L-arabinose. Saccharomyces cerevisiae is a glucose-fermenting yeast that is traditionally used for ethanol production. Fermentation of L-arabinose is also possible after metabolic engineering. Transport into the cell is the first and rate-limiting step for L-arabinose metabolism. The galactose permease, Gal2p, is a non-specific, endogenous monosaccharide transporter that has been shown to transport L-arabinose. However, Gal2p-mediated transport of L-arabinose occurs at a low efficiency. In this study, homologous modeling and L-arabinose docking were used to predict amino acids in Gal2p that are crucial for L-arabinose transport. Nine amino acid residues in Gal2p were identified and were the focus for site-directed mutagenesis. In the Gal2p transport-deficient chassis cells, the capacity for L-arabinose transport of the different Gal2p mutants was compared by testing growth rates using L-arabinose as the sole carbon source. Almost all the tested mutations affected L-arabinose transport capacity. Among them, F85 is a unique site. The F85S, F85G, F85C, and F85T point mutations significantly increased L-arabinose transport activities, while, the F85E and F85R mutations decreased L-arabinose transport activities compared to the Gal2p-expressing wild-type strain. These results verified F85 as a key residue in L-arabinose transport. The F85S mutation, having the most significant effect, elevated the exponential growth rate by 40%. The F85S mutation also improved xylose transport efficiency and weakened the glucose transport preference. Overall, enhancing the L-arabinose transport capacity further improved the L-arabinose metabolism of engineered S. cerevisiae.Efficient and cost-effective bioethanol production from lignocellulosic materials requires co-fermentation of the main hydrolyzed sugars, including glucose, xylose, and  L-arabinose  was verified as the main L-arabinose transporter; Hxt9p and Hxt10p also exhibited limited L-arabinose transport capacity in some strains  from Scheffersomyces stipitis, Stp2p (Km 4.5 mM and Vmax 0.6 nmol/min/mg dry mass) from Arabidopsis thaliana, KmAXT1p (Km 263 mM and Vmax 57 nmol/min/mg dry mass) from Kluyveromyces marxianus, PgAXT1p (Km 0.13 mM and Vmax 18 nmol/min/mg dry mass) from Pichia guilliermondii, Mgt05860p, Mgt05293p, and Mgt04891p from Meyerozyma guilliermondii, LAT-1 (Km 58.1 mM and Vmax 116.7 mmol/h/g dry mass) from Neurospora crassa, Tct1p from Trichosporon cutaneum, Stp1p from Trichoderma reesei, and MtLAT-1 (Km 29.4 mM and Vmax 10.3 mmol/h/g dry mass) from Myceliophthora thermophila . The site-directed mutations of Gal2 were constructed by a fusion PCR strategy based on the overlap extension PCR  and 5 g L-1 ammonium sulfate . To cultivate the yeast and maintain the required plasmids, the medium was supplemented with the appropriate carbon source and complete supplement mixture  . For strains containing the KanMX4 marker, the medium was supplied with 200 μg mL-1 G418 sulfate . Plasmids were amplified in E. coli strain DH5α , which was grown on Luria-Bertani (LB) medium with 200 μg mL-1 ampicillin.The yeast synthetic complete (SC) medium used in this study contains 1.7 g L-1 glucose for 24 h, and then shifted into medium containing 5 g L-1 glucose and 15 g L-1L-arabinose for 48 h. After that, the cells were collected and used for batch cultivation in the respective SC medium containing 20 g L-1L-arabinose at an initial OD600 of 1. To cultivate BSW4EYX-based strains, single colonies were pre-incubated in SC medium containing 20 g L-1 maltose for 24 h, and then the cells were collected and used for batch cultivation in the respective SC medium containing 20 g L-1 glucose, 20 g L-1 xylose, or 20 g L-1L-arabinose at an initial OD600 of 1 or 5. All the yeast strains were batch cultivated in 40 mL cultures in 150 mL aerobic triangular flasks or air-limited flasks at 30°C, 200 r min-1. All E. coli strains were cultured at 37°C, 200 r min-1.To cultivate BSW3AP-based strains, single colonies were preincubated in SC medium containing 20 g LE. coli homolog of the glucose transporters GLUT1-4  . The nd 4GC0)  served and 4GC0)  was usedsoftware .L-arabinose, xylose, or glucose transport, we utilized site-directed random mutagenesis. The variety of the residues were introduced by the design of primers, with the coded three bases replaced by NNN  was measured by a BioPhotometer plus  and was used to determine the strain growth. The growth capacities of strains were determined by the exponential growth rates  prominence LC-20A , which has a refractive index detector RID-10A  and an Aminex HPX-87P ion exchange column , was used to determine the concentration of the above chemicals at 80°C with a mobile phase of water at a flow rate of 0.6 mL min-1, as previously reported . It was clear that BSW5AP-A lost the ability to grow on L-arabinose. After overexpressing Gal2 by plasmid in BSW5AP, the BSW5AP-AGal2 strain regained the ability to grow on L-arabinose; however, the exponential growth rate of BSW5AP-AGal2 was lower than BSW3AP, which may have resulted from the