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Background The necessity to develop high lipid-producing microalgae is emphasized for the commercialization of microalgal biomass, which is environmentally friendly and sustainable. Nannochloropsis are one of the best industrial microalgae and have been widely studied for their lipids, including high-value polyunsaturated fatty acids (PUFAs). Many reports on the genetic and biological engineering of Nannochloropsis to improve their growth and lipid contents have been published. Results We performed insertional mutagenesis in Nannochloropsis salina, and screened mutants with high lipid contents using fluorescence-activated cell sorting (FACS). We isolated a mutant, Mut68, which showed improved growth and a concomitant increase in lipid contents. Mut68 exhibited 53% faster growth rate and 34% higher fatty acid methyl ester (FAME) contents after incubation for 8 days, resulting in a 75% increase in FAME productivity compared to that in the wild type (WT). By sequencing the whole genome, we identified the disrupted gene in Mut68 that encoded trehalose-6-phosphate (T6P) synthase (TPS). TPS is composed of two domains: TPS domain and T6P phosphatase (TPP) domain, which catalyze the initial formation of T6P and dephosphorylation to trehalose, respectively. Mut68 was disrupted at the TPP domain in the C-terminal half, which was confirmed by metabolic analyses revealing a great reduction in the trehalose content in Mut68. Consistent with the unaffected N-terminal TPS domain, Mut68 showed moderate increase in T6P that is known for regulation of sugar metabolism, growth, and lipid biosynthesis. Interestingly, the metabolic analyses also revealed a significant increase in stress-related amino acids, including proline and glutamine, which may further contribute to the Mut68 phenotypes. Conclusion We have successfully isolated an insertional mutant showing improved growth and lipid production. Moreover, we identified the disrupted gene encoding TPS. Consistent with the disrupted TPP domain, metabolic analyses revealed a moderate increase in T6P and greatly reduced trehalose. Herein, we provide an excellent proof of concept that the selection of insertional mutations via FACS can be employed for the isolation of mutants with improved growth and lipid production. In addition, trehalose and genes encoding TPS will provide novel targets for chemical and genetic engineering, in other microalgae and organisms as well as Nannochloropsis.

Chlorella spp. are green algae that are found across wide-ranging habitats from deserts to arctic regions, with various strains having adapted to survive under diverse environmental conditions. In this study, two novel Chlorella strains (ABC-002, ABC-008) were isolated from a freshwater lake in South Korea during the winter season and examined for possible use in the biofuel production process. The comparison of ABC-002 and ABC-008 strains with Chlorella vulgaris UTEX265 under two different temperatures (10°C, 25°C) revealed their cold-tolerant phenotypes as well as high biomass yields. The maximum quantum yields of UTEX25, ABC- 002, and ABC-008 at 10°C were 0.5594, 0.6747, and 0.7150, respectively, providing evidence of the relatively higher cold-resistance capabilities of these two strains. Furthermore, both the biomass yields and lipid content of the two novel strains were found to be higher than those of UTEX265; the overall lipid productivities of ABC-002 and ABC-008 were 1.7 ~ 2.8 fold and 1.6 ~ 4.2 fold higher compared to that of UTEX265, respectively. Thus, the high biomass and lipid productivity over a wide range of temperatures indicate that C. vulgaris ABC-002 and ABC-008 are promising candidates for applications in biofuel productions via outdoor biomass cultivation.

International audience; Nannochloropsis species, unicellular industrial oleaginous microalgae, are model organisms for microalgal systems and synthetic biology. To facilitate community-based annotation and mining of the rapidly accumulating functional genomics resources, we have initiated an international consortium and present a comprehensive multi-omics resource database named Nannochloropsis Design and Synthesis (NanDeSyn; http://nandesyn.single-cell.cn). Via the Tripal toolkit, it features user-friendly interfaces hosting genomic resources with gene annotations and transcriptomic and proteomic data for six Nannochloropsis species, including two updated genomes of Nannochloropsis oceanica IMET1 and Nannochloropsis salina CCMP1776. Toolboxes for search, Blast, synteny view, enrichment analysis, metabolic pathway analysis, a genome browser, etc. are also included. In addition, functional validation of genes is indicated based on phenotypes of mutants and relevant bibliography. Furthermore, epigenomic resources are also incorporated, especially for sequencing of small RNAs including microRNAs and circular RNAs. Such comprehensive and integrated landscapes of Nannochloropsis genomics and epigenomics will promote and accelerate community efforts in systems and synthetic biology of these industrially important microalgae.

Development of transgenic strains of microalgae is necessary to obtain microalgal strains that are capable of producing high levels of oils and a variety of bio-products. However, microalgae have a much lower transformation efficiency in comparison to other micro-organisms due to the presence of thick cell walls. Here, a two-pronged approach for optimizing the electroporation parameters combined with the application of secondary metabolites from myxobacteria to weaken the cell wall was used to maximize the transformation efficiency of Nannochloropsis salina (Eustigmatophyceae). When the electroporation parameters were set at 50 pulses of square waves at a field strength of 12,000 V cm−1, a transformation efficiency of up to 53 per 108 cells was achieved for N. salina in the early exponential phase of growth. Next, cultivation of the microalgae using bacterial conditioned F2N medium containing 10% supernatant of the myxobacteria strain DZ2 resulted in a moderate inhibition of the growth rate and distinctive morphological changes. Flow cytometry confirmed an increase in the overall cell size as well as granularity. When the bacterial medium conditioned cells were employed for transformation under optimized electroporation conditions, the transformation efficiency improved 2.71-fold up to 144 per 108 cells. Taken together, a combinatory approach for electroporation using high field strength square wave pulses and conditioning cells with myxobacterial supernatant is a promising method for the efficient genetic transformation of microalgae.

Even though there has been much interest in genetic engineering of microalgae, its progress has been slow due to the difficulty and limitation of available techniques. Currently, genetic modification in most microalgal strains is confined to single gene transformation. Here, a multigene expression system for the oleaginous model strain Nannochloropsis salina was developed with glycine-serine-glycine spacer linked 2A self-cleaving peptides (2A) for the first time. An efficiency test of the four most widely used 2As revealed that two different types of 2As T2A and E2A have the best performance in N. salina with a maximum cleavage rate of nearly 45%. The system was able to express the linked sequence of the selection marker shble and the fluorescence protein sfCherry with intact functions. Because 2A enabled multigene expression in the single cassette form, the use of 2A also reduced the vector size, which along with the stronger promoter resulted in a 9-fold increase in the transformation efficiency. Furthermore, confirmative screening accuracy of more than 90% was observed. Hence, the 2A applied vector system is expected to be beneficial in microalgal research field because it enables multigene expression as well as offering improved transformation and screening efficiency.

Fructophilic behavior in microalgae is a rare trait that could benefit biorefineries by enabling substitution of carbon source with fructose, and our previous study identified that Ettlia sp. prefers fructose relative to glucose. In this study, by analyzing the transcription levels of genes related to sugar transport and the glycolysis pathway, the fructose utilization of Ettlia sp. was investigated. In a fructose-containing medium, the expression levels of fructokinase (EttFRK3) and glucokinase (EttGCK1 and EttGCK2) genes were significantly upregulated in heterotrophic cultivation of Ettlia sp. under fructose supplementation conditions. Further, a sugar transporter (EttSTF11) was significantly upregulated by 3.2-fold in one day, and this increase was analogous to the specific growth rate exhibited by the species. Subsequent cultivation tests with multi-sugar sources also showed a significant upregulation of EttSTF11 relative to other treatments without fructose. A phylogenetic tree derived from the analysis of different transporters of interest identified that EttSTF11 was adjacent to reference fructose transporters with a high bootstrap value of 71. Given that the transmembrane domains of EttSTF11 were analogous to those of reference fructose transporter genes, EttSTF11 appeared to play a critical role in fructose consumption and metabolism in Ettlia sp. This article is protected by copyright. All rights reserved.

Transgene expression in microalgae can be hampered by transgene silencing and unstable expression due to position effects. To overcome this, "safe harboring" transgene expression system was established for Nannochloropsis. Initially, transformants were obtained expressing a sfGFP reporter, followed by screening for high expression of sfGFP with fluorescence-activated cell sorter (FACS). 'T1' transcriptional hotspot was identified from a mutant showing best expression of sfGFP, but did not affect growth or lipid contents. By using a Cas9 editor strain, FAD12 gene, encoding Δ12-fatty acid desaturase (FAD12), was successfully knocked-in at the T1 locus, resulting in significantly higher expression of FAD12 than those of random integration. Importantly, the "safe harbored" FAD12 transformants showed four-fold higher production of linoleic acid (LA), the product of FAD12, leading to 1.5-fold increase in eicosapentaenoic acid (EPA). This safe harboring principle provide excellent proof of the concept for successful genetic/metabolic engineering of microalgae and other organisms.

Background Chlorophylls play important roles in photosynthesis, and thus are critical for growth and related metabolic pathways in photosynthetic organisms. They are particularly important in microalgae, emerging as the next generation feedstock for biomass and biofuels. Nannochloropsis are industrial microalgae for these purposes, but are peculiar in that they lack accessory chlorophylls. In addition, the localization of heterologous proteins to the chloroplast of Nannochloropsis has not been fully studied, due to the secondary plastid surrounded by four membranes. This study addressed questions of correct localization and functional benefits of heterologous expression of chlorophyllide a oxygenase from Chlamydomonas (CrCAO) in Nannochloropsis. Results We cloned CrCAO from Chlamydomonas, which catalyzes oxidation of Chla producing Chlb, and overexpressed it in N. salina to reveal effects of the heterologous Chlb for photosynthesis, growth, and lipid production. For correct localization of CrCAO into the secondary plastid in N. salina, we added the signal-recognition sequence and the transit peptide (cloned from an endogenous chloroplast-localized protein) to the N terminus of CrCAO. We obtained two transformants that expressed CrCAO and produced Chlb. They showed improved growth under medium light (90 μmol/m2/s) conditions, and their photosynthetic efficiency was increased compared to WT. They also showed increased expression of certain photosynthetic proteins, accompanied by an increased maximum electron-transfer rate up to 15.8% and quantum yields up to 17%, likely supporting the faster growth. This improved growth resulted in increased biomass production, and more importantly lipid productivity particularly with medium light. Conclusions We demonstrated beneficial effects of heterologous expression of CrCAO in Chlb-less organism N. salina, where the newly produced Chlb enhanced photosynthesis and growth. Accordingly, transformants showed improved production of biomass and lipids, important traits of microalgae from the industrial perspectives. Our transformants are the first Nannochloropsis cells that produced Chlb in the whole evolutionary path. We also succeeded in delivering a heterologous protein into the secondary plastid for the first time in Nannochloropsis. Taken together, our data showed that manipulation of photosynthetic pigments, including Chlb, can be employed in genetic improvements of microalgae for production of biofuels and other biomaterials.

Microalgae are promising alternative feedstocks that can be used to produce biofuels and replace conventional fossil fuels. In order to increase the lipid production of microalgae, strain development via genetic engineering is required. To date, metabolic engineering studies have mainly focused on lipid synthesis pathways. However, the complicated metabolic pathways that exist in many organelles along with limitations of genetic engineering tools have made it difficult to obtain desirable microalgal strains. As an alternative strategy, we tried to increase carbon flux into fatty acid synthesis by overexpressing the NADP-dependent malic enzyme, NsME1, in Nannochloropsis salina. We found that the biomass and fatty acid methyl ester (FAME) contents were increased in NsME1 overexpressing transformants, such that the FAME yield of the top-producing NsME1 transformant was 53% higher than the wild type. To understand the effects of NsME1 on lipid production, we analyzed the total carbon concentration and NADPH/NADP ratio, which were found to be enhanced in the transformants. We also investigated mRNA expression levels of genes involved in C4-like carbon concentrating mechanism and fatty acid synthesis by quantitative real-time PCR, and confirmed their positive contribution to fatty acid production. Taken together, our results demonstrate that overexpression of NsME1 could simultaneously improve the carbon concentration and reducing power in cells, thereby increasing the lipid and FAME yields of Nannochloropsis salina. We suggest that NsME1 can serve as a promising genetic target for enhancing lipid-based bio-products in Nannochloropsis and potentially other industrial microalgae.

Microalgae have great potential for the production of biofuels due to the ability of the organism to accumulate large quantities of storage lipids under stress conditions. Mitogen activated protein kinase (MAPK) signaling cascades are widely recognized for their role in stress response signal transduction in eukaryotes. To assess the correlation between MAPK activation and lipid productivity, Chlamydomonas reinhardtii was studied under various concentrations of NaCl. The results demonstrated that C. reinhardtii exhibits elevated levels of extracellular-signal regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) activities after undergoing osmotic stress, as well as an increase in cellular lipid content. To establish a more direct causal link between both kinases and lipid productivity, C. reinhardtii was subjected to biochemically induced regulation of ERK and JNK pathways. Activating the MEK-ERK pathway via C6 ceramide treatment increased ERK activation and lipid production simultaneously, while PD98059 mediated inhibition of the pathway yielded opposite results. Interestingly, suppression of the JNK pathway with SP600125 resulted in a substantial decrease in cell viability under osmotic stress. These results suggest that ERK and JNK MAP kinases have important roles in microalgal lipid accumulation and cell growth under osmotic stress, respectively.

The production performance and egg quality traits among five strains of Korean native chickens (KNC) were evaluated in conventional cages. A total of 240 KNC were housed in a controlled environment. Each strain had 12 replicates with 4 chickens per cage. Feed intake, body weights, egg production and egg quality were measured at 24, 28 and 32 of weeks. Egg quality parameters were analyzed using 150 eggs. Results indicated significant (P 0.05) for feed efficiency. The difference among those KNC strains on egg shell color, egg shell strength and egg shell density were not different (P>0.05) at the age of week 24 while it was significant (P 0.05) on egg length and egg shape index from five strains of KNC. The significant difference (P 0.05) at the age of 32 weeks. Regarding internal quality parameters, albumen height and Haugh unit were significantly (P 0.05). Based on the egg weight and the production performance, GS-10 KNC strain was superior when compared with the other strains.

Highlights • Heterologous sfCherry protein was expressed in N. salina for the first time. • N. salina was transformed by particle bombardment. • Integration site of the transgene on the genome was determined by RESDA PCR. • Expression of sfCherry was confirmed by a western blotting and confocal microscopy., Oleaginous microalgae of the Nannochloropsis genus are considered excellent candidates for biofuels and value-added products owing to their high biomass productivity and lipid content. Here, we report the first overexpression and detection of a heterologous sfCherry fluorescent protein in Nannochloropsis salina in order to develop a transformation toolbox for future genetic improvements. Particle bombardment was employed for transformation, and expression of Shble under the control of TUB and UEP promoters, cloned from N. salina, was used to confer resistance to Zeocin antibiotics, resulting in 5.9 and 4.7 transformants per 108 cells, respectively. Stable integration of the markers into the genome was confirmed using a restriction enzyme site-directed amplification (RESDA) PCR. The expression of sfCherry fluorescent protein was confirmed by Western blot analysis and confocal microscopy. These results suggest new possibilities of efficient genetic engineering of Nannochloropsis for the production of biofuels and other biochemicals.

The oleaginous microalga Nannochloropsis sp. has been spotlighted as a promising candidate in genetic engineering research for biodiesel production. However, one of the major bottlenecks in the genetic manipulation against Nannochloropsis sp. is low transformation efficiency. Based on the idea that they grow rapidly in broth culture, the effect of conditioned medium on colonization and transformation efficiency of Nannochloropsis salina was investigated. Cells grown on agar plates with 20-40% conditioned medium produced colonies that were approximately 2.3-fold larger than cells grown without conditioned medium. More importantly, the transformation efficiency was about 2-fold greater on plates with 30% conditioned medium relative to those without conditioned medium. In addition, FAME productivity in liquid cultures with 100% conditioned medium increased up to 20% compared with cultures of control medium. These results suggest that conditioned medium can be applied for efficient transformation and cost-effective cultivation of N. salina for biodiesel production.

Background Microalgal strain development through genetic engineering has received much attention as a way to improve the traits of microalgae suitable for biofuel production. However, there are still some limitations in application of genetically modified organisms. In this regard, there has been recent interest in the isolation and characterization of superior strains naturally modified and/or adapted under a certain condition and on the interpretation of phenotypic changes through the whole genome sequencing. Results In this study, we isolated and characterized a novel derivative of C. reinhardtii, whose phenotypic traits diverged significantly from its ancestral strain, C. reinhardtii CC-124. This strain, designated as CC-124H, displayed cell population containing increased numbers of larger cells, which resulted in an increased biomass productivity compared to its ancestor CC-124. CC-124H was further compared with the CC-124 wild-type strain which underwent long-term storage under low light condition, designated as CC-124L. In an effort to evaluate the potential of CC-124H for biofuel production, we also found that CC-124H accumulated 116 and 66% greater lipids than that of the CC-124L, after 4 days under nitrogen and sulfur depleted conditions, respectively. Taken together, our results revealed that CC-124H had significantly increased fatty acid methyl ester (FAME) yields that were 2.66 and 1.98 times higher than that of the CC-124L at 4 days after the onset of cultivation under N and S depleted conditions, respectively, and these higher FAME yields were still maintained by day 8. We next analyzed single nucleotide polymorphisms (SNPs) and insertion/deletions (indels) based on the whole genome sequencing. The result revealed that of the 44 CDS region alterations, 34 resulted in non-synonymous substitutions within 33 genes which may mostly be involved in cell cycle, division or proliferation. Conclusion Our phenotypic analysis, which emphasized lipid productivity, clearly revealed that CC-124H had a dramatically enhanced biomass and lipid content compared to the CC-124L. Moreover, SNPs and indels analysis enabled us to identify 34 of non-synonymous substitutions which may result in phenotypic changes of CC-124H. All of these results suggest that the concept of adaptive evolution combined with genome wide analysis can be applied to microalgal strain development for biofuel production. Electronic supplementary material The online version of this article (10.1186/s13068-017-1000-0) contains supplementary material, which is available to authorized users.

Background Genetic engineering of microalgae is necessary to produce economically feasible strains for biofuel production. Current efforts are focused on the manipulation of individual metabolic genes, but the outcomes are not sufficiently stable and/or efficient for large-scale production of biofuels and other materials. Transcription factors (TFs) are emerging as good alternatives for engineering of microalgae, not only to increase production of biomaterials but to enhance stress tolerance. Here, we investigated an AP2 type TF Wrinkled1 in Arabidopsis (AtWRI1) known as a key regulator of lipid biosynthesis in plants, and applied it to industrial microalgae, Nannochloropsis salina. Results We expressed AtWRI1 TF heterologously in N. salina, named NsAtWRI1, in an effort to re-enact its key regulatory function of lipid accumulation. Stable integration AtWRI1 was confirmed by RESDA PCR, and its expression was confirmed by Western blotting using the FLAG tag. Characterizations of transformants revealed that the neutral and total lipid contents were greater in NsAtWRI1 transformants than in WT under both normal and stress conditions from day 8. Especially, total lipid contents were 36.5 and 44.7% higher in NsAtWRI1 2–3 than in WT under normal and osmotic stress condition, respectively. FAME contents of NsAtWRI1 2–3 were also increased compared to WT. As a result, FAME yield of NsAtWRI1 2–3 was increased to 768 mg/L/day, which was 64% higher than that of WT under the normal condition. We identified candidates of AtWRI1-regulated genes by searching for the presence of the AW-box in promoter regions, among which lipid metabolic genes were further analyzed by qRT-PCR. Overall, qRT-PCR results on day 1 indicated that AtWRI1 down-regulated TAGL and DAGK, and up-regulated PPDK, LPL, LPGAT1, and PDH, resulting in enhanced lipid production in NsAtWRI1 transformants from early growth phase. Conclusion AtWRI1 TF regulated several genes involved in lipid synthesis in N. salina, resulting in enhancement of neutral lipid and FAME production. These findings suggest that heterologous expression of AtWRI1 TF can be utilized for efficient biofuel production in industrial microalgae. Electronic supplementary material The online version of this article (doi:10.1186/s13068-017-0919-5) contains supplementary material, which is available to authorized users.

Genome editing techniques are critical for manipulating genes not only to investigate their functions in biology but also to improve traits for genetic engineering in biotechnology. Genome editing has been greatly facilitated by engineered nucleases, dubbed molecular scissors, including zinc-finger nuclease (ZFN), TAL effector endonuclease (TALEN) and clustered regularly interspaced palindromic sequences (CRISPR)/Cas9. In particular, CRISPR/Cas9 has revolutionized genome editing fields with its simplicity, efficiency and accuracy compared to previous nucleases. CRISPR/Cas9-induced genome editing is being used in numerous organisms including microalgae. Microalgae have been subjected to extensive genetic and biological engineering due to their great potential as sustainable biofuel and chemical feedstocks. However, progress in microalgal engineering is slow mainly due to a lack of a proper transformation toolbox, and the same problem also applies to genome editing techniques. Given these problems, there are a few reports on successful genome editing in microalgae. It is, thus, time to consider the problems and solutions of genome editing in microalgae as well as further applications of this exciting technology for other scientific and engineering purposes.

Ability to increase the lipid production in microalgae is one of the heavily sought-after ideas to improve the economic feasibility of microalgae-derived transportation fuels for commercial applications. We used the oxidative stress by TiO2 nanoparticles, a well-known photocatalyst, to induce lipid production in microalgae. Chlorella vulgaris UTEX 265 was cultivated under various concentrations of TiO2 ranging from 0.1 to 5 g/L under UV-A illumination. Maximum specific growth rate was affected in responding to TiO2 concentrations. In the presence of UV-A, chlorophyll concentration was decreased at the highest concentration of TiO2 (5 g/L TiO2) by oxidative stress. The fatty acid ethyl ester (FAME) composition analysis suggested that oxidative stress causes the accumulation and decomposition of lipids. The highest FAME productivity was 18.2 g/L/d under low concentrations of TiO2 (0.1 g/L) and a short induction time (two days). The controlled condition of TiO2/UV-A inducing oxidative stress (0.1 g/L TiO2 and two days induction) could be used to increase the lipid productivity of C. vulgaris UTEX 265. Our results show the possibility of modulating the lipid induction process through oxidative stress with TiO2/UV-A.

Background Microalgae are considered promising alternative energy sources because they consume CO2 and accumulate large amounts of lipids that can be used as biofuel. Nannochloropsis is a particularly promising microalga due to its high growth rate and lipid content, and the availability of genomic information. Transcription factors (TFs) are global regulators of biological pathways by up- or down-regulation of related genes. Among these, basic helix-loop-helix (bHLH) TFs regulate growth, development, and stress responses in plants and animals, and have been identified in microalgae. We identified two bHLH TFs in the genome of N. salina CCMP1776, NsbHLH1, and NsbHLH2, and characterized functions of NsbHLH2 that may be involved in growth and nutrient uptake. Results We obtained NsbHLH2 overexpressing transformants of N. salina CCMP1776 by particle bombardment and confirmed that these were stable transformants. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting using antibodies against the FLAG tag that was attached at the end of the coding sequence confirmed the expression of the NsbHLH2 protein under various culture conditions. The qRT-PCR results also indicated that the endogenous and transgenic expression of NsbHLH2 was reduced under stressed conditions. Overexpression of NsbHLH2 led to increased growth rate in the early growth period, and concomitantly higher nutrient uptake, than wild type (WT). These enhanced growth and nutrient uptake resulted in increased productivities of biomass and FAME. For example, one of the transformants, NsbHLH2 3–6, showed increased biomass productivity by 36 % under the normal condition, and FAME productivity by 33 % under nitrogen limitation condition. Conclusively, the improved growth in the transformants can be associated with the enhanced nutrient uptake. We are currently assessing their potential for scale-up cultivation with positive outcomes. Conclusion Overexpression of NsbHLH2 led to enhanced growth rate and nutrient uptake during the early growth phase, and increased biomass and FAME productivity, especially in the later period under normal and stressed conditions. Based on these results, we postulate that NsbHLH2 can be employed for the industrial production of biodiesel from N. salina. Electronic supplementary material The online version of this article (doi:10.1186/s13068-015-0386-9) contains supplementary material, which is available to authorized users.

This review provides an overview of the published data on the canola meal and its suitability for duck as an alternative plant-origin protein source to soybean meal. Canola meal is a legume origin protein source containing comparable amino acid profile to soybean meal and rich in essential minerals and vitamins. Nonetheless, it is known to contain less in energy content than soybean meal. Factors like field conditions and processing methods creates compositional variations among canola meal. Presence of anti-nutritional factors such as phenolic substances, phytate and glucosinolates which are known to reduce growth performance in livestock animals, are the major drawbacks for canola meal to be a competitive plant-origin protein source in the feed industry. This review is focused to address i) nutritional characteristics and feeding value of canola meal for ducks and ii) impacts of feeding canola meal on performances of ducks.

Recombinant human erythropoietin (rhEPO) has been successfully used for correcting renal anemia. However, recent studies have raised some concerns about the safety of rhEPO treatment due to its immunogenic side effect - pure red cell aplasia (PRCA). We now report a case of development of anti-EPO neutralizing antibodies (Abs) implicated in thrombocytopenia as well as erythrocytopenia. A 35-year-old man had a history of administering rhEPO (epoetin alfa, epoetin beta and darbepoetin alfa) for 2years to treat renal anemia. The hematological parameters were collected. Anti-EPO, anti-platelet, and anti-thrombopoietin (TPO) Ab assays were performed to test the presence of autoreactive Abs. After performing antibody assays due to severe resistance to rhEPO treatment, a high titer of anti-EPO neutralizing Abs was detected. However, unexpectedly, this patient also showed thrombocytopenia rather than PRCA. We investigated the cause of the marked thrombocytopenia and found anti-TPO Abs in patient serum. To our best knowledge, this is the first report of the development of anti-TPO Abs during rhEPO treatment for anemia.