A novel avian influenza virus (A/wild duck/Korea/K102/2018) (H2N9) was isolated from wild birds in South Korea in 2018, and phylogenetic and molecular analyses were conducted on complete gene sequences obtained by next-generation sequencing. Phylogenetic analysis indicated that the hemagglutinin (HA) and neuraminidase (NA) genes of the A/wild duck/Korea/K102/2018 (H2N9) virus belonged to the Eurasian countries, whereas other internal genes (polymerase basic protein 1 (PB1), PB2, nucleoprotein (NP), polymerase acidic protein (PA), matrix protein (M), and non-structural protein (NS)) belonged to the East Asian countries. A monobasic amino acid (PQIEPR/GLF) at the HA cleavage site, E627 in the PB2 gene, and no deletion of the stalk region in the NA gene indicated that the A/wild duck/Korea/K102/2018 (H2N9) isolate was a typical low pathogenicity avian influenza (LPAI). Nucleotide sequence similarity analysis of HA revealed that the highest homology (98.34%) is to that of A/duck/Mongolia/482/2015 (H2N3), and amino acid sequence of NA was closely related to that of A/duck/Bangladesh/8987/2010 (H10N9) (96.45%). In contrast, internal genes showed homology higher than 98% compared to those of other isolates derived from duck and wild birds of China or Japan in 2016–2018. The newly isolated A/wild duck/Korea/K102/2018 (H2N9) strain is the first reported avian influenza virus in Korea, and may have evolved from multiple genotypes in wild birds and ducks in Mongolia, China, and Japan. Influenza viruses are classified based on 18 hemagglutinin (HA) and 11 neuraminidase (NA) surface proteins into a large variety of subtypes, some of which are a public health threat . The widThe prevalence of H2 subtype AIV is much lower than that of other subtypes such as H5, H7, and H9,  but H2 iBased on three patients infected by human H7N9, the constructed phylogenetic trees of NA genes showed H7N9, H2N9, or H11N9 as its origin. However, the Korean H7 subtype was suggested as the origin of highly pathogenic avian influenza (HPAI) H7N9 2013 . Therefore, continuous surveillance of H2 subtype AIV in Korea is imperative.To date, H2N9 has been absent in Korea but avian LPAI H7N9 has been recently detected in migratory birds ,16. FurtTherefore, continuous surveillance of H2N9 and LPAI H7N9 is highly recommended to predict the potential spread of HPAI H7N9 in Korea. Other subtypes , have been co-circulating in Korea ,19,20. TFor AIV surveillance in wild birds, AIV genomic analysis by next-generation sequencing (NGS) is ideal, as the mutation rate of influenza A viruses has been traditionally determined by sequencing different cDNA clones obtained from multiple plaques descending from a plaque-purified influenza A virus . In addiIn the present study, we describe the characterization of a H2N9 isolate from a wild duck in Korea for the first time. The complete genomic sequence of this isolate was obtained using NGS and its genetic/antigenic variation was analyzed.A total of 500 fresh fecal droppings of wild birds were sampled and pooled in groups of up to three using sterile swabs in Korea on December 11, 2018. The samples were stored at 2 to 8 °C and shipped to the laboratory within 12 h for further analysis.The fecal samples were resuspended in phosphate buffered saline (PBS) containing antibiotic solution (100 U/µL of penicillin and 100 mg/µL of streptomycin)  and clarified by centrifugation (3000 rpm for 10 min at 4 °C). Supernatants were filtered through a hydrophilic polyethersulfone membrane   to remove eukaryotic cells and bacteria. Processed samples were inoculated into the allantoic cavities of ten-day-old embryonated chicken eggs and incubated at 37 °C for 2–3 days. Allantoic fluids were harvested and viral titers were determined using hemagglutination assays as previously described .Viral RNA was extracted from allantoic fluid (640 hemagglutination units (HAU)/mL) using a total RNA extraction kit  according to the manufacturer’s instructions. Finally, RNA was eluted in 60 μL of RNase-free water, and after addition of 20 U of RNase inhibitor , was stored at −80 °C until further use. In the negative control, sterile water was added instead of the specimen.c oxidase I (COI) using COI sequences from all 260 bird species as previously described [To evaluate influenza growth and determine the subtype, conventional real-time RT-PCR for influenza A virus that amplifies the matrix gene coding sequence was performed using total RNA following the WHO guidelines . Host idescribed . The hos® . The cDNA library of influenza RNA was generated using QIAseq FX Single Cell RNA Library Kit . cDNA concentration was measured using LightCycle qPCR , and library size was checked using Agilent High Sensitivity D5000 ScreenTape System .NGS was conducted by GnCBio  using HiSeq X as previously reported ,27. BrieRaw sequence reads were quality-trimmed using Trim Galore!  (q = 20). Influenza and non-influenza viruses were classified by DeconSeq (iden = 60) using a database created by downloading influenza virus sequences from the National Center for Biotechnology Information (NCBI) databank. The homology of the segments 4 (HA), 5 (NA), and 8 (NS1), known to have high variability among influenza virus segments, was examined by Basic Local Alignment Search Tool for nucleotides (BLASTn) in the influenza virus database. Among the results, the most homologous and the most common sequences were selected as the reference of this study. Sequence reads classified as influenza virus were mapped to the selected reference sequence using the gsMapper program . Consensus sequences obtained by mapping were corrected for sequence errors using a proofread program and open reading frames (ORF) were predicted using ORFfinder (NCBI) .® (NCBI). Phylogenetic trees were generated by the neighbor-joining method using MEGA6 . Bootstrap values were calculated based on 1000 alignment replicates.The closest relatives of the viral genes sequenced in this study were identified using the BLAST function in GenBankVirus isolation was performed from fresh fecal samples of migratory birds in Korea, and an AIV was confirmed from a wild duck (A/wild duck/Korea/K102/2018 (H2N9)) isolate. This isolate was collected from wild bird feces in the Gyeongbuk area  on December 11, 2018. Anas platyrhynchos, A. platyrhynchos x A. crecca, A. poecilorhyncha, and Tadorna tadorna (The host was identified as  tadorna . Due to The H2N9 AIV from the wild duck samples (A/wild duck/Korea/K102/2018 (H2N9)) was isolated by inoculating the virus in embryonated chicken eggs, followed by hemagglutination assays (640 HAU/mL in stock) and subtype PCR. The alignment lengths for each small data set were PB2, 2280 nucleotides (nt); PB1, 2278 nt; PA, 2121 nt; HA, 1688 nt; NP, 1482 nt; NA, 1384 nt; M, 980 nt; and NS, 822 nt. In the sequence analysis, the number of AIV reads was 2,186,688, which accounted for 10.37% of the raw NGS data. Consensus contigs for eight segments of the AIV were obtained by data assembly and the error correction process. Investigation with ORFfinder (NCBI) confirmed that these contigs all have a complete ORF . DetailePhylogenic analysis for the eight genes of the AIV (A/wild duck/Korea/K102/2018) (H2N9) was performed to assess their genetic relationships with those of domestic poultry and wild birds in Korea and neighboring countries, using data from NCBI. All phylogenic analyses of each gene are shown in the separate The HA gene of this virus was similar to that of the H2N3 strain isolated in Mongolia (2015), whereas the NA gene of our isolate was similar to that of the H10N9 strain isolated in Bangladesh (2010). The viral genes PB2 and PA were 98.9% and 99.49% identical to the PB2 and PA genes of A/duck/Jiangsu/SE0261/2018 (H5N3). The remaining genes from our isolate  showed a close relationship with A/Duck/Dongting/D76-1/2016 (H5N7), A/duck/Hokkaido/X9/2016 (H8N4), A/duck/Chongqing/S4362/2017 (H5N3), and A/wild bird/Jiangxi/P419/2016 (H6N8), respectively. The percent sequence homology for each gene segment from A/wild duck/Korea/K102/2018 (H2N9) compared to the closest genetic relative is shown in The receptor-binding site (RBS) of HA was assessed by analyzing mutations that change host tropism from bird to human . The RBSThe HA cleavage site of the A/wild duck/Korea/K102/2018 (H2N9) virus contained the PQIEPR↓GLF (↓ denotes cleavage site) sequence, with a monobasic amino acid  in the HA cleavage site, a signature of LPAI . Comparing the receptor-binding sequence of the HA1 gene of our isolate with those of Hong Kong (H2N9) isolates and the human H2N2 isolate, A/Korea/426/1968(H2N2) showed that the amino acid residues at positions 138, 190, 194, and 225 (H3 numbering) are similar. However, the amino acid sequence of avian H2N9 isolates, including our strain, is different from that of human isolates at positions 226 and 228 (H3 numbering) because only human isolates (H2N2) exhibit different amino acids in 220 loop (residues 221 to 228) . AccordiThe NA stalk truncation of 20 amino acids increases virulence in mice and ferrets ,34,35. OHowever, other mutations at positions 26, 107, 144, 146, and 224 in the NA gene, which increases virulence in mice and mammals, were found in our isolate and the reference strains . InteresShanghai/2013(H7N9) NA gained a substitution of the amino acid at position 294 from R to K among four human H2N9 strains and our isolate showed K. This implies the potential susceptibility of our isolate to anti-influenza drugs. Mutation in the ribonucleoprotein complex  could have increased the replication efficiency of the virus in a different host. Mutations in the internal genes were compared with those of H2N9 in wild birds to assess new variations and notably different residues were identified, as shown in The PB1 protein residues D/A3V, V13P, R207K, and S375N/T are known to increase polymerase activity and virulence in mammals ,43,44,45Two substitutions in the NP protein, V41I and D210E, contribute to viral transmissibility in mammalian cells . MoreoveIn addition, similar to the 1918 H1N1 pandemic strain and avian H5N1 strain, our H2N9 isolate had a PDZ domain-binding at the C-terminus of NS1, reflecting a potential adaptation in mice. Mutated residues of NA and six internal genes are summarized in In wild birds, AIVs co-circulate and their reassortments can cause severe outbreaks in poultry and other animals by crossing the species barrier. HPAI evolve from LPAI, and one of most notable viral strains is Chinese H7N9 virus, which evolved from LPAIs  . The othThe peninsular Republic of Korea is crossed by the East Asian Australasian and West Pacific Flyways and is near the Central Asian Flyway. This creates diversity in migratory birds and in AIVs. In our study, phylogenetic analysis demonstrated that the HA and NA genes of A/wild duck/Korea/K102/2018 (H2N9) belonged to the Eurasian lineage, with HA and NA genes showing the closest relationship to A/duck/Mongolia/482/2015 (H2N3) and A/duck/Bangladesh/8987/2010 (H10N9), respectively. In contrast, the internal genes  were of East Asian lineage, with the six gene segments of our H2N9 isolate being most closely related to those in other countries such as China  and Japan (H8N6). Among the internal genes, only the PB1 gene of our H2N9 strain was grouped with Korean AIV strains (A/wild waterfowl/Korea/F14-5/2016(H6N1), A/wild waterfowl/Korea/F7-18/2018 (H4N8), and A/mallard/Korea/F94-16/2017 (H4N6). The remaining genes were absent from the Korea strain groups.R↓GLF  is typically activated by a trypsin-like serine protease and this requirement restricts the tropism of influenza viruses to the types of tissues that express the proteases for activation, indicating that this isolate is an LPAI with a monobasic cleavage motif [The monobasic cleavage motif PQIEPge motif .RRRKKR↓G of the HA belongs to the consensus site cleaved by furin, a ubiquitously expressed mammalian enzyme that allows viruses to multiply in mammals rapidly, leading to high pathogenicity of AIV [On the contrary, the multi-basic cleavage motif PQGEy of AIV ,54.R↓GLF in the cleavage site between HA1 and HA2 of A/wild duck/Korea/K102/2018 (H2N9) was not different from that of an H2N2 isolate from a human sample (A/Korea/426/1968) showing PQIESR↓GLF, a monobasic cleavage site. Human A/Korea/426/1968 may achieve adaptation in humans by substitution of different amino acids in the 220 loop  are highly conserved . In humaA deletion in the NA stalk region at position 69–73 that increases virulence in mice was not found in our strain and the Hong Kong strains, whereas this deletion is present in human strains (A/Shanghai/1/2013/H7N9 and A/Anhui/1/2013/H7N9). Interestingly, the mutations M26I, 107V, R144K, and N144S, which may increase virulence in mammals, were found in both our isolate and the reference strain of H2N9. The A/wild duck/Korea/K102/2018 (H2N9) harbored no mutation at position E627K of PB2, which plays an important role in the adaption of AIVs to mammals. These results imply that the avian A/wild duck/Korea/K102/2018 (H2N9) may not exhibit host tropism to humans.In contrast, other mutations that enhance polymerase activity and increase virulence in mammals were commonly present in both our H2N9 isolate and the references isolates. Consequently, the genotype of the A/wild duck/Korea/K102/2018 (H2N9) strain might be the result of reassortment events that occurred during wild bird migration.To date, H2N9 has never been discovered in Korea, and to the best of our knowledge, this is the first study to describe a reassortant avian H2N9 virus from wild duck fecal samples. Although H2N9 belongs to LPAI, it is still necessary to monitor its presence in Korea because it is known to be one of the multiple reassortments involved in the induction of HPAI H7N9 from LPAI H7N9 in humans .Although there was no outbreak of H7N9 in Korea, the avian LPAI H7N9 virus was introduced into Korea ,16. TherThere is growing evidence of AIV reassortment at stopover sites, where migratory flyways intersect and thus, the frequency of intercontinental reassortment is likely a reflection of overlapping migratory pathways ,59,60. IIn conclusion, LPAI A/wild duck/Korea/K102/2018 (H2N9) is the first reported isolate in Korea and may contain properties that would enhance virulence in the mammalian host. Owing to potential reassortments between H2N9 and other pre-existing viral subtypes in Korea, surveillance and characterization of all these viral subtypes is essential for management of AIV health risk in the early stages. Therefore, continuous surveillance and genetic analysis to characterize LPAI viruses, including the H7, H9, H11, and H2 subtypes of AIV are strongly encouraged.


Trypanosoma cruzi populations than in their respective susceptible counterparts (BZS and 17WTS). To confirm the role of eIF5A in BZ resistance, we transfected BZS and 17WTS with the wild-type eIF5A or mutant eIF5A-S2A . Upon overexpressing eIF5A, both susceptible lines became approximately 3- and 5-fold more sensitive to BZ. In contrast, the eIF5A-S2A mutant did not alter its susceptibility to BZ. These data suggest that BZ resistance might arise from either decreasing the translation of proteins that require eIF5A, or as a consequence of differential levels of precursors for the hypusination reactions , both of which alter BZ’s effects in the parasite.Eukaryotic initiation factor 5A (eIF5A) is a conserved protein with an essential role in translation elongation. Using one and two-dimensional western blotting, we showed that the eIF5A protein level was 2-fold lower in benznidazole (BZ)-resistant (BZR and 17LER)  Trypanosoma cruzi is the etiological agent of Chagas disease (American trypanosomiasis), which affects 6 to 7 million people worldwide, mainly in Latin America.T. cruzi. BZ exerts its action via reductive stress, which causes deleterious effects on the DNA, proteins, and lipids of the parasite.T. cruzi clones showed that BZ metabolites have high mutagenic activity.The protozoan parasite T. cruzi are still poorly understood, demonstrating the need of selecting new targets for Chagas disease chemotherapy. Several studies have identified different proteins, such as old yellow enzyme (NADPH dehydrogenase), iron-superoxide dismutase, tryparedoxin peroxidase, ascorbate peroxidase, hexose transporter, cyclophilin, lipoamide dehydrogenase, and aldo-keto reductase, which may contribute to BZ-resistance mechanisms in T. cruzi.-Drug resistance is a serious public health problem in many countries. NFX and BZ resistance mechanisms in eIF5A is a highly conserved protein in eukaryotic organisms from archaebacteria to mammals. This factor participates in apoptosis, cell cycle control, and RNA decay. It was initially identified as a translation initiation factor; however, more recent studies revealed its role in translation elongation and termination.Trypanosoma brucei growth, possibly by preventing the synthesis of flagellar proteins, which are rich in polyproline residues.T. cruzi and are therefore rate limiting for parasite growth and survival, including their incorporation in trypanothione for redox metabolism and for the hypusination of eIF5A for translation activity.In trypanosomes, eIF5A is essential for T. cruzi populations selected by gradual exposure to BZ.,T. cruzi populations.Therefore, in the present study, we determined whether the expression levels of eIF5A are related to the susceptibility to BZ of Homo sapiens eIF5A sequence, showed a high degree of conservation . The evolutionary relationships among eIF5A sequences were inferred using the maximum likelihood method in PhyMLH. sapiens. Despite the high similarity among all eIF5A trypanosomatids sequences, the tree topology clearly separated Leishmania from Trypanosoma species.eIF5A amino acid sequences are relatively conserved among trypanosomatids, with few modifications. An amino acid sequence alignment performed using an online version of MAFFT,T. cruzi population selected in vivo for BZ resistance (BZR), which was derived from the Y strain, and its susceptible counterpart (BZS).T. cruzi epimastigote forms. A population with in vitro-induced BZ resistance (17LER) derived from the Tehuantepec cl2 strain and its susceptible counterpart (17WTS)In the present study, we used a 9 parasites) were lysed in buffer containing 8 M urea, 2 M thiourea, 4% CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate), 50 mM dithiothreitol, 20 mM Tris-HCl, pH 7.4, and Complete Mini Protease Inhibitor Cocktail . After centrifugation at 20000 × g for 1 h, the supernatants (protein extracts) were quantified using the Bradford method and subjected to western blotting analysis using a specific antibody against T. cruzi eIF5A (anti-TceIF5A).T. cruzi samples analysed (T. cruzi heat shock protein 70), used as a normalisation antibody for the western blotting. Densitometry analysis showed that the eIF5A protein level was 2-fold lower in BZR and 17LER compared with that in their respective susceptible counterparts, BZS and 17WTS.Epimastigotes in the exponential growth phase  using the protocol described by Rêgo et al.T. cruzi eIF5A (TceIF5A) in resistance to BZ, 1 x 108 parasites from the BZ-susceptible populations (BZS and 17WTS) were transfected with 100 μg of the plasmids p33-TceIF5A (expressing the wild-type TceIF5A) or p33-TceIF5A-S2A .T. cruzi transfected with plasmids p33-TceIF5A or p33-TceIF5A-S2A mutant. This could be a consequence of the overexpression of eIF5A protein in these samples or related to posttranslational modifications of eIF5A  or transfected with the plasmids p33-TceIF5A (BZS/17WTS eIF5A) or p33-TceIF5A-S2A (BZS/17WTS eIF5A-S2A mutant) were subjected to BZ susceptibility assays. Parasites were incubated in LIT medium at 2 × 106 cells/mL in 24-well plates in the absence or presence of increasing BZ concentrations (2.5 to 20 μM) for 96 h. The effective concentration necessary to decrease growth by 50% (EC50) was determined using a model Z1 Coulter Counter . EC50 values were obtained from three independent measurements in triplicate, using the linear interpolation method. The results showed that the BZ EC50 of untransfected BZS and 17WTS T. cruzi populations was 25 and 20 μM, respectively  than in their respective susceptible counterparts BZS and 17WTS. In addition, the overexpression of eIF5A made these T. cruzi populations more susceptible to BZ. Importantly, overexpression of the eIF5A-S2A mutant did not alter the susceptibility these parasites to BZ, indicating that the phosphorylation of the serine residue modulates eIF5A activity, favouring the BZ-susceptibility phenotype. Chung et al.Our results showed that the protein levels of eIF5A were 2-fold lower in benznidazole-resistant Leishmania braziliensis demonstrated that the elongation factor 2 (EF2) presented lower abundance in antimony-resistant L. braziliensis samples than in the susceptible samples,Our previous phosphoproteomic study in Alternatively, it is important to consider that the amount of hypusinated eIF5A protein could be directly associated with the levels of polyamines and consequently of trypanothione, a key component in the detoxification BZ.T. cruzi populations, indicating the multiplicity of mechanisms that contribute to the drug resistance phenotype.,,T. cruzi through the participation of eIF5A in the benznidazole-susceptibility mechanism.Importantly, several genes are involved in and are differentially expressed during BZ-selected and BZ-natural resistance in


Stimulated by retinoic acid gene 8 (Stra8), which is required for entry of germ cells into meiotic prophase I. Canonically, the biological activities of RA are mediated by nuclear RA receptors. Recent studies in somatic cells found that RA noncanonically stimulates intracellular signal transduction pathways to regulate multiple cellular processes. In this study, using a germ cell culture system, we investigated (1) whether RA treatment activates any mitogen-activated protein kinase (MAPK) pathways in fetal germ cells at the time of sex differentiation, and (2) if this is the case, whether the corresponding RA-stimulated signaling pathway regulates Stra8 expression in fetal germ cells and their entry into meiosis. When XX germ cells at embryonic day (E) 12.5 were cultured with RA, the extracellular-signal-regulated kinase (ERK) 1/2 pathway was predominantly activated. MEK1/2 inhibitor (U0126) treatment suppressed the mRNA expressions of RA-induced Stra8 and meiotic marker genes  in both XX and XY fetal germ cells. Furthermore, U0126 treatment dramatically reduced STRA8 protein levels and numbers of meiotic cells among cultured XX and XY fetal germ cells even in the presence of RA. Taken together, our results suggest the novel concept that the RA functions by stimulating the ERK1/2 pathway and that this activity is critical for Stra8 expression and meiotic progression in fetal germ cells.In murine fetal germ cells, retinoic acid (RA) is an extrinsic cue for meiotic initiation that stimulates transcriptional activation of the  Primordial germ cells (PGCs) are the embryonic precursors of oogonia and prospermatogonia in mammals. In mouse fetuses, early-stage PGCs continue to proliferate mitotically and migrate through the somatic tissues to eventually colonize the gonads at approximately embryonic day (E) 10.5. In the gonads, fetal germ cells are induced to undergo sex differentiation depending on the somatic gonadal environment rather than on their sex chromosome constitution. A common feature of female-specific sex differentiation in developing ovaries is entry into meiosis. Thus, in an ovarian environment, XX germ cells immediately enter meiotic prophase I at E12.5–13.5 and proceed to the diplotene stage by E17.5 . After btrans retinoic acid (RA) has been widely recognized as a key regulator of entry into meiosis in both male and female germ cells . Si. Si49]. Stra8 and initiate meiosis [We have previously reported that XY germ cells isolated at E13.5 autonomously progress through the male pathway in culture without somatic support . Our preNext, we examined the direct interaction between RA-stimulated ERK1/2 activity and STRA8 expression in XY germ cells. XY germ cells at E13.5 were cultured for 48h under the same four basic conditions shown in Stra8 and four meiotic marker  transcript levels in E13.5 XY germ cells cultured for 48 or 72h under the four different conditions (Stra8 transcription to 5.4x higher than the control (p < 0.001). However, U0126 significantly suppressed RA-induced Stra8 transcript levels (to approximately 50% of that under the RA condition) (p < 0.05). U0126 treatment suppressed Stra8 expression relative compared to the control germ cells (p > 0.05). After 72h of culture, RA treatment maintained a similar Stra8 transcript level to 48h. RA-induced Stra8 expression was also significantly diminished by the U0126 treatment (RA+U0126). In contrast, the expression levels of meiotic marker genes at 48h were only slightly elevated in the presence of RA, and there was no significant difference compared to their controls, respectively  (p < 0.05–0.001). Importantly, U0126 treatment dramatically suppressed all marker gene expressions induced by RA compared to the controls (p < 0.01–0.001). On the contrary, Mvh expression under the four culture conditions was very similar at 48h (p > 0.05) (Stra8 and meiotic marker genes in XY germ cells.We next examined nditions . After 4ectively . After 7 > 0.05) . At 72h, > 0.05) . These dvs. control). These data confirm that the RA-stimulated ERK1/2 pathway regulates RA-stimulated STRA8 expression in E13.5 XY germ cells in culture.We further tested whether the RA-stimulated ERK1/2 pathway regulates STRA8 protein expression and entry into meiosis in E13.5 XY germ cells . Sorted To determine if the RA-stimulated ERK1/2 pathway impacts meiotic initiation in XY germ cells, E13.5 XY germ cells were cultured under the same four conditions. After culture, samples of 200–300 XY germ cells were collected and immunostained with γH2AX antibody  to deterStra8 expression and entry into meiosis in fetal germ cells. We found that RA treatment selectively stimulates the ERK1/2 pathway in XX germ cells at E12.5. We further demonstrated that RA-induced Stra8 expression (at both the mRNA and protein levels) and several meiotic marker expressions were significantly suppressed by inhibiting the RA-stimulated ERK1/2 pathway in cultured germ cells, regardless of their sexual phenotype. Our findings suggest a new concept that Stra8 expression may be controlled not only by the canonical activity of RA via nuclear RARs, but also by the noncanonical activity of RA via the ERK1/2 pathway in fetal germ cells.Recent studies in somatic cells have proposed that RA noncanonically activates intracellular signal transduction pathways to regulate transcription of RA target genes ,50,51. Ic-fos gene expressions to induce neuronal differentiation [c-fos and c-jun [Our initial interest was whether the noncanonical route of RA activity originally evidenced by somatic cell studies is also utilized in fetal germ cells for meiotic initiation or not. Interestingly, treatment with RA predominantly enhanced the ERK1/2 pathway, but not the p38 MAPK, JNK or AKT pathways in E12.5 XX germ cells in culture. Recent studies in the somatic cells have revealed a novel biological activity of RA via signal transduction pathways, suggesting that RA induces the selective activation of multiple signal transduction pathways ,29. In pntiation . A previntiation . In A126nd c-jun . It was nd c-jun . In the nd c-jun . Importand c-jun . Our preStra8 expression (both at the mRNA and protein levels) in E12.5 XX germ cells and E13.5 XY germ cells in culture. Thus, our results are consistent with the novel idea that the RA-stimulated ERK1/2 pathway contributes to Stra8 expression in fetal germ cells. Using postnatal mouse spermatogonia, Pellegrini et al. (2008) determined that both RA and Kit Ligand (KL) increased the mRNA expression of Stra8 and Dmc1 [Stra8 expression, their study strongly supports the idea that noncanonical activity of RA controls meiotic initiation. Very recently, it was reported that inactivation of Wdr62, which encodes a scaffold protein in the JNK signaling pathway, caused meiotic initiation defects in mice [Stra8 and meiotic marker expressions, suggesting Wdr62 is involved in RA-induced meiotic initiation via activating JNK pathway [Stra8 contains two putative RAREs (DR2 and IR5) within a 400 bp promoter region upstream from the transcription start site [Stra8 transcriptional expression [Stra8 promoter region in the postnatal testes at 8 dpp [Stra8 expression is canonically mediated by nuclear RARs in germ cells. In the fetal ovary at E13.5, in contrast, a similar ChIP study reported that the binding affinity of RARs  to RAREs in the Stra8 promoter is either very low or not detectable [Stra8 and meiotic initiation in isolated germ cells and gonadal germ cells in vitro [Stra8 expression. Actually, previous studies in somatic cells have suggested that expression of RA target genes is regulated by RA-stimulated signal transduction cascades. Thus, in both mouse embryonic fibroblasts and human breast cancer cells, RA treatment activates the p38 MAPK pathway to enhance transcription of Cyp26A1, one of the RA target genes [RARβ2, which may be involved in epigenetic events, such as chromatin modification and remodeling [Stra8 transcriptional activation in female gonads [Stra8 transcription was prematurely activated at E11.5, and meiotic-related genes were expressed earlier than usual. These authors speculated that PRC1 maintains repression of Stra8 and early meiosis programs until RA signaling has reached a certain threshold in female gonads [Stra8 expression appears to be epigenetically regulated via histone modifications and chromatin accessibility, which may be associated with the function of RA-induced signal transduction cascades.We found that the ERK phosphorylation inhibitor U0126) effectively suppressed RA-induced and Dmc1 . By inhiand Dmc1 . Althoug in mice . The fet pathway . This is pathway ,57. As a effectivpression . Using aat 8 dpp . These rtectable . Howeverin vitro ,43. Baseet genes . In embrmodeling . The molmodeling ,35,58–60modeling ,61. PRCsmodeling . Therefoe gonads . In PRC1e gonads . In concStra8 expression at both the mRNA and protein levels, resulting in regulating entry into meiosis. Thus, we propose the novel concept that RA signaling functions via an alternate signal transduction cascade to control Stra8 expression and meiosis, independent of the canonical pathway mediated by nuclear RARs Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S4 Table(PDF)Click here for additional data file.S5 Table(PDF)Click here for additional data file.S6 Table(PDF)Click here for additional data file.S7 Table(PDF)Click here for additional data file.S8 Table(PDF)Click here for additional data file.S9 Table(PDF)Click here for additional data file.S10 Table(PDF)Click here for additional data file.S11 Table(PDF)Click here for additional data file.S12 Table(PDF)Click here for additional data file.S13 Table(PDF)Click here for additional data file.S14 Table(PDF)Click here for additional data file.S15 Tableβ-ACTIN-normalized phospho/total protein ratio was set as the O.D. value of each phosphorylated protein. The O.D. value of STRA8 was determined by normalizing to the β-ACTIN. The data represent the mean ± SD of two independent WB experiments.For signal transduction pathways, the (PDF)Click here for additional data file.S1 FigTo evaluate the effect of U0126 on the ERK1/2 phosphorylation, XX germ cells at E12.5 were cultured with a MEK inhibitor (U0126) at different concentrations  for 1h. After culture, germ cells were subjected to Western blotting to quantify the ERK1/2 phosphorylation. The levels of protein bands were quantified by densitometry and represented on the top of each band as numerical values that calculated relative to the control set as 1.0.(PDF)Click here for additional data file.S2 Figmouse Vasa homolog (Mvh). Results were normalized to the β -actin transcript expression. All expression values were calculated relative to control levels set at 1.0. Data represent the mean ± SEM (n = 3). (B) Sorted XX germ cells at E12.5 were cultured under four different conditions  for 24 and 48h. After culture, the cells were subjected to qPCR analysis for Mvh. Each gene expressions were normalized to the β-actin expression. All expression values were calculated relative to control levels set at 1.0. Data represent the mean ± SEM (n = 4).(A) Female gonads at E12.5 were cultured with 50 μM U0126 (U0126) or without treatment (control) for 24 or 48h. After culture, germ cells were collected to analysis the transcript expressions of (PDF)Click here for additional data file.S3 FigMvh. The expression levels were normalized to β -actin mRNA expression. All expression values were calculated relative to control levels set at 1.0. Data represent the mean ± SEM (n = 3–4). # p < 0.05 vs. RA.Isolated E13.5 XY germ cells were cultured under four different conditions  for 48 and 72h. After culture, the cells were subjected to qPCR analysis to determine the transcript levels of (PDF)Click here for additional data file.


Microarray data were validated by quantitative PCR, confirming the differential expression of 12 known AIRE-regulated genes. Comparison of AIRE-dependent differential expression in our cell line model with murine datasets identified 447 conserved genes with a number of transcription regulatory interactions, forming several key nodes, including STAT1, which had over 30 interactions with other AIRE-regulated genes. As STAT1 mutations cause dominant chronic mucocutaneous candidiasis and decreased STAT1 levels in monocytes of autoimmune polyglandular syndrome 1 (APS-1) patients, it was important to further characterize AIRE–STAT1 interactions. TEC 1A3AIREhi were treated with the STAT1 phosphorylation inhibitors fludarabine and LLL3 showed that phosphorylated STAT1 (p-STAT1) was not responsible for any of the observed differential expression. Moreover, treatment of TEC 1A3 AIREhi with STAT1 shRNA did not induce any significant variation in the expression of unphosphorylated STAT1 (U-STAT1) downstream genes, suggesting that these genes were directly regulated by AIRE but not via U-STAT1. The novel model system we have developed provides potential opportunities for further analysis of the pathogenesis of (APS-1) and the wider roles of the AIRE gene.Autoimmune regulator (AIRE) regulates promiscuous expression of tissue-restricted antigens in medullary epithelial cells (mTEC) of the thymus. To understand the diverse effects of AIRE, it is crucial to elucidate the molecular mechanisms underlying the process of AIRE-regulated gene expression. In this study, we generated a recombinant AIRE expression variant of the TEC 1A3 human cell line, TEC 1A3 AIRE APS-1 iific TSA , 18. Amoific TSA . For insific TSA  and calcific TSA . NLRP5 iific TSA . Autoantific TSA . Autoantific TSA . These aific TSA , 24. In ific TSA . T1D occpatients , 27. APSpatients . Interstpatients , 30. APSpatients . The trapatients . Hedstrapatients  reportedAire knockout mice, we have been able to explore the biological effects of Aire at the systemic, tissue, and cellular level and the thymi from these murine models have been used to explore the biochemical properties of Aire, particularly for determining the downstream targets of aire-regulated gene expression (ARGE)  , 34–39. n origin . Our aiml-glutamine (Lonza) supplemented with 10% fetal calf serum (Lonza). Medium for cell line variants were supplemented with either zeocin or hygromycin B, dependent on the cell line variant.The TEC 1A3 cell line was originally established from fetal and postnatal human thymus tissue by Toribio’s group in 1994 . TEC 1A3LacZeo vector following the flp-in system manual . In brief, this process involved the transfection of TEC 1A3 cells with a pFRT/LacZeo vector using FuGENE6 reagent (Promega). Cells were grown in media supplemented with 250 µg/ml zeocin to select for those containing the pFRT/LacZeo vector integrated into the genome, with cloning rings used to isolate foci to collect individual cell lines. Southern blotting was used to screen the different cell lines to identify those containing only a single integrant of the pFRT/LacZeo vector. Relative expression efficiency from the FRT region for each TEC 1A3/pFRT/LacZeo cell line generated was estimated by comparing β-galactosidase activity using the β-gal assay kit (Invitrogen). The cell line with the highest β-galactosidase activity was chosen to be the TEC 1A3 flp-In host cell line for subsequent reactions.A TEC 1A3 cell line containing a single FRT (Flp recombinase target site) was generated using the pFRT/Autoimmune regulator cDNA was PCR amplified from a pET31/rAIRE expression vector  using the primers AAAAAAGCTAGCCGCCACCATGGCGACGGACGCGGCGCTA and GCTACAGGGCCCTCAATGATGATGATGATGATG.hi variants).The amplified cDNA was inserted into the multiple-cloning site of the pcDNA5/FRT vector using the NheI and ApaI restriction sites, generating the pcDNA5/FRT/rAIRE expression vector. Both the TEC 1A3 flp-in host cells and T-REx 293 cells were seeded into 6-well plates to reach 60–70% confluence after 24 h. The cells were co-transfected with 1 µg plasmid DNA (the Flp recombinase expression vector pOG44 with the pcDNA5/FRT/rAIRE expression vector at 20:1 ratio) using 3 µl FuGENE6 reagent (Promega) per transfection. After 24 h, cells were washed with PBS then maintained in fresh media supplemented with hygromycin B at 150 µg/ml to select for cells containing integrated pcDNA5/FRT/rAIRE expression vector (AIRECell pellets were resuspended in 2× loading buffer  and proteins separated by SDS-PAGE . After electrophoresis, gel was transferred to PVDF membrane using iblot system (Invitrogen). Primary antibodies used were 1:1,000 polyclonal goat anti-aire-1 (D-17) IgG  and 1:500 polyclonal goat anti-actin (I-19) IgG . The secondary antibody used was 1:2,500 polyclonal rabbit anti-goat IgG conjugated with peroxidase , visualized using EZ-ECL chemiluminescent reagent . The PhosphoPlus Stat1 (Tyr701) antibody kit was used to detect STAT1 and p-STAT1 (Tyr701) in samples.lo cells and three separate cultures of TEC 1A3 AIREhi cells using the SV Total RNA isolation kit (Promega). The quality and quantity of the RNA from all samples was assessed . Linear amplification of each RNA sample was undertaken following the Eberwine procedure (® 3′ IVT Express Kit (Affymetrix), and checked again for quality and quantity. Fifteen microgram of amplified cRNA from the three TEC 1A3 AIRElo cells and three TEC 1A3 AIREhi cells were fragmented and each hybridized individually to a separate Human Genome U133 Plus 2.0 GeneChip (Affymetrix), as specific in manufacturer’s protocols, and subsequently underwent washes. Chips were then stained, scanned, and signal intensities for each transcript produced using GeneChip Operating Software. These data (uploaded to GSE 35244) and data generated from wild-type and rAire+ HEK 293 cells [GSE24733  <5%] to determine genes that showed significant differential expression in the AIREhi (rAire+ in HEK) cells compared to AIRElo (Aire−/wild-type in HEK) cells.RNA was extracted from cell pellets from three separate cultures of TEC 1A3 AIRErocedure  using thGSE24733 ] were anlo and TEC 1A3 AIREhi cells used for microarray analysis was carried out the same way.RNA was extracted from cells using trizol and purified from the trizol-cell lysate using the Direct-Zol miniprep kit (Zymo Research), before being used for cDNA synthesis using the Superscipt III First-Strand Synthesis System (Invitrogen) with random hexamer primers. cDNA synthesis for all three RNA samples from TEC 1A3 AIREPower SYBR® Green PCR (Applied Biosystems) and each of the primer pairs listed in Table t values determined for the other genes tested. The ΔΔCt method was used to calculate the change in relative expression for each gene between AIRElo and AIREhi cells, or between untreated and treated AIREhi cells.Triplicate QPCR reactions were carried out for each sample using RT-PCR was carried out using AmpliTaq Gold 360 (Applied Biosystems) under standard reaction conditions using 1 µl human adult thymus cDNA  and primers at a final concentration of 0.5 µM. The standard PCR program from the AmpliTaq Gold 360 product manual was used, with an annealing temperature of 60°C and 38 cycles. Gene-specific primer sequences are given in Table Genespring GX v11.0 (Agilent Technologies) was again used to analyze data generated on medullary thymic epithelial cells from wild-type and Aire knockout mice of the C57BL/6, BALB/c, and NOD strains [GSE8564  and GSE1t-test (P ≤ 0.05) to determine genes that showed significant differential expression in the AIREhi (wild-type mice) cells compared to AIRElo (knockout mice) cells. Genespring GX was used to compare individually each analyzed murine dataset with our analyzed TEC 1A3 data set to determine gene conservation across two data sets. Individual conserved gene lists were exported, compiled, and filtered to show only genes with differential expression reported for the human dataset and at least three of the four murine datasets, with each reported gene showing a fold-change ≥ 1.2 for probes from at least three different datasets.These datasets, along with our normalized TEC1A3 cell line dataset (GSE35244), were then analyzed by Student’s The lists of AIRE-regulated genes from both our TEC 1A3 data set and from the conserved gene list were analyzed by DAVIDThe STAT1 phosphorylation inhibitors fludarabine phosphate (Sigma) and LLL3, a kind gift from Dr. Pui-Kai Li, College of Pharmacy, Ohio State University, were dissolved in DMSO and used to supplement cell culture media to the final given concentrations.Two 21-nucleotide sequences targeting human STAT1  were subcloned in the pLVTHM genome vector (Addgene plasmid 12247) according to the manufacturer’s protocol. Briefly, siSTAT1-c48 sense oligonucleotide 5′-CGCGTCCCCGCAGGTTCACCAGCTTTATGATTCAAGAGATCATAAAGCTGGTGAACCTGCTTTTTGGAAAT-3′ and siSTAT1-c48 antisense oligonucleotide 5′-CGATTTCCAAAAAGCAGGTTCACCAGCTTTATGATCTCTTGAATCATAAAGCTGGTGAACCTGCGGGGA were annealed, and siSTAT1-c660 sense oligonucleotide 5′-CGCGTCCCCGCTGAATGTCACTGAACTTACTTCAAGAGAGTAAGTTCAGTGACATTCAGCTTTTTGGAAAT-3′ and siSTAT1-c660 antisense oligonucleotide 5′-CGATTTCCAAAAAGCTGAATGTCACTGAACTTACTCTCTTGAAGTAAGTTCAGTGACATTCAGCGGGGA were also annealed, and each set of annealed oligonucleotides separately cloned into the MluI/ClaI digested vector.This approach allowed generation of a stem-loop-stem small hairpin RNA to effectively reduce PTEN expression levels . Self-inhi cells were transduced with either lentiviral vectors carrying the STAT1 knockdown small interfering RNA (multiplicity of infection = 100) or the scrambled small interfering RNA (multiplicity of infection = 100). Transduced cells were cultured for 7 days, before being harvested for analysis by QPCR and western blotting.TEC 1A3 AIRELacZeo vector and subsequent antibiotic selection using zeocin of those which had the vector integrated into their genome (data not shown)  and a Flp recombinase expression vector (pOG44). Antibiotic selection conditions were again used to select for successful recombinants, using hygromycin B instead of zeocin, with initial AIRE expression in these cell lines determined by western blotting  and the rAIRE expression TEC 1A3 cell line (AIREhi).Native AIRE expression in the TEC 1A3 cell line was found to be too low for reliable measurement when tested using western blotting and quantitative PCR (QPCR) (data not shown). Therefore, AIRE knockdown was excluded as an appropriate experimental option and a rAIRE expression variant of this cell line model was generated instead. The flp-in system (Invitrogen) of site-specific recombination was used to integrate a rAIRE expression vector into the genome of the TEC 1A3 cell line to minimize experimental variation, which could result from transient transfection  . This fig Figure A and QPCg Figure B. Subseqlo and TEC 1A3 AIREhi cell lines was determined using the Affymetrix Human Genome U133 Plus 2.0 array. To compare our data with previously published murine data, we applied stringent conditions with Benjamini–Hochberg correction, FDR < 5%, and fold-change ≥ 1.2 to determine gene-differentially expressed with P ≤ 0.05. There were 555 probes differentially expressed  that did not have a corresponding gene annotated. Differential expression was identified on several other probes that corresponded to the same gene. Overall, this data set indicates differential expression for 482 different genes, with 353 upregulated and 129 downregulated in response to AIRE expression. This represents a larger repertoire, with a higher stringency of ARGs validation than has been previously reported using a human model (Table CCL5) and downregulated gene (MAGEB2), alongside AIRE and a selection of the genes found to be expressed in an AIRE/AIRE-dependent manner in this study and at least one other study. Thymic expression was also confirmed for most of these genes by RT-PCR using commercially available human thymic cDNA  were also differentially expressed in two murine data sets .The transcriptional profile of the TEC 1A3 AIREel Table , 48, 49.lo cell line variant  and, ignoring outliers, the expression values are positively skewed (skewness = 4.25), whereas the downregulated genes were not skewed as heavily (skewness = −1.492). The positively skewed distribution of expression values for AIRE upregulated genes in the AIRElo cell line suggests that AIRE preferentially upregulates expression for genes that would otherwise be expressed at more basal levels (“inactive” genes), rather than those whose expression is already elevated (“active” genes). Subsequent analysis using the DAVID bioinformatics resource , suggesting that specific pathways are not targeted by AIRE/ARGE.The relative fold change in expression for our dataset, when compared against the expression levels in the AIREt Figure , shows tresource  highlighAire knockout mice on the B6, Balb/c, and NOD genetic backgrounds. These four different murine data sets  were then individually compared to the repertoire of probes showing AIRE-dependent differential expression in our TEC 1A3 cell line model . This enabled us to determine which of the identified human probes had at least one murine probe corresponding to a homologous gene also showing Aire-dependent differential expression. The four separate comparisons were then compiled, revealing over 2,000 human probes with a corresponding murine probe in at least one of the murine data-sets (P < 0.0095), but more importantly, we identified a conserved list of 428 different human genes, which were found to have AIRE-dependent differential expression in our TEC 1A3 data-set, had a homologous gene in at least three of the murine data sets and fold-change ≥ 1.2 for probes from at least three of the data-sets mutations also develop CMC and autoimmunity  still has transcriptional regulatory activity, albeit affecting a lower number of genes than its phosphorylated form. We, therefore, tested for STAT1 phosphorylation in our model and whether inhibition of STAT1 phosphorylation or knock down of STAT1 expression affected the expression of conserved ARGs that were linked to the STAT1 node in the EdgeExpress analysis (STAT1 edges). Most of the STAT1 molecules detected in TEC1 A3 were unphosphorylated. Cells treated with inhibitors of STAT1 phosphorylation, fludarabine, or LLL3, showed no difference in STAT1 between treated and untreated cells, nor in intracellular STAT1 phosphorylation status, as most STAT1 detected was unphosphorylated. The absence of phosphorylated STAT1 (p-STAT1) in TEC 1A3 cells was also observed in HaCat and Jurkat cells as well as in normal skin , suggesting that p-STAT1 is not present in normal physiological conditions and the phosphorylation of STAT1 is associated with the reaction to inflammation.via upregulated expression of an intermediate transcription factor. Surprisingly, DUSP4, which is a conserved gene downregulated by AIRE and reported to interact functionally with STAT1 (We knocked down the expression of STAT1 using two different lentivirus delivered anti-STAT1 shRNAs (shRNAc48 and shRNAc660) as well as scrambled shRNA as a negative control. We used QPCR to determine the change in relative expression level of STAT1, and when normalized to β-Actin expression, STAT1 shRNAc660 reduced the expression of STAT1 by more than fourfold with a lesser reduction in STAT1 expression detected when cells were transduced with STAT1 shRNAc48. Western blot analysis confirmed this reduction in STAT1 expression with both anti-STAT1 shRNAs whereas scrambled STAT1 shRNA showed no reduction of STAT1 protein compared to untreated cells (data not shown). Thus, we used STAT1 shRNAc660 to analyze the regulation of STAT1 downstream genes. Knowing that U-STAT1 is the main form of STAT1 present in our TEC 1A3 model, we analyzed the expression of a set of genes previously shown to be regulated by U-STAT1, including the AIRE-upregulated IFIT1, IFIT3, IFI44, IRF7, NIPA1, OAS1, and OAS2 , 60 as wth STAT1 , showed To test this hypothesis, we selected 18 genes known to be downregulated by AIRE and STAT1. All these were among the conserved ARGs except CAV2 and VCL which were found among ARG in TEC1 A3 only. The knockdown of STAT1 revealed a mixed pattern. There were genes whose expression had been greatly affected and, therefore, seem dependent on U-STAT1, such as CC chemokine-binding protein 2 (CCBP2), growth arrest-specific protein 7 (GAS7), and sorting nexin 10 (SNX10) with an increase of 1.8, 2.0, and 3.8 fold, respectively. In contrast, expression of the other genes tested were either unchanged ; slightly increased (REPS2); or slightly decreased . This suggests that the regulation of these genes is either completely or relatively independent of U-STAT1 (Figure hi. This is not surprising; like in TEC 1A3, most of genes encoding APS-1 antigens were not differentially expressed in APS-1 mouse models (Our cell line model has shown that AIRE up- and downregulates the expression of a number of genes in a ratio of approximately 2:1, as previously observed analysis of ARGE using microarrays , 38, 39.e models , 34–39. e models . This vae models .hi cells (Another limitation of our model is the number of AIRE-regulated genes. More than 2,300 genes have been shown to be upregulated over twofold in mTEChi cells , but onlSTAT1 GOF mutations also develop CMC and autoimmunity (via the STAT1 node indirectly to affect the differential expression of the non–node genes downstream in the proposed network. This concurs with the results of Danso-Abeam et al. (Patients with immunity , 57. Theimmunity . In contimmunity . Therefom et al.  who alsom et al.  has beenm et al. . It is ivia intermediate transcription factors according to the hierarchical transcription factor model to regulate the thousands of genes promiscuously expressed as a result of AIRE activity. However, as the range of ARGs has been found to differ across individual mTECs (via another functionally related molecule. Such a relationship is not clear for other genes, including SNX10 with expression levels approximately fourfold greater when STAT1 was knocked down. SNX10 is believed to be a regulator of macrophage polarization (via an intermediate transcription factor node in the proposed network. Interestingly, a recent study showed that APS-1 patients have 20% decrease of total STAT1 protein in human monocytes (Evidence is beginning to accumulate, that when looking at global ARGE patterns from pooled cells, AIRE does not operate al mTECs , it is pal mTECs . This isrization . SNX10 hrization . CCBP2 arization  whereas rization . Little onocytes . Our finonocytes , as wellonocytes .hi compared to APS-1 mouse models, the system we have presented here is robust and offers a new tool to dissect the molecular mechanism underlying ARGE.Despite the relatively modest number of AIRE-regulated genes detected in TEC 1A3 AIREConceived and designed the experiments: TL, MA, and RT-A. Performed the experiments: TL. Analyzed the data: TL and RT-A. Contributed reagents/materials/analysis tools and wrote the paper: TL, AGM, AJM, MA, and RT-A. Designed the study and put experimental plan for it: TL and RT-A.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Triticum aestivum L. chromosomes involved in early responses to short-term water-deficit stress . In the present study, intervarietal substitution lines of drought-tolerant 'Saratovskaya 29' and sensitive 'Janetzkis Probat' wheat cultivars were examined. We studied the biochemical plant response system and conducted an analysis of catalase, ascorbate peroxidase and guaiacol peroxidase activities, levels of lipid peroxidation and changes in relative water content. Our results determined that the first reaction was a significant increase in guaiacol peroxidase (GPX) activity. However, the strongest impact on plant responses was found for catalase (CAT), which caused a significant decrease in lipid peroxidation (LPO) levels. Our findings indicate that chromosomes 5A, 4B, 6B and 7D are associated with early responses to short-term osmotic stress in wheat.Water deficit induces reactive oxygen species (ROS) overproduction, which in turn inhibits plant growth and development. High concentrations of ROS disrupt the osmotic balance in plant cells and alter membrane integrity. Chromosomes carrying structural or regulatory genes must be detected to better understand plant response mechanisms to stress. The aim of our study was to identify  Triticum aestivum L.) is widely cultivated in every geographical region in the world next to rice and maize [Cereals are the primary food source for the growing world population, especially in developing countries . Common nd maize . The wornd maize ,4. From nd maize . Droughtnd maize ,7. An innd maize . The durnd maize .2O2 is detoxified by peroxidases (APX and GPX) and catalases (CAT). CAT eliminates H2O2 by converting it into water and molecular oxygen; APX is the first enzyme of the ascorbate-glutathione cycle responsible for H2O2 detoxification whereas GPX decomposes H2O2 by oxidizing antioxidants [Triticum aestivum under different environmental conditions . Maneratures ,25,26,27eratures ,29,30. Ieratures . Genes etauschii  and on cnococcum . Genes anococcum . MoreoveThe aim of our study was to examine biochemical changes, including antioxidant enzyme activities, relative water content (RWC) levels and membrane damage reflected by lipid peroxidation under water-deficit conditions. Moreover, from intervarietal single chromosome substitution lines (ISCSLs), we identified chromosomes associated with early responses to short-term osmotic stress in wheat.Triticum aestivum L.) were examined in this study  of wheat (is study . 'Sarato-2 s-1). Five-day-old seedlings were subjected to osmotic stress by applying 10% of PEG-6000 to MS solution. Wheat seedlings were sampled after 1, 3 and 6 hours of stress treatment and then immediately frozen and stored at -80°C for further analysis. Plants grown in MS medium without PEG were used as a control.Grains were disinfected for 4 hours in chlorine gas. To induce germination, grains were incubated for 48 hours at 4°C and then placed on Petri dishes containing filter paper soaked with distilled water for 2 days in the dark at 24°C. Germinating seedlings were transferred to plastic pots containing MS medium. Plants were then grown in hydroponic culture in a growth chamber for 5 days under control conditions  was added to a mixture containing 3 cm3 of 1% phosphoric acid, 1 cm3 of 0.6% aqueous solution of thiobarbituric acid and 0.1 cm3 of aqueous FeSO4× 7 H2O (2.8 mg cm3–1). The samples were incubated at 100°C for 1 h and then quickly cooled on ice. Then, 4 cm3 of butanol-1 was added to each sample. After centrifugation at 3,000 g for 10 min, TBARS absorbance was measured at 532 and 600 nm . TBARS concentrations were calculated using a coefficient of extinction of 156 × mM-1 cm-1 and were expressed as μmol/g fresh weight (FW).Measurements of thiobarbituric acid reactive substance (TBARS) content were collected using Uchiyama and Mihara’s method . The hom2O2 degradation, which was identified as a decrease in absorbance at 240 nm (extinction coefficient of 39.4 mM−1 cm−1) [2O2 solution in sodium phosphate buffer (pH 7.8) and 50 mm3 of plant extract in a final volume of 3 cm3. Enzyme activity was expressed as μmol H2O2 mg protein-1 min-1.CAT activity was measured by estimating H−1 cm−1) . The rea3) consisted of 50 mM phosphate buffer (pH 7.5), 0.5 mM ascorbate, 0.1 mM EDTA and 0.1 cm3 of enzyme extract. The reaction was initiated by adding 0.1 cm3 of 0.1 mM. H2O2. The decrease in absorbance as a result of ascorbate oxidation was measured at 290 nm (extinction coefficient of 2.8 mM cm-1). APX activity was expressed as μmol ascorbate oxidized mg min-1 protein-1.Measurements of APX (EC 1.11.1.11) activity were performed following Nakano and Asada’s method . The rea3) contained 50 mM phosphate buffer (pH 7.5), 0.1 cm3 of 0.15% H2O2 and 0.1 cm3 of plant extract. The reaction was initiated by adding 0.1 cm3 of 56 mM guaiacol. The increase in absorbance as a result of guaiacol conversion into its oxidized form (tetraguaiacol) was measured at 470 nm. GPX activity was determined using the extinction coefficient for tetraguaiacol (26.6 mM-1 cm-1).GPX activity (EC 1.11.1.9) was assayed based on the modified method described by Balakhnina et al. . The reaRelative water content (RWC) was measured in control and stressed seedlings as described earlier ,40,41. FThe experiments were repeated in two full independent biological replications and each data point was measured as the mean of three technical replications (n = 6). A one-way analysis of variance (ANOVA) and Tukey’s posttest were used to compare the means of controls and stress-treated plants across the same substitution lines. Dunnett's test was applied to estimate significant differences between the tested lines and S29; differences measured at p < 0.05 were considered significant. Correlations of biochemical changes were estimated using Pearson’s correlation coefficient. In order to determine the main sources of variability, principal component analysis (PCA) method was applied. All data were analyzed using Statistica 13.1 (Dell Software).Water stress induced lipid peroxidation was observed in most of the tested lines and was expressed by higher levels of TBARS content . Only S2The same characteristic patterns of CAT activity were observed in the majority of lines . The iniThe results of the APX activity analysis indicate that significant changes were only observed for lines with substituted A genome chromosomes (except for 3A and 5A), 2D and S29 . HoweverThe analysis of GPX activity did not reveal any characteristic trends . For linFurthermore, the performed analysis shows that CAT activity is positively correlated with APX and negatively correlated with GPX and LPO .The obtained results do not show any significant changes in RWC for the tested lines treated with 10% PEG for 6 h. Water content levels were similar across all lines and varied between 85% and 95%. All changes in RWC were not statistically significant. No differences between the donor (S29) and tested lines were detected .The PCA analysis revealed that APX activity is the least influential in determining the PC1 while CAT activity plays the most important role . The firEremosparton songoricum [Setaria italica [Pisum sativum L. [Cleome gynandra [Setaria italica L. [Triticum aestivum responses to long-term drought [The formation of ROS is responsible for the stress-induced peroxidation of lipids and for membrane damage. Some compounds react with thiobarbituric acid (TBA), e.g., MDA, and form products called thiobarbituric acid reactive substances (TBARS) . Total qngoricum , Setaria italica , barley  italica , Pisum stivum L. . Our stugynandra  or for agynandra . Moreovegynandra  or Setaralica L. . Several drought ,49. PrevEremosparton songoricum under drought conditions [2O2 accumulation. The antioxidant system has not been yet activated. The same trend has also been observed in the 'Rum' cultivar of barley during long periods of stress [Our study demonstrates that major patterns of CAT activity undergo a significant decline after 1 and 3 h followed by an increase. Similar observations have been made for nditions  and for nditions . In the f stress . In our f stress  and for f stress .A different pattern of CAT activity was observed for substitution lines 4A, 6A, 4B and 7D after long-term periods of water shortage relative to the recipient . Based oPisum sativum L. [Koeleria macrantha [Phaseolus vulgaris [2O2 scavenging. This scavenging system can prevent increases in H2O2 levels in the cytosol during periods of optimum growing conditions and under certain plant-stress conditions as H2O2 concentrations increase. This lack of change in enzyme activity can be attributed to a high level of activity observed in plants under control conditions. Moreover, Mittler and Zilinskas [2O2 forming under these stress conditions. However, based on previous reports, we believe that prolonged drought treatment should cause an increase in APX activity, which was observed in wheat after 6 d [We did not observe any changes in APX activity for most of the tested lines. Our results confirm the results of an earlier study performed on drought-sensitive wheat cultivars subjected to mild (-0.4 MPa) osmotic stress for 8 d . Moreovetivum L. , maize [tivum L. , Koeleriacrantha , rice [5acrantha , Phaseolvulgaris , or the vulgaris  in the fvulgaris , APXs arilinskas  found thfter 6 d  and in tfter 6 d . In our fter 6 d , increasfter 6 d  and wheafter 6 d . Osipovafter 6 d  observedBrassica after 3 h of heat stress [Vigna aconitifolia [Eremosparton songoricum [In our study, we did not find major trends of GPX activity. Variable patterns of GPX activity have also been observed in different genotypes of wheat  and maizt stress . We founitifolia  and Eremngoricum . Accordingoricum , peroxidngoricum ,50 and wngoricum ,58,59 sungoricum  suggestengoricum , CAT andngoricum  suggesteBrassica [Kentucky bluegrass [Eremosparton songoricum [Kentucky bluegrass [The results of our analysis reveal a significant correlation between CAT and APX but a negative one between CAT and GPX. A negative correlation between CAT and GPX has also been found for maize , wheat  and BrasBrassica . AccordiBrassica  and in Kluegrass . The prengoricum  and Kentluegrass .Cleome gynandra [Phaseolus acutifolius [Arabidopsis thaliana [In the current study, we did not observe any significant changes in RWC over 6 h of osmotic stress. Moreover, no significant differences in RWC were observed among the tested lines. From the obtained results and previous studies, we suggest that 6 h of 10% PEG treatment should not lead to physiological changes in plants attributed to alterations in RWC. Our results are in line with many reports on wheat ,56 and ogynandra  and Phastifolius . Accorditifolius , 3 days tifolius  and 5 datifolius . From prtifolius  proved ttifolius . RWC is tifolius  and maiztifolius . Based otifolius . In our thaliana  and tomathaliana  under drIn the present study, we identified chromosomes associated with early wheat responses to short-term stress from a set of S29 substitution lines (JP) showing differing degrees of tolerance to water deficit. We found that the substitution of chromosomes 6B and 7D spurred the most significant changes in LPO to the recipient. It indicates that chromosomes 1A, 4A, 5A, 4B and 7D include genes involved in CAT activity. Genes involved in GPX activity are associated with chromosomes belonging to homologous groups 5  and 4 (4B and 4D) and to chromosomes 6A, 2B and 7B.Our study reveals a complex system of plant responses to short-term water-deficit stress. The first reaction of the tested wheat lines included an increase in GPX activity. However, catalase had the strongest impact on plant responses, as it caused a significant decline in LPO levels. In the current study, we found that chromosomes 5A, 4B, 6B and 7D are associated with early wheat responses to short-term stress. The substitution of these chromosomes spurred the most significant changes in the activity of the tested enzymes and in lipid peroxidation relative to S29. Therefore, structural and regulatory genes involved in rapid plant reactions may be positioned on these chromosomes.S1 FigChanges of lipid peroxidation expressed in terms of TBARS concentration in lines with substitution of A genome chromosomes (a-f) compared to S29 during 1, 3 and 6 h of 10% PEG treatment and in non-exposed plants. Bars represent 95% confidence intervals (CI). *indicates significant differences compared to S29 at p<0.05 according to Dunnett's test.(TIF)Click here for additional data file.S2 FigChanges of lipid peroxidation expressed in terms of TBARS concentration in lines with substitution of B genome chromosomes (a-f) compared to S29 during 1, 3 and 6 h of 10% PEG treatment and in non-exposed plants. Bars represent confidence interval (CI). *indicates significant differences compared to S29 at p<0.05 according to Dunnett's test.(TIF)Click here for additional data file.S3 FigChanges of lipid peroxidation expressed in terms of TBARS concentration in lines with substitution of D genome chromosomes (a-f) compared to S29 during 1, 3 and 6 h of 10% PEG treatment and in non-exposed plants. Bars represent confidence interval (CI). *indicates significant differences compared to S29 at p<0.05 according to Dunnett's test.(TIF)Click here for additional data file.S4 FigChanges of a) lipid peroxidation expressed in terms of TBARS concentration b) CAT activity c) APX activity d) GPX activity in JP compared to S29 during 1, 3 and 6 h of 10% PEG treatment and in non-exposed plants. Bars represent confidence interval (CI). *indicates significant differences compared to S29 at p<0.05 according to Dunnett's test.(TIF)Click here for additional data file.S5 FigChanges of CAT activity in lines with substitution of A genome chromosomes (a-f) compared to S29 during 1, 3 and 6 h of 10% PEG treatment and in non-exposed plants. Bars represent 95% confidence intervals (CI). *indicates significant differences compared to S29 at p<0.05 according to Dunnett's test.(TIF)Click here for additional data file.S6 FigChanges of CAT activity in lines with substitution of B genome chromosomes (a-f) compared to S29 during 1, 3 and 6 h of 10% PEG treatment and in non-exposed plants. Bars represent confidence interval (CI). *indicates significant differences compared to S29 at p<0.05 according to Dunnett's test.(TIF)Click here for additional data file.S7 FigChanges of CAT activity in lines with substitution of D genome chromosomes (a-f) compared to S29 during 1, 3 and 6 h of 10% PEG treatment and in non-exposed plants. Bars represent confidence interval (CI). *indicates significant differences compared to S29 at p<0.05 according to Dunnett's test.(TIF)Click here for additional data file.S8 FigChanges of APX activity in lines with substitution of A genome chromosomes (a-f) compared to S29 during 1, 3 and 6 h of 10% PEG treatment and in non-exposed plants. Bars represent 95% confidence intervals (CI). *indicates significant differences compared to S29 at p<0.05 according to Dunnett's test.(TIF)Click here for additional data file.S9 FigChanges of APX activity in lines with substitution of B genome chromosomes (a-f) compared to S29 during 1, 3 and 6 h of 10% PEG treatment and in non-exposed plants. Bars represent confidence interval (CI). *indicates significant differences compared to S29 at p<0.05 according to Dunnett's test.(TIF)Click here for additional data file.S10 FigChanges of APX activity in lines with substitution of D genome chromosomes (a-f) compared to S29 during 1, 3 and 6 h of 10% PEG treatment and in non-exposed plants. Bars represent confidence interval (CI). *indicates significant differences compared to S29 at p<0.05 according to Dunnett's test.(TIF)Click here for additional data file.S11 FigChanges of GPX activity in lines with substitution of A genome chromosomes (a-f) compared to S29 during 1, 3 and 6 h of 10% PEG treatment and in non-exposed plants. Bars represent 95% confidence intervals (CI). *indicates significant differences compared to S29 at p<0.05 according to Dunnett's test.(TIF)Click here for additional data file.S12 FigChanges of GPX activity in lines with substitution of B genome chromosomes (a-f) compared to S29 during 1, 3 and 6 h of 10% PEG treatment and in non-exposed plants. Bars represent confidence interval (CI). *indicates significant differences compared to S29 at p<0.05 according to Dunnett's test.(TIF)Click here for additional data file.S13 FigChanges of GPX activity in lines with substitution of D genome chromosomes (a-f) compared to S29 during 1, 3 and 6 h of 10% PEG treatment and in non-exposed plants. Bars represent confidence interval (CI). *indicates significant differences compared to S29 at p<0.05 according to Dunnett's test.(TIF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.


Maize seedlings are constantly exposed to inorganic phosphate (Pi)-limited environments. To understand how maize cope with low Pi (LP) and high Pi (HP) conditions, physiological and global proteomic analysis of QXN233 genotype were performed under the long-term Pi starvation and supplementation.We investigated the physiological response of QXN233 genotype to LP and HP conditions and detected the changes in ion fluxes by non-invasive micro-test technology and gene expression by quantitative real-time polymerase chain reaction. QXN233 was further assessed using vermiculite assay, and then proteins were isolated and identified by nano-liquid chromatography-mass spectrometry.+ and Pi, and Na+ efflux was enhanced under HP condition. Furthermore, a total of 681 and 1374 were identified in the leaves and roots, respectively, which were mostly involved in metabolism, ion transport, and stress response. Importantly, several key Pi transporters were identified for breeding potential. Several ion transporters demonstrated an elaborate interplay between Pi and other ions, together contributing to the growth of QXN233 seedlings.A negative relationship was observed between NaThe results from this study provide insights into the response of maize seedlings to long-term Pi exposure.The online version of this article (10.1186/s12953-018-0147-3) contains supplementary material, which is available to authorized users. Phosphorus (P) is an essential macronutrient required for normal plant growth and reproduction. P is involved in various metabolism and biological processes, including nucleic acid and protein synthesis, membrane integrity, photosynthesis, respiration, energy metabolism, hormone regulation, stress tolerance, and disease resistance , 2. HoweZea mays L.), many ZmPHTs genes had been identified, and some transcription factors (TFs) were also found to be involved in the transcriptional regulation of Pi transporters, including myeloblastosis viral oncogene homolog (MYB)-type and basic Helix-Loop-Helix (bHLH)-type , and A0A1D6HFX1 . The expression levels of these genes were examined by qRT-PCR. The results showed that ZmPHT1, ZmPHT2, and ZmPHT9 increased in leaves or roots under LP condition while decreased under HP condition, consistent with the results of proteomic analysis  transporters, were also identified. Similarly, some of them presented opposite expression patterns under LP and HP conditions Table . For car+ transporters, A0A1D6N218 (fold change = 1.42 in roots) and W5UB74 (fold change = 1.26 in roots), were elevated under LP condition, but B6T7A1 (voltage-gated K+ channel beta subunit) and A0A1D6DSW6 and A0A1D6JIE4 , responsible for K+ transport and exclusion, were also induced. Moreover, two nitrate transporters, A0A1D6KAA6  and A0A1D6N629 , were detected in roots under LP condition. Many ABC transporters were also significantly altered in roots. Among them, A0A1D6NEN5 (ABC transporter B family member 9) and B4FBM3 (ABC transporter G family member 6) showed opposite expression patterns in roots’ response to LP and HP conditions  was both downregulated in the leaves and roots under LP condition but upregulated in the roots under HP condition. Q6I681 [ascorbate (As)-specific transmembrane electron transporter 1] was up- and downregulated in roots under LP and HP conditions, respectively. Under LP condition, A0A096TR23 [molybdate (Mo) transporter 2] was highly expressed in the leaves and roots and A0A1D6PKM7 [boron (B) transporter 4] was lowly expressed in the leaves. These results indicated that high As and Mo and low B contents are required by plants under Pi deficiency , B4F9L6 and A0A1D6KE94 (purple acid phosphatases), and A0A1D6HFX1 (SPX domain protein) associated with Pi assimilation and regulation, were increased under LP stress but decreased under HP supplementation, indicating that these DEPs play key roles in Pi homeostasis for maize coping with different Pi levels. These foundings will improve our understanding on how maize seedlings regulate DEPs to respond to different Pi environments. The key DEPs are also candidate proteins that may aid in the selection and breeding of LP-tolerant maize genotype.Additional file 1:Figure S1. Phenotypic responses of QXN233 genotype to LP or HP condition. QXN233 grown under the different Pi-treated conditions for 10 days (a) via a vermiculite assay or for 20 days (b) via a hydroponic assay. Bar = 5 cm, Bar = 2 cm. Figure S2. Phenotypic responses of QXN233 genotype to LP or HP condition. QXN233 grown under the different Pi-treated conditions for 25 days (a) via a vermiculite assay. Bar = 10 cm. Table S1. Primers used in qRT-PCR. Table S2. Quantitative analyses of plant height and the width and length of the longest leaf in QXN233 after 30 days under 0 mM Pi or 3 mM Pi via vermiculite assay. Values represent means ± SEM of three replicates. Asterisks indicate a significant difference between the Pi-treated and control groups . Table S3. DEPs of QXN233 identified under low or high Pi (LP or HP) compared with the normal condition via the proteomic analysis (Ratio |0 Pi or 3 Pi/Control| > 1.2 and P < 0.05). The red and green markers presented the upregulated and downregulated values of DEPs, respectively. Table S4. Dataset.xlsx. (ZIP 4300 kb)


A 17-month-old boy in India with severe acute respiratory infection was laboratory confirmed to have avian influenza A(H9N2) virus infection. Complete genome analysis of the strain indicated a mixed lineage of G1 and H7N3. The strain also was found to be susceptible to adamantanes and neuraminidase inhibitors. Low-pathogenicity avian influenza A(H9N2) viruses have a wide host range, and outbreaks in poultry have been recorded since the 1990s in China –associated deaths among children <2 years of age. A total of 2,085 nasopharyngeal swabs from children with severe or fatal pneumonia were transported to India’s National Institute of Virology to test for influenza, RSV, and other respiratory viruses. A nasopharyngeal swab from a 17-month-old boy received on February 12, 2019, tested positive by PCR for influenza A(H9N2) virus.>20 seconds. He was fully immunized for his age, with bacillus Calmette–Guérin, diphtheria, hepatitis B, poliovirus, and measles vaccines. Both length and weight for age were less than −3 SD. History of travel with his parents to a local religious gathering 1 week before symptom onset was elicited. The father had similar symptoms on return from the gathering but could not undergo serologic testing because of his migrant work. No history of poultry exposure was elicited. The child received an antibacterial drug and antipyretics and recovered uneventfully.The child, a resident of Melghat, had fever, cough, breathlessness, and difficulty feeding for 2 days after illness onset on January 31, 2019. His high intermittent grade fever had no diurnal variation and no association with rash or mucocutaneous lesions. Examination revealed a conscious, restless child with a respiratory rate of 48 breaths/min and lower chest wall in-drawing with intermittent absence of breathing for We tested the clinical sample using duplex real-time PCR for influenza A/B, H3N2, and 2009 pandemic H1N1 viruses; RSV A/B; human metapneumovirus; parainfluenza virus types 1–4; rhinovirus; and adenovirus. The sample was strongly positive for influenza A virus  but negative for seasonal influenza viruses and all respiratory viruses. Real-time PCR analysis for avian influenza viruses H5N1, H7N9, H10N8, and H9N2 revealed positivity for H9N2 virus . We confirmed this result by sequencing the matrix (M) and hemagglutinin (HA) genes of the isolate, designated A/India/TCM2581/2019/(H9N2); the M gene (260 bp) had 97.27% nucleotide identity with A/chicken/India/99321/2009(H9N2), and the HA gene  had 96.93% nucleotide identity with A/chicken/India/12CL3074/2015(H9N2). https://www.illumina.com) and a de novo assembly program (CLC Genomics Software 10.1.1 [https://megasoftware.net) with a Tamura-Nei nucleotide substitution model including 1,000 replicates bootstrap support   Table 1.–3 substitutions/site/year.Bayesian evolutionary analyses using BEAST version 1.8.1  Figure 2In conclusion, multiple introductions of H9N2 viruses in poultry have been observed in India. The identification of a human case of H9N2 virus infection highlights the importance of systemic surveillance in humans and animals to monitor this threat to human health.Additional information about laboratory-confirmed avian influenza A(H9N2) virus infection, India, 2019.


P ≤ 0.01) for the assessed traits. Further, AMMI analysis of variance revealed highly significant (P ≤ 0.001) differences among genotypes, environments and G × E interaction effects for all the studied traits. Both AMMI and GGE biplot analyses identified the following promising genotypes: G2 (Resisto × Ukerewe), G3 (Ukerewe × Ex-Msimbu-1), G4 (03-03 x SPKBH008), G12 (Ukerewe × SPKBH008) and G18 (Resisto × Simama) with high yields, high dry matter content and SPVD resistance across all test environments. The candidate genotypes are recommended for further stability tests and release in Tanzania or similar environments.Genotype-by-environment interaction analysis is key for selection and cultivar release, and to identify suitable production and test environments. The objective of this study was to determine the magnitude of genotype-by-environment interaction (GEI) for storage root yield, yield-related traits and sweet potato virus disease (SPVD) resistance among candidate sweet potato genotypes in Tanzania. Twenty-three newly bred clones and three check varieties were evaluated across six diverse environments using a randomized complete block design with three replications. The Additive Main Effect and Multiplicative Interaction (AMMI) and genotype and genotype-by-environment (GGE) biplot analyses were used to determine GEI of genotypes. Genotype, environment and GEI effects were highly significant ( It is a rich source of carbohydrates, vitamins A and C, fiber and minerals   and geno22.1The study was conducted in six diverse and sweet potato growing environments in Tanzania namely: Gairo, Kilombero Agricultural Training Research Institute (KATRIN), Sokoine University of Agriculture (SUA), Sugarcane Research Institute (SRI), Chambezi and Mkuranga. The sites represent low to high altitudes with varied agro-ecological conditions. The study sites are among the major sweet growing areas and are hotspot areas for SPVD in Tanzania. The description of experimental sites and chemical composition of the soils at each site is presented in 2.2The experimental genotypes and check varieties were evaluated using a randomized complete block design with three replications at each site. Experimental plots consisted of two rows of 6 m for each genotype. The intra-row and inter-row spacing were 0.3 m and 1 m, respectively. Four to six node cuttings were planted on ridges. Agronomic practices such as weeding and fertilization were followed per recommendation for sweet potato production in Tanzania.2.3At harvest, storage roots were grouped into marketable and un-marketable, counted and their fresh weight (kg) per plot was recorded and later converted to tonnes per hectare (tons/ha). This provided storage root yield per hectare. The number of roots was expressed on a plant basis. From each plot, a sample of three to four storage roots were collected for dry matter content (DMC) determination. DMC was determined as described by Reactions to SPVD were assessed visually at 60, 90 and 120 days after planting using a 1 to 5 scale, where 1 = no visible symptoms, 2 = mild symptoms , 3 = moderate symptoms (mosaic symptoms on leaves), 4 = severe symptoms (mosaic symptoms with plants showing stunted growth) and 5 = very severe symptoms of purpling/yellowing of leaves, severe leaf distortion, reduced leaf size and severe stunting . The gen2.3.12.3.1.1The data collected for number of storage roots, storage root yield, dry matter content and SPVD across six environments sites were subjected to combined analysis of variance (ANOVA) using Statistical Analysis System version 9.2 . Genotyp2.3.1.2th edition ; kth PC; and ith genotype and jth environment, respectively for PCk; i in environment j. AMMI and GGE biplot were performed using GenStat 17th edition  = ronments . The modronments  based on edition .33.1P ≤ 0.001) differences among the six test environments and the tested genotypes (P ≤ 0.001) genotype by environment interaction effect was observed for all studied traits and SPVD resistance implying differential genotypic performances across environments. Further, the AMMI analysis of variance in the current study revealed great contribution of environments (64%) and GE interactions (25%) for variation in storage root yield compared with the main effect of genotypes (11%). Analysis of variance for number of roots per plant, storage root yield, dry matter content and resistance to SPVD showed highly significant . This genotype (G20) performed better than other genotypes in four environments namely E2 (SRI), E3 (KATRIN), E4 (SUA) and E6 (Mkuranga) with mean root number of 7.7, 7.1, 8.2 and 5.8, respectively. Further, genotypes such as G22, G3 and G2 also ranked best across all environments with mean roots per plant of 5.3, 4.5 and 4.6 in that order. These genotypes  also performed better in four environments namely E2 (SRI), E3 (KATRIN), E4 (SUA) and E6 (Mkuranga). The number of roots per plant in the selected clones is higher than some sweet potato landrace varieties and improved genotypes currently cultivated in Tanzania . Marked GGE biplot analysis showing the relative performance of sweet potato genotypes for number of roots per plant across environments is presented in , G22 was identified as the most desirable genotype for number of roots per plant due to its close proximity to the ideal variety (tip of arrow head on the vector through the average environment coordination in the center of inner circle).The average-environment coordination (AEC) view of the GGE biplot comparing sweet potato genotypes across environments is shown in 3.2.2Mean fresh root yield ranged from 7.5 to 17.2 tons/ha for G24 and G5, respectively with overall mean of 10.7 tons/ha (data not shown). More than 46% of the genotypes yielded above the overall mean with G5, G11 and G23 recording the highest storage root yields of 17.2, 13.8 and 13.5 tons/ha, respectively. Storage root yield of the identified clones is higher than those recently developed in Rwanda  and sweeThe GGE biplot showing the “which–won- where” for storage root yield is presented in The GGE biplot identified two mega-environments with E1 (Gairo) constituting one mega-environment and E2 (SRI), E3 (KATRIN), E4 (SUA), E5 (Chambezi) and E6 (Mkuranga) composing the second mega-environment , implyinThe AEC comparison using GGE biplot in the current study identified E6 (Mkuranga) and E2 (SRI) as the best representative or “ideal” environment for discriminating the tested genotypes with respect to storage root yield . An “ide3.2.3Dry matter content (DMC) varied from 30.3 to 40.8% for genotypes G25 and G26 (check varieties), respectively, with an average of 36%. Among the newly bred genotypes, G1, G2, G5, G14 and G22 recorded the lowest DMC of 33.7, 32.9, 33.0, 32.6 and 33.6%, respectively which were below the average DMC. Conversely, G10, G13, G15 and G19 recorded high DMC of 38.1, 37.6, 37.6 and 38.7%, respectively. These are recommended for breeding for enhanced dry matter content. Breeding for high dry matter content is an important goal in sweet potato improvement programmes. In the present study, DMC of sweet potato clones was higher than those reported for sweet potato genotypes released previously in sub-Saharan Africa . Test enGGE biplots showing “which-won-where” or which genotypes are best for which environment for dry matter content (DMC) is presented in The AEC comparison view GGE biplot identified KATRIN (E3) and Chambezi (E5) as “ideal” environments for discriminating genotypes with respect to DMC, whereas environments such as E1 (Gairo) and E4 (SUA) as “non-ideal” environments . Similar3.2.4The SPVD scores for the six environments varied from 1.17 to 3.11 corresponding for genotypes G4 and G25, respectively. About 62% of the genotypes recorded SPVD scores less than the average across all the six environments. Genotypes such as G4, G5, G7, G8, G9, G11, G12, G13, G17, G18, G21, G23 and G26 exhibited SPVD values of ≤1.5, suggesting their resistance to SPVD. The level of SPVD resistance among the presently tested sweet potato clones is comparable and in some instances higher than previously released commercial cultivars and clones .The GGE biplot showing environments and respective sweetpotato genotypes for resistance to sweet potato virus disease is presented in The AEC view of the GGE biplot further indicated that KATRIN (E3) and SUA (E4) were identified as the best representative environments for discriminating genotypes with respect to SPVD, whereas E1 (Gairo) and E6 (Mkuranga) and E1 (Gairo) were the poorest . Genotyp4The current study determined the magnitude of genotype-by-environment interaction and stability for number of roots per plant, storage root yield, dry matter content and reaction to sweet potato virus disease among newly developed sweet potato genotypes in eastern Tanzania. Candidate sweet potato genotypes G2 (Resisto × Ukerewe), G3 (Ukerewe × Ex-Msimbu-1), G4 (03-03 x SPKBH008), G12 (Ukerewe × SPKBH008) and G18 (Resisto × Simama) are good yielders with high dry matter content and SPVD resistance across all test environments. The candidate genotypes are recommended for further stability tests and release in Tanzania or similar environments.Stephan Ngailo: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper.Hussein Shimelis, Julia Sibiya, Kiddo Mtunda: Conceived and designed the experiments; Analyzed and interpreted the data; Wrote the paper.Jacob Mashilo: Analyzed and interpreted the data; Wrote the paper.Alliance for a Green Revolution in Africa (AGRA) through financial support of the study through the African Centre for Crop Improvement (ACCI).This work was supported by the The authors declare no conflict of interest.No additional information is available for this paper.


Urinary extracellular vesicles (EV) could be promising biomarkers for urological diseases. In this retrospective feasibility study, we conducted biomarker screening for early stage bladder cancer using EV mRNA analysis.Biomarker candidates were identified through RNA-seq analysis of urinary EV from patients with non-muscle invasive bladder cancer (N=3), advanced urothelial cancer (N=3), no residual tumor after TURBT (N=2), and healthy and disease controls (N=4). Diagnostic performance was evaluated by RT-qPCR in a larger patient group including bladder cancer (N=173), renal pelvis and ureter cancer (N=33), no residual tumor and non-cancer disease control (N=36).SLC2A1, GPRC5A and KRT17 were overexpressed in pT1 and higher stage bladder cancer by 20.6-fold, 18.2-fold and 29.5-fold, respectively. These genes allowed detection of non-muscle invasive bladder cancer  as well as pT2 and higher muscle invasive bladder cancer (AUC: 0.72 to 0.90). Subgroup analysis indicated that these markers could be useful for the detection of cytology-negative/-suspicious and recurrent bladder cancers.Urinary EV Three urinary EV mRNA were discovered to be elevated in bladder cancer. Urinary EV mRNA are promising biomarkers of urothelial cancer and worth further investigation. National Cancer Institute estimated that there will be approximately 74,000 new bladder cancer cases and 14,000 deaths in the United States alone . Other uCurrent gold standard of bladder cancer detection is cystoscopy with urine cytology. The sensitivity and specificity of cystoscopy is 87% and 100%, respectively . Other uExtracellular vesicles (EV) are known to be released into the urinary space from all the areas of the nephrons by encapsulating the cytoplasmic molecules of the cell of origin . SeveralOur previous studies employed an EV mRNA assay to conduct biomarker screenings for hematologic, kidney and ovarian disorders, where the assay performance such as sensitivity, linearity, and reproducibility was optimized and characterized –12. In tCEACAM7, CRH, FABP4, GPRC5A, HSD17B2, KRT17, LINC00967, OLFM3, P4HA1, SLC2A1, TMEM45A, and TMPRSS4 , renal pelvis cancer , no residual tumor after transurethral resection of bladder tumor (TURBT) , and healthy  and disease controls DC, N=1) and applied to RNA-seq analysis  were assayed by RT-qPCR in 254 urine samples including 173 bladder cancer patient urine samples  and not differentially expressed among any diagnostic groups such as cancer type, bladder cancer stage and grade, therefore ALDOB was selected as a reference gene.Bladder cancer marker candidates as well as reference genes , pT1 , pT2 and higher stages , G2  and G3 bladder cancer  compared to the non-BC control groups (DC and NRT) , pT1 , G2  and G3 bladder cancer  therefore may be useful to supplement SLC2A1 especially for the detection of pTis bladder cancer , pT2 and higher stages , G2  and G3 bladder cancer , which was consistent with the expression of SLC2A1 and GPRC5A  Figure . GPRC5A r Figure . KRT17 eA Figure .SLC2A1 outperformed urine cytology and bladder tumor antigen (BTA) especially for the detection of all the stages (AUC 0.70 vs. 0.57 to 0.63), pTa (AUC 0.64 vs. 0.49 to 0.56) and pT1 bladder cancer (AUC 0.86 vs. 0.60 to 0.82). GPRC5A also outperformed these conventional bladder cancer markers for the detection of pTis (AUC 0.80 vs. 0.54 to 0.78) and KRT17 for the detection of pT2 and higher stage bladder cancer (AUC 0.90 vs. 0.66 to 0.72).The diagnostic performance of these urinary EV mRNA for the detection of bladder cancer was evaluated using ROC curve analysis by comparing various stages/grades of bladder cancer to the control groups (DC and NRT) Figure . UrinarySLC2A1 was able to detect cytology-negative/suspicious bladder cancer (N=115) with AUC 0.68 ) since 87% of the pTa samples were lower grades (G1/G2) although those cancers have a low risk for recurrence, while high-risk pTa/G3 bladder cancer may be detected with better diagnostic performance ).In this study, RNA-seq analysis of urinary EV from bladder cancer patients was performed and indicated cancer- and immune system-related functions were activated in BC urinary EV. Interestingly immune system-related functions were activated in NRT urine although urine was collected more than four months after BCG intravesical treatment or without any immune related treatment. This may suggest that their bladder environments may be exposed to carcinogenic compounds constantly and cause high recurrence of cancer however further studies are necessary to exclude other possibilities. From the RNA-seq data, the top 12 bladder cancer marker candidates were identified and further analyzed in a separate cohort of 254 urine samples by RT-qPCR. We selected three promising urinary EV markers, SLC2A1 (solute carrier family 2 member 1) encodes glucose transporters 1 (Glut1). Glut1 is overexpressed in many types of cancers including urothelial cancers and involved with glucose uptake to support the growth and proliferation of cancer cells [SLC2A1 expression may be associated with the prognosis of urothelial cancer patients. GPRC5A (G protein-coupled receptor class C group 5 member A) encodes retinoic acid-induced protein 3 (RAI3), which is associated with many types of cancers [KRT17 (keratin 17) has been reported to be overexpressed not only in bladder cancer [KRT17 stimulates the Akt/mTOR pathway and upregulates SLC2A1 and glucose uptake which facilitates tumor growth [SLC2A1, GPRC5A and KRT17 in bladder cancer urine, however further study is necessary to determine if these EV mRNA are originated from tumors or associated with the overexpression of corresponding proteins in tumors.These three genes and corresponding proteins have been studied well in several types of cancers. er cells . In a laer cells . Therefo cancers . RAI3 be cancers . On the  cancers , liver [ cancers , gastric cancers  and panc cancers  is assocr cancer  but alsor cancer , cervicar cancer , oral [2r cancer , esophagr cancer , pancrear cancer  and color cancer , and assr cancer  and cervr cancer . In oralr growth . These sSLC2A1 and KRT17 in bladder tumors are moderately correlated with the ‘basal’ markers such as KRT16, KRT6A, KRT6C, KRT5, CDH3, KRT6B, and KRT14, while the expression level of GPRC5A is weakly correlated with the ‘luminal’ markers such as KRT19 and ERBB3 . Patients eligible for the study were suspected urothelial cancer patients based on the presence of hematuria, irritable bladder symptoms, or the test result of cystourethroscopy, computed tomography and/or abdominal echogram. Up to 15 mL spot urine was collected prior to TURBT with informed consent at our facility between May 2014 and May 2016  using the delta Ct method. Data analysis was performed using R. Statistical significance was obtained by Welch’s t-test with p value < 5%. Diagnostic performance was evaluated and compared to urine cytology and bladder tumor antigen assays by the area under the curve (AUC) in ROC curve analysis. Optimum threshold was obtained at the nearest point of the ROC curve to the top-left corner, and used to calculate sensitivity and specificity to characterize diagnostic performance of marker candidates.Urine samples were obtained from urothelial cancer and other patients and healthy donors N=254) (Table 54 (Table