different expression strategy. We verified that our strains exhibited Gal2p-dependent L-arabinose transport, and we successfully generated the L-arabinose transport-deficient chassis BSW5AP.To construct L-arabinose binding was constructed according to the outward-facing and partly occluded conformation of the E. coli xylose permease XylEp . The polar or aromatic amino acid residues in different transmembrane sequences (TMSs) of Gal2p were chosen for more in-depth study, including F85, T89, F223, N346, N347, F350, Y351, N376, and Y446.The homologous model of Gal2p for se XylEp . The homL-arabinose, xylose, or glucose transport, we used random and site-directed mutagenesis to introduce amino acid substitutions. The exponential growth rates of the expressing BSW5AP strains during aerobic cultivation were used to determine the change in growth capacity (Table 4).To investigate the function of the selected amino acid residues of Gal2p in either L-arabinose. However, changes to the polar amino acids S, G, C, T, Y, or N of F85 somehow increased the exponential growth rates. Furthermore, the exponential growth rates of the F85S, F85G, F85C, and F85T mutants on L-arabinose significantly increased by 40, 32, 20, and 16%, respectively, compared with the strain with wild-type Gal2p. As such, F85 was identified as a very important amino acid residue for L-arabinose transport . For site T89, except for the slight increase in exponential growth when changed to an I residue, changes to H, R, and K all resulted in a decrease or loss of growth capacity on L-arabinose. Another important amino acid residue for L-arabinose transport of Gal2p is F223. When F223 was changed to Q, C, L, and S, the exponential growth rates increased, and F223Q significantly improved by 16%. We changed the amino acids N346 and N347 together and found that, even when changed to small side chain amino acids A and G, respectively, the L-arabinose transport capacity of Gal2p was not remarkably reduced. Random mutation of amino acids F350 and Y351 resulted in a loss in the L-arabinose transport capacity of Gal2p. Mutations to the amino acid residue N376 in TMS 8 of Gal2p also presented complex effects on L-arabinose transport. Changing N376 to Y, R, and K, resulted in loss of Gal2p transport capacity. The mutations N376T, N376G, N376C, N376S, N376A, N376F, and N376I decreased the exponential growth rates by 16, 16, 16, 24, 28, 64, and 68%, respectively. Mutating the Y446 residue to A, I, C, or S resulted in a loss of transport capacity. This result indicated that Y446 is very conservative and is necessary for L-arabinose transport in Gal2p.A total of 10 substitution mutants were identified at the selected F85 site. When F85 was changed to a charged E or R, Gal2p lost the transport capacity of L-arabinose transport capacity of positive mutations F85S, F85G, F85C, and F85T of Gal2p was further examined by L-arabinose metabolism. The L-arabinose metabolism capacity was evaluated by oxygen-limited batch cultivation. The L-arabinose metabolizing and ethanol producing were deeply concerned. The growth curves and L-arabinose metabolic trend are presented in Figure 3.The enhanced L-arabinose metabolic efficiency positively correlated with the exponential growth rates on L-arabinose (Table 4). F85S and F85G exhibited rapid growth rates on L-arabinose and utilization of L-arabinose. During the 76 h fermentation, the Gal2p-expressing strain utilized 4.5 g L-arabinose and consumed only 22%. The L-arabinose utilization of F85S and F85G increased to 8.3 and 7.3 g, and the consumption amounts were 42 and 37%, respectively. Compared with the wild-type Gal2p-expressing strain, the consumption of L-arabinose by the F85S and F85G mutants increased by 84 and 62%, respectively. Furthermore, the F85S and F85G mutants produced 1.4 and 1.1 g L-1 more ethanol than the wild-type Gal2p-expressing strain.The L-arabinose transport capacity of Gal2p. The F85 site of Gal2p is also very conservative in the L-arabinose transporters expressed in S. cerevisiae and is located in the “G-G/F-XXX-G” motif in the first transmembrane span . The universal meaning of this key site was presented.From the results above, we can predict that the change in F85 to polar amino acids will improve the S. cerevisiae can transport many sugars, including glucose, xylose and L-arabinose  and xylose-containing medium . Unlike mutation F223Q, the F85G, F85S, F85T, and F85C mutations all decreased the glucose transport efficiency by 67, 45, 38, and 19%, respectively. This result indicated that the benzene ring of F85 in Gal2p might be important for glucose transport. For xylose transport of Gal2p, the F85C, F85S, and F85T mutations improved the xylose transport efficiency, and the exponential growth rates increased by 29, 23, and 15%, respectively. However, the exponential growth rate of the F85G mutant decreased by 19%, and F223Q did not change the growth efficiency on xylose.Endogenous Gal2p of rabinose . To testf xylose . The expL-arabinose, we then transformed the plasmid YIp5-ara (Table 1) to the BSW4EYX strains expressing F85S or Gal2p. During a 96 h fermentation for the expressing strains, the mutation F85S decreased the glucose utilization efficiency , obviously increased the xylose consumption amount  by 1.1 g L-1, and also increased the L-arabinose consumption amount  by 1 g L-1, compared with the original Gal2p. After about 40 h cultivation, the F85S expressing strain could exhibit more significant co-utilization of xylose and L-arabinose with glucose, and the ethanol production was 10 g L-1 which was consistent with that of Gal2p.To further test the effect of the best positive mutation F85S on the co-utilization and co-fermentation of glucose, xylose, and L-arabinose and xylose in lignocellulosic materials is becoming more important for renewable fuel production. The transport efficiency of the two pentoses needs to be increased in the metabolic process of microbial cell factories. Gene knockout and overexpression studies verified the necessary function of Gal2p for L-arabinose transport in S. cerevisiae  were shown to significantly affect the L-arabinose transport activity of Gal2p. Meanwhile, compared with directed evolution, site-directed mutagenesis was verified in this study to be a simple and effective method to identify the crucial amino acid residues of pentose transporters.Our group formerly constructed an efficient t BSW3AP . After dof XylEp  was usedFigures 2C,D) is now verified to have important effect on L-arabinose transport. Changing F85 to polar amino acids S, G, C, T, Y, or N somehow increased the L-arabinose transport activity of Gal2p, otherwise, changing to charged amino acids E or R resulted in a loss of the transport capacity of L-arabinose. This result suggests that the polar chain in this site is useful for L-arabinose binding for processing, but charge forces interferes this process. It is noteworthy that F85S significantly enhanced the exponential growth rates on L-arabinose, and the L-arabinose consumption amounts could be improved by 84%. This mutation also increased the exponential growth rates on xylose by 23%, and consequently decreased the glucose transport preference. The corresponding site of F85 in HXT7p is F79, and the mutant HXT7 (F79S) was previously reported to improve the xylose uptake rate .The F85 position in TMS1 of Gal2p (ake rate . Furtherransport . This reL-arabinose transport of Gal2p. When F223 was changed to Q, C, L, and S, the exponential growth rates of the mutants increased, and F223Q significantly improved the exponential growth rate by 16%. Although the F223Q mutation increased the L-arabinose transport capacity, the mutation did not affect xylose or glucose transport. The mutations at residues T89 and N376 significantly decreased the growth of these mutants on L-arabinose, suggesting that these residues are also important for L-arabinose transport. In addition, N376 was formerly shown to affect the xylose transport affinity and preference of Gal2p (L-arabinose transport capacity. The phenyl structure of the sites F350 and Y351 might play an important role for L-arabinose exclusion in transport process of Gal2p. Y446 is also highly conserved and necessary for L-arabinose transport of Gal2p, as changes to A, I, C, or S, all resulted in loss of L-arabinose transport capacity. Y446 was also previously reported to be essential for galactose recognition by Gal2p (F223 is another important amino acid residue for of Gal2p . Random of Gal2p , resulteby Gal2p .L-arabinose, although the co-fermentation efficiency of xylose and L-arabinose was not high enough and the ethanol production was not significantly increased. F85S partly alleviated the glucose suppression, although the glucose inhibitory effect on xylose and L-arabinose utilization still existed as reported (S. cerevisiae for pentose metabolism.The best mutation F85S of Gal2p was tested to improve the co-utilization of glucose, xylose, and reported . Moreovereported . F85S ofL-arabinose transport were predicted and then studied by site-directed mutagenesis. A total of five mutations, F85S, F85G, F85C, F85T, and F223Q, significantly enhanced the L-arabinose transport activity of Gal2p. Meanwhile, F85C, F85S, and F85T mutants also exhibited increased xylose transport capacity. The F85 site was shown to be a key residue in improving the L-arabinose transport activity of Gal2p, especially when changed to polar amino acids. To the best of our knowledge, this is the first study to identify the crucial amino acid residues of the L-arabinose transporter in S. cerevisiae. The positive pentose transport mutations, especially F85S of Gal2p, were useful for further improving the utilization efficiency of engineered S. cerevisiae for lignocellulosic ethanol production.In this study, the crucial amino acid residues of Gal2p for CW, XB, and YD designed the study. CW, CQ, YL, SW, and JM performed the laboratory work and analyzed the data. CW wrote the manuscript. YD, QZ, YS, and BD advised the manuscript. CW, XB, and YD supported the study.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.


While rephenotyping mutants from this study, the authors found that a subclone with a frameshift was used in the construction of the authors’ rpb1 D716K mutant and six D716K derived double mutants. In addition, they did not observe lethality of rpb1 E712A. The authors are issuing a correction to rectify this mistake.This Research Article reports that lethality was observed for rpb1 D716K and rpb1 E712A are viable  and the single substitution mutants T834P, S1091A, S1091E, S1091C, K1092A, K1092D, K1093M.Panel D in These changes do not affect the main conclusion of the article, but affect the interpretation of a small subset of D716K or E712A related mutants which are reported in the Results and Discussion. The authors have added an interpretation of transcription-related phenotypes of D716K and its relevant double mutants, and corrected an interpretation of E712A-containing double mutants in comparison to the E712A single substitution mutant. The corrected text is provided below.S and MPAS) in E1307K but not E1307A, suggesting that E1307K gained an interfering interaction to destabilize the open TL state. In contrast, mutations in the residues E712 and D716 did not confer transcription-related phenotypes.“Functional interactions among residues can be explored by the similarity between single substitution variants and the phenotypes of double mutants. We first sought evidence that variants in potential TL interaction partners could confer similar GOF or LOF phenotypes. In the simulation, K1092 switched interaction partners between two funnel helix residues D716 and E712 [18], and other charged residues were either observed or simulated to interact with S1091, K1092 or K1093 . TherefoR) when combined with other S1091 alleles , although the D716A/K or S1091A/E single substitutions did not confer strong transcription-related phenotypes  and phenotypically inert E712A/R (funnel helix), along with multiple allele specific genetic interactions ”.S8 Figx-y plot showing the lack of correlation between helical propensity change and phenotypic score on MPA, a good indicator of altered transcription activity. 120 variants from the TL tip region (top panel) and 104 variants from the same region but excluding V1094 mutants (bottom panel) are shown, with linear regression fit of the data shown in black lines. (B-E) Complementation abilities of TL tip  variants, tip proximal D716 (B), E712 (C), R1281 (D), E1307 (E) variants and the corresponding double mutants were determined by plasmid shuffling assays. (F) Transcription-related phenotypes of TL tip and the TL-proximal charged residue variants. S1091C, K1093M and E1307K confer MPAS phenotypes, and K1093M additionally confers an Spt- phenotype, while others alone don’t confer any strong transcription-related phenotypes.(A) (TIF)Click here for additional data file.S9 FigGenetic interactions between tip variants and nearby charged residues D716 (A), E712 (B), R1281 (C) and E1307  variants detected by alterations in transcription-related phenotypes. Relevant single-substitution mutant phenotypes from (TIF)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


Bats are an extremely successful group of mammals and possess a variety of unique characteristics, including their ability to co-exist with a diverse range of pathogens. The major histocompatibility complex (MHC) is the most gene dense and polymorphic region of the genome and MHC class II (MHC-II) molecules play a vital role in the presentation of antigens derived from extracellular pathogens and activation of the adaptive immune response. Characterisation of the MHC-II region of bats is crucial for understanding the evolution of the MHC and of the role of pathogens in shaping the immune system.Pteropus alecto), providing the first detailed insight into the MHC-II region of any species of bat. Twelve MHC-II genes, including one locus (DRB2) located outside the class II region, were identified on a single scaffold in the bat genome. The presence of a class II locus outside the MHC-II region is atypical and provides evidence for an ancient class II duplication block. Two non-classical loci, DO and DM and two classical, DQ and DR loci, were identified in P. alecto. A putative classical, DPB pseudogene was also identified. The bat’s antigen processing cluster, though contracted, remains highly conserved, thus supporting its importance in antigen presentation and disease resistance.Here we describe the relatively contracted MHC-II region of the Australian black flying-fox  contains supplementary material, which is available to authorized users. Bats are the second most species-rich group of mammals after rodents, accounting for approximately 20% of all classified living mammals . Bats arBartonella sp and Leptospira, have been reported in a variety of bats [Plasmodium, Polychromophilus, Nycteria, and Hepatocystis) have also been identified across both suborders of bats [Pseudogymnