 which included 27 pancreatic cancer tissues of patients with histological grades G1, G2, and G3. As shown in Fig. FTH1 and FTL with higher-grade tumors, but they did not reach statistical significance. Additionally, we analyzed a dataset from Ishikawa et al.30 which included 49 sample tissues with 25 normal and pancreatic cancer patients at stages 0 (n = 3), I (n = 3), III (n = 2), IVA (n = 13), and IVB (n = 3). mRNA expressions of neither FTH1 nor FTL were significantly associated with clinical stages of pancreatic cancer  participating in ferroxidase activity and being devoted to rapid uptake and release, while ferritin light polypeptide (FTL) functions in the long-term storage of iron32. A gene expression analysis, analyzing 10 normal tissues and 51 pancreatic cancer tissues from TCGA datasets, was performed to investigate whether iron homeostasis genes contributed to the association between ferritin and pancreatic cancer risks. We first used a heatmap analysis to visualize key iron metabolism genes, the expressions of which correlated with the histological grades (control and G1–G3) of pancreatic cancer  and high expression of MYC were associated with advanced grades of pancreatic tumors  of pancreatic cancer , CBS (cystathionine-β-synthase), FDFT1 (farnesyl diphosphate farnesyl transferase 1), LPCAT3 (lysophosphatidylcholine acyltransferase 3), RPL8 , TP53, and TTC35 (tetratricopeptide repeat domain 35), the expressions of which statistically significantly differed between normal tissues and pancreatic tumors of higher histological grades , whereas MYC was positively correlated with FTL  in the control group. In any grade of pancreatic tumor tissues, however, no statistically significant correlation was found between either FTH1 or FTL and an iron homeostasis gene set. Although more data are needed to confirm the association, our result suggests that FTH1 and FTL may be involved in iron homeostasis for maintaining normal intracellular iron levels in cells.We conducted a correlation analysis to investigate whether iron homeostasis genes, on Table . From a 36. Hence, we also determined whether the FTH1 or FTL gene was correlated with a set of ferroptosis genes that were revealed to be highly associated with increased risks for pancreatic cancer. The correlation analysis from TCGA dataset showed that FTH1 displayed significant positive correlations with the CBS , LPCAT3 , and TTC35  genes in the control group, while it was strongly negatively correlated with the CBS , LPCAT3 , and TP53  genes in pancreatic tumor tissues , FDFT1 , RPL8 , and TP53  in the control group, whereas it was strongly negatively correlated with ALOX15 , CBS , FDFT1 , TP53  and TTC35  in pancreatic tumor tissues  and heterogeneity  cells35. Thus, we retrieved data from Oncomine and TCGA dataset to identify iron homeostasis and ferroptosis genes that may mediate the association between ferritin and increasing pancreatic cancer risks  expressions, which ultimately increases the intracellular iron pool45. Our study, however, revealed that high expression of the MYC gene was linked to an increased risk of pancreatic cancer, but it was not significantly correlated with either the FTH1 or FTL gene in different tumor grades, implying that MYC may be involved in regulating iron hemostasis in pancreatic cancer but not through modulating ferritin activity.The exact underlying mechanisms for the detrimental effects of ferritin on pancreatic cancer remain unknown; however, some studies proposed that the role of ferritin in regulating iron homeostasis may be associated with increasing risks of pancreatic cancerCBS and TP53 gene levels with advanced grades of pancreatic cancer, although statistical significance was shown in either G2 and/or G3 of pancreatic cancer compared to the controls. CBS is a gene that mediates ferroptosis inhibition by regulating the trans-sulfuration pathway, which rescues cells from erastin-induced ferroptosis46. Surprisingly, we found that CBS expression was significantly low in G3 compared to the controls. Moreover, a correlation analysis of CBS and FTH1 showed a significant positive correlation in the controls but a negative correlation in G2 pancreatic tumors. These findings suggest that CBS may stimulate the ferroptotic pathway in pancreatic cancer cells through different mechanisms, possibly involving an interplay between CBS and FTH1. A tumor suppressor gene, TP53, was also notably downregulated in G2 and G3 pancreatic tumors, whereas significant negative regulation of the FTH1 and FTL genes was found in different histological grades. A previous study also suggested crosstalk between TP53 and iron regulators, in which anticancer effects of iron chelators, such as deferoxamine and triapine, upon iron depletion are mediated in part by suppressing TP53 activation47. It was also reported that TP53 is recruited on the FTH1 promoter by NF-Y which subsequently represses ferritin expression48. A recent study suggested that ferroptosis is a major component of TP53 in mediating tumor suppression47. Our study demonstrated that the TP53 level in pancreatic cancer was highly associated with FTH1 and FTL, suggesting that TP53 cooperates with these genes in modulating pancreatic carcinoma. As TP53 appears to modulate both iron metabolism and ferroptosis, a comprehensive understanding of TP53 in modulating ferroptosis needs to be further elucidated, which is required to target pancreatic cancer as a therapeutic strategy.Several ferroptosis genes were closely correlated with advanced tumor grades, and their expressions were most significantly altered in G3 pancreatic cancer. There was a trend of decreasing RLP8 and TTC35 are known as mitochondrial genes that were shown to suppress erastin-induced ferroptosis50. Our study found that RLP8 and TTC35 genes were significantly increased in advanced grades of pancreatic tumor tissues compared to the controls, suggesting that these genes are key negative regulators that not only increase aggressiveness in pancreatic cancer cells but also contribute to ferroptosis resistance. Moreover, we showed that correlations between expressions of FTH1 and the ferroptosis regulators, FDFT1 and LPCAT3, may be linked to initiation of pancreatic tumors  as a target protein of FIN56, which promotes degradation of the lipid repair enzyme, GPX4, and induces accumulation of lipid peroxidation51. LPCAT3 is the gene that is involved in phospholipid synthesis and induces ROS accumulation to drive ferroptotic cell death17. Collectively, our analysis of TCGA dataset suggested that several candidate genes may be associated with the onset and progression of pancreatic cancer.Both rs Table . FDFT1 e52. To reduce the risk of false-negative results, we conducted a meta-analysis which included 315 subjects from six eligible articles, to overcome the limitation of a small sample size in our case–control study. Second, methodological variations among the included studies may have led to statistical heterogeneity. Five articles measured ferritin concentration by IRMA or 2-site IRMA. whereas one study conducted by Hamazoe et al. measured SF by RIA, which may have led to a sensitivity difference between the assays54. In addition, there was a lack of obstructive jaundice information of PDAC patients. Previous studies emphasized that the presence of jaundice improves the biomarker specificity for PDAC patients56. Considering jaundice as a possible confounding factor for PDAC biomarker discovery, further studies are required to examine whether the presence of obstructive jaundice in PDAC patients may influence the level of serum ferritin55.There are several limitations of our study. First, there were only 68 Taiwanese participants used for the analysis, which may lead to potential type II errors57. We also aimed to update a previous finding of related topic with the inclusion of new cohort study, since former six studies that have compared SF levels in pancreatic cancer patients and controls were published in either late 1980s or early 1990s. Further to this, we utilized Oncomine database as a tool to identify several genes that modulate ferritin and predict a poorer prognosis of pancreatic cancer, presenting new possibilities for research and ultimate utilization for cancer therapies. More studies, however, are needed to elucidate the regulation of ferritin in pancreatic tumors.However, despite these limitations, our study is the first population-based case–control study that presented SF levels of PDAC patients and healthy controls in Taiwan, including a pooled-analysis of six studies to further strengthens the evidence present on the association between SF and pancreatic cancer. The present results are consistent with a recent meta-analysis study from Lin et al., which showed an elevated pretreatment SF is associated with poorer survival outcomes in hepatobiliary and pancreas (HBP) cancer patients, emphasizing the possibility of utilizing SF as the prognostic value for pancreatic cancerThe study was conducted in accordance with the Declaration of Helsinki. The tissues were obtained from Taipei Medical University Joint Biobank and study was conducted according to the ethical approval by the Ethics Committee of Taipei Medical University .22, but the number of subjects and the measured SF levels in both healthy controls and pancreatic cancer patients differ between the two studies; thus, we included these publications as two separate studies.A search of PUBMED and EMBASE was conducted by two investigators who reviewed studies published up to 1 March 2020. The search was restricted to the English and Chinese languages and human subjects, but one article written in Japanese was later included from a hand search. The search keywords included: “serum ferritin” OR “ferritin” AND “pancreatic cancer”. Search questions were based on the PICO framework: 1. an original research article published in a peer-reviewed journal; 2. the population included adults with pancreatic cancer who were aged ≥ 18 years; 3. the intervention was the serum level of ferritin; 4. the comparison intervention was non-pancreatic cancer subjects, controls or healthy subjects; and 5. the intervention outcome included pancreatic cancer occurrence. Retrieved records were sent to Endnote©  and duplicates were removed. Articles were excluded if they did not meet the predefined inclusion criteria, did not include human subjects, were a review or case report, and did not provide SF levels for both pancreatic cancer subjects and healthy controls. We acknowledge that two publications included in the meta-analysis were from the same authors59. Each study was rated as being weak (1 point), moderate (2 points), or strong (3 points) for risk of bias within six domains: study bias, study design, confounders, blinding, data collection methods, and withdrawals and drop-outs. In the case of discrepancies, discussion and a detailed examination of the full text was performed , using the Effective Public Practice Project (EPHPP) Quality Assessment Tool33. We estimated a pooled standard mean difference (SMD) and the 95% confidence interval (CI) on the basis of both fixed-effect and random-effect models. The standard deviation (SD) was calculated as the standard error (SE) using the equation: SD = SE * n = population size). Between-study heterogeneity was calculated using the Cochrane Q-test, and values were evaluated as tau2, Chi2, and I2, where a higher I2 value indicates high heterogeneity between studies. Generally, I2 values of 25%, 50%, and 75% respectively indicate low, moderate, and high heterogeneity. Publication bias was examined using a funnel plot and Egger’s test. Further, we evaluated the potential sources of heterogeneity by fitting linear meta-regression models60.A meta-analysis was conducted using R Studio 3.5.3. Preoperative serum samples were obtained from Taipei Medical University Joint Biobank, including 34 Taiwanese patients with PDAC and 34 age-matched and gender-matched healthy controls, whose age ranged 41–82 years. Tumor grades and stages were reviewed by senior gastrointestinal pathologists according to the 7th edition of American Joint Committee on Cancer/International Cancer Control (AJCC/UICC). Clinical data on gender, age, tumor location, TNM tumor stage, histologic type and grade, and pathological features were obtained from the TMU Joint Biobank. Five patients (14.7%) were in grade 1 (G1), 20 patients (58.8%) were in grade 2 (G2), and nine patients (26.5%) were in grade 3 (G3). No patients were in histological grade 4 (G4). Briefly, a 10-mL sample of peripheral blood was collected from PDAC patients and healthy controls in a tube that contained separating gel and clot activator. Test tubes were centrifuged at 3400 rpm for 7 min, and serum was aliquoted after the clot was removed. Serum samples were then stored at − 80 °C until being analyzed. SF was assessed by an electrochemiluminescence immunoassay (ECLIA) and was quantified with a Roche Modular P800 analyzer .33 using the packages meta61 (v4.9.4) and metafor62 (v2.0.0) on the dataset from TCGA cohorts and the Oncomine database. In total, 10 normal pancreatic tissue samples and 51 pancreatic tumor tissues were included for the gene set analysis; tumor tissues were then sub-grouped into grade 1 (G1), grade 2 (G2), or grade 3 (G3) according to histological diagnoses. Differential expression was calculated based on z-scores showing up regulated (red) and down regulated (green) genes clustered in heatmaps. Statistical analyses were performed with GraphPad prism 8.4.1. Gene sets associated with histologic tumor grades of pancreatic cancer were statistically evaluated using a two-way analysis of variance (ANOVA) with Tukey’s post-hoc tests.A gene set analysis was performed using R Studio 3.5.3p value of < 0.05 was considered to indicate significance. Significance in all figures is indicated as follows: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.All analyses were performed using GraphPad prism 8.4.1. A Supplementary Information.


The sensitivity, specificity, positive predictive value, and positive likelihood ratio of the SIRS criteria for serious infection was 45.9%, 69.4%, 43.5%, 71.4%, 1.5, and 0.8, respectively. Logistic regression showed that male gender, body temperature ≥ 38.5 °C, heart rate ≥ 178 bpm, and age ≤ 50 days were significant predictors. (4) Conclusions: The performance of the SIRS criteria for predicting serious infections among febrile young infants was poor.(1) Background: Young infants have a high risk of serious infection. The Systematic Inflammatory Response Syndrome (SIRS) criteria can be useful to identify both serious bacterial and viral infections. The aims of this study were to evaluate the diagnostic performance of the SIRS criteria for identifying serious infections in febrile young infants and to identify potential clinical predictors of such infections. (2) Methods: We conducted this prospective cohort study including febrile young infants (aged < 90 days) seen at the emergency department with a body temperature of 38.0 °C or higher. We calculated the diagnostic performance parameters and conducted the logistic regression analysis to identify the predictors of serious infection. (3) Results: Of 311 enrolled patients, 36.7% ( Young infants aged < 90 days) have a high risk of developing serious infections (SIs) due to their greater susceptibility to pathogens and problems with clinical examination that may cause potential infections to be overlooked ,2. Tradi0 days haThe concept of sepsis is useful in this context because it is defined by the systemic inflammatory response syndrome (SIRS) in the presence of a suspected or proven infection, i.e., patients with sepsis can be suffering either from a serious bacterial or viral infection ,17. The Tokyo Metropolitan Children’s Medical Center is a pediatric tertiary care center in Tokyo, Japan with about 38,000 annual pediatric visits to the ED. While the initial management of patients is usually provided by pediatric or emergency medicine residents, board-certificated emergency medicine physicians or pediatricians supervise patient care .We conducted this prospective cohort study from 1 August 2014 through 30 September 2016. This study was initiated before the updated clinical policy and guidelines for febrile young infants of the American College of Emergency Physicians and American Academy of Pediatrics were published [We also followed up on all patients in our cohort by speaking with their guardians via telephone to assess for 28-day mortality.We collected information on patient demographics including patient age, sex, history, vital signs, physical examination findings, laboratory examination results, definitive diagnosis, and outcomes. The vital sign and laboratory variables related to SIRS criteria  were evaluated at admission to the ED. The diagnosis data of the patients were obtained during the clinical course of the disease and finally defined at the time of telephone follow-up for assessing the 28-day outcome.For this study, we adopted the definition of SIRS provided by the International Pediatric Sepsis Conference Guideline . In our We defined a serious infection in the following manner by applying the definition used in previous studies that covers both bacterial and viral infections ,21,22.(1)Septicemia (including bacteremia and viremia): pathogenic bacteria isolated from a blood culture or a pathogenic virus detected by real-time PCR.(2)Meningitis: identification of bacteria or a virus in cerebrospinal fluid.(3)Pneumonia: infiltrate on chest X-ray.(4)Soft tissue infection: acute suppurative inflammation of subcutaneous tissue.(5)Urinary tract infection: >positive urine culture with a single species of pathogen and systemic symptoms such as fever.(6)Bronchiolitis due to RSV or human metapneumovirus requiring emergency intervention and/or evidence of organ failure.We used the above definition of bronchiolitis to select severe cases. ‘Emergency intervention’ was defined as a clinical state requiring emergency supplemental oxygen or fluid resuscitation of more than 20 ml/kg in the ED indicative of respiratory distress/failure or compensatory/hypotensive shock according to the pediatric sepsis guidelines . Other eViruses were detected by a rapid antigen test  or real-time PCR. Patients with lower respiratory infections were tested for RSV, human metapneumovirus, and the influenza virus by rapid antigen test. Viremia and/or central nervous system infections due to parechovirus or enterovirus were diagnosed by routinely performing real-time PCR for all hospitalized patients in whom the focus of infection was unknown.p < 0.05 was considered statistically significant. Diagnostic performance parameters including sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and positive/negative likelihood ratio (LR+/−) of the SIRS criteria were calculated for SI. Multivariate logistic regression analysis was performed to identify the predictors of SI based on an extensive review of past studies and their clinical implications. To broaden their clinical application, continuous variables were converted into categorical variables by determining each variable’s cut-off point prior to logistic regression, and the optimal cutoff point for each variable was calculated using the receiver operating characteristic curve for SI.Data were analyzed using the SPSS software package, version 23 . Standard descriptive statistics were reported with the mean and SD for continuous variables and frequency and percentage for categorical variables. The chi-squared and Student’s t-test were conducted for statistical analysis, and a two-sided Of the 327 eligible patients, sixteen were excluded due to a previous visit to our ED, receiving some form of intervention or insufficient data . The remOf all the patients, 177 (56.9%) were hospitalized in a ward, and seven (2.3%) were admitted to the pediatric intensive care unit. No patient died within 28 days following the first visit to our ED.The sensitivity, specificity, PPV, NPV, LR+, and LR− of the SIRS criteria for SI was 47.2%, 68.0%, 37.2%, 76,3%, 1.5 , 1.1–2.0), and 0.8 , respectively .p = 0.101). The proportion of males was significantly higher among SI patients than among non-SI patients . Regarding the vital signs, SI patients presented a significantly higher body temperature  and tachycardia . Although the respiratory rate was similar between the two groups, the patients with SI showed lower oxygen saturation  than the patients without SI. The majority of patients in the SI group showed poor perfusion in their extremities  and prolonged capillary refilling time . While there was no difference in the WBC count between the groups, the percentage of lymphocytes was significantly smaller  in the SI group. No differences were found in the percentage of monocytes and the venous lactate value between the groups  2.45, 95% CI 1.41–4.28), body temperature ≥ 38.5 °C , heart rate ≥ 178 bpm , and age ≤ 50 days  were identified as significant predictors of serious infection , specificity (68.0%), and LR+ (1.5) failed to provide clinicians with adequate information to choose appropriate acute care for febrile young infants. A study of adult sepsis showed that diagnostic performance measures such as sensitivity and specificity of the SIRS criteria were inferior to those of SOFA . FurtherOur study also identified clinical predictors of SI in febrile young infants that can be grouped under demographics and vital signs. In the demographic findings, we identified younger age (≤50 days), indicative of the immune system immaturity in young infants, as a predictive factor. Male gender was also an independent predictor, possibly due to the fact that uncircumcised male infants have a higher risk of urinary tract infection. Lack of circumcision can be a cause of SI because East Asian cultures normally do not circumcise newborns . Among tWe therefore assumed that the low predictive value of the respiratory rate and WBC count in the SIRS criteria lowered the diagnostic performance of the SIRS criteria for identifying SI in young infants. Furthermore, it should be noted that patients’ demographic variables and vital signs are the most significant predictive factors because clinicians are able to obtain these data within the short period of time allowed for patient care in the ED.This study has limitations. As this was a prospective study conducted at a single center in Japan, the etiology of the fever in young infants might differ from that in other regions, especially the Western countries. For example, newborns are rarely circumcised in Japan, with the result that the prevalence of urinary tract infections among young infants might be higher than in cultures which practice neonatal circumcision. However, pediatric societies in North America make allowances for the cultural background of the child and his family and do not routinely recommend circumcision anymore . Next, wIn conclusion, the SIRS criteria were ineffective in identifying serious infections in febrile infants younger than 90 days. However, such infections can be identified based on predictors such as age, gender, tachycardia, and high-grade fever.


Oxidative stress (OS) is detrimental to the production of reactive oxygen species. It affects poultry performance, health status, welfare, and meat and eggs quality. Many methods of reducing oxidative stress have been considered; however, due to the birds’ welfare and the consumers’ safety, natural substances (antioxidants) have mainly been taken into consideration. The current data show that feeding broiler chickens Ross 308 with either a dietary supplementation of vinegar (5 and 10 mL/kg of diet) or rocket salad (2 and 3 g/kg of diet) could mitigate the experimentally induced oxidative stress level. Positive changes were demonstrated by the improvement of the serum oxidation system, which led to increases in the birds’ productivity and carcass quality, and modulations of intestine histomorphological characteristics. Thus, vinegar and rocket salad might be promising natural alternatives for costly feed additives under the various types of stresses or acute environmental conditions.p ≤ 0.05) OS through improving the birds’ survivability, body weight gain, feed conversion ratio, and carcass yield. The highest villus height and villus height/crypt depth ratio of the duodenum were achieved by Vi2, Ro1-2, and NC (p ≤ 0.05). The villus surface area and muscular layer thickness were smallest in the PC, while they did not differ significantly in other groups (p > 0.05). Similar relationships were found in serum superoxide dismutase, glutathione peroxidase, hydroperoxide, and malondialdehyde; however, higher doses of both additives were more effective. It seems that liquid vinegar and rocket salad could have beneficial influences on the antioxidant status of birds.The purpose of the study was to investigate the ameliorative effect of dietary rocket salad and apple cider vinegar on the oxidative stress (OS) status of broilers. Specifically, 720 Ross 308 chicks were divided into six groups: negative and positive controls . The experimental groups Vi1 and Vi2 were fed with feed mixture moisturized with 5 and 10 mL of vinegar/kg, and Ro1 and Ro2 were fed with 2 and 3 g of rocket salad/kg, respectively. The birds’ productivity, oxidative serum parameters, and morphometric indices of the gastrointestinal tract were registered at 6 weeks of rearing. The vinegar or rocket salad additive had powerful potentials to significantly suppress ( The detrimental effect of oxidative stress (OS) to the production of reactive oxygen species can be prevented or alleviated by natural substances that are called antioxidants. OS affects avian performance, health status, welfare, and final products’ quality (meat and eggs) ,2. Antio3COOH) is the predominant ingredient in apple cider vinegar, which is characterized by its antimicrobial and anticoccidial activity [Vinegar is a flavoring condiment and acidifier made by alcoholic fermentation from diverse sugary and starchy materials, and it is widely used for cooking purposes. Acetic acid , commonly known as arugula, is one of the annual herbs of the Brassicaceae family. It is characterized by its pungent spicy flavor and taste, which can be used for cooking purposes fresh or after preparation in steam [Rocket salad  supplemented in diets or using apple cider vinegar mixed with diets to alleviate the OS of broiler chickens fed with lead acetate as an oxidative promoter. The effect of this process on productive performance, gut histomorphology, and oxidation parameters of blood serum was investigated.It was hypothesized that, due to their antioxidative properties, both rocket salad and vinegar may also be useful in oxidative stress reduction in poultry. However, according to our findings, there have been very limited reports about using rocket salad or vinegar mixed in poultry diets, especially concerning their antioxidative activity, particularly in poultry. Therefore, the aim of the current work was to unveil the possible antioxidative activity of rocket salad . With regard to animal welfare and ethical principles, all experimental animal treatments were approved by the Scientific Committee of Department of Animal Production Techniques, Technical College of Al-Musaib Al-Furat Al-Awset Technical University .Chicks were fed ad libitum on an isonitrogenous and isocaloric diet based on soybean and corn  with frePowdery lead acetate  was used as an oxidative factor to artificially induce OS in broilers by adding it daily to drinking water ,21.Apple cider vinegar and rocket salad used as antioxidant materials in diets were both commercially available and purchased from the local market. Apple cider vinegar (5% acetic acid) was in liquid form, which was manufactured by Food Canning Company, Karbala, Iraq. It was applied as an antioxidative material via moisturization with the diet by the mixing method of (volume:weight) either 5 or 10 mL per kg of diet. The dietary moisturization with vinegar was performed daily to preserve its pureness property and prevention from volatilization.Eruca sativa Mill.) was made from seeds of plants obtained from the commercial market. It was tightly packaged in a black polyethylene bag after mixing in feed by 2 and 3 g of rocket salad/kg. The chemical composition of rocket salad  were recorded weekly and presented accumulatively, including body weight, weight gain, feed intake, and feed conversion ratio (FCR). Water intake and survivability were also calculated. The production efficiency factor (PEF) as an indicator of the economic profit of trial was calculated based on Lemme et al. [The averages of productive characteristics . All sections of the gut starting from the esophagus to the rectum were measured using a vernier caliper and tape measure. To measure the crop, proventriculus, and gizzard length, a tape measure was used for this purpose, while the lengths of the duodenum, jejunum, ileum, ceca, and rectum sections were measured by a vernier caliper. The relative weights of all visceral organs  were calculated after registering the absolute weights using an accurate-sensitivity (0.01 g) digital scale . Then, 1 cm of duodenum  was removed and placed in a plastic container containing 10% formalin for fixation and then dehydrated and embedded with paraffin. Later, a 4 μm thickness was taken and exposed to staining with hematoxylin and eosin to process the investigation of villi and crypts dimensions and muscular layer thickness. The histological analysis was performed from 4 birds per replicate of treatment with 2 sections per bird, and 10 well-oriented villi and crypts from the duodenum of each slide were measured (20 slides per bird). Then, the average value for each bird was used for further statistical analysis (n = 16/treatment). All methods of investigation were carried out according to the procedures reported by Rubio et al. [The slaughtering of 4 birds per replicate was carried out on the 6th week via cervical dislocation and then decapitation. The birds had limited access to feed for 8 h before the slaughter. The carcass yield and proportions of carcass cuts and abdominal fat out of live body weight were registered. Samples of the gastrointestinal tract were collected to analyze their morphometric parameters . To separate serum, blood samples were centrifuged for 15 min at 3000 RPM, and then the serum samples were preserved at −25 °C until analysis. Indicators of lipid peroxidation in serum involving malondialdehyde (MDA) and hydroperoxide (LOOH) were determined according to Salih et al. [Blood samples were placed in a gel separator tube after being collected from a neck vein during slaughtering  under effect of treatment (j);Yµ—the general average of the studied trait;i—the effect of treatment;tij—random error.ep ≤ 0.05) in body weight at 6 weeks, weight gain and feed intake at 1–6 weeks, and survivability in favor of all experimental groups and NC compared to PC. All experimental groups also demonstrated a significant decrease (p ≤ 0.05) in feed conversion ratio at 1–3 weeks and 1–6 weeks compared to PC.The data presented in p ≤ 0.05) in experimental groups and NC (p ≤ 0.05) in abdominal fat ratio in the same groups compared with PC. In comparison with PC, Vi1, Vi2, and Ro1 exhibited high thigh ratios (p ≤ 0.05), while Vi1, Vi2, Ro1, and Ro2 exhibited high drumstick ratios (p ≤ 0.05). However, increases in wings, back, and neck ratios were registered in PC compared to other groups.The production efficiency factor, carcass yield, and breast ratio were significantly higher (s and NC  with thep ≤ 0.05) in Ro2 in comparison to the group with induced oxidative stress. No significant differences among groups were found in other anatomical traits.It is clear from p ≤ 0.05) in Vi2, Ro1, Ro2, and NC, and there was an increase (p ≤ 0.05) in villus width, villus surface area, and muscular layer thickness in all experimental groups and NC compared to PC. The Vi2, Ro1, Ro2, and NC registered an increase (p ≤ 0.05) in villus height/crypt depth with a lack of differences among groups in crypt depth.Regarding p ≤ 0.05) in blood serum LOOH and MDA levels, although in the same groups, the blood serum SOD, GPx, and FRAP were high (p ≤ 0.05) compared to in PC. Moreover, Vi1, Vi2, Ro1, and NC had a higher (p ≤ 0.05) blood serum CAT in relation to PC  were noticed [All the feed additives performed positive roles to maintain the oxidative status and suppress OS through boosting the antioxidative enzymes  and FRAP while lowering lipid peroxidation markers (LOOH and MDA) in serum. The antioxidant enzymes are essential for modifying harmful oxygen molecules into nontoxic molecules, which inhibits the peroxidation of lipids . The mec2O2 . No data2O2  found de2O2 . In ram 2O2 . Under t noticed .The current data prove that the feeding of broiler chickens Ross 308 with either a dietary moisturization with vinegar (5 and 10 mL/kg of diet) or dietary supplementation of rocket salad (2 and 3 g/kg of diet) could mitigate the oxidative stress induced experimentally by lead acetate in drinking water. Positive changes were demonstrated by the improvement of the serum oxidation system, which, in turn, led to increases in birds’ productivity and carcass quality, and the modulation of intestine histomorphological characteristics. Thus, vinegar and rocket salad might be natural promising alternatives for costly feed additives during the various types of stresses or acute environmental conditions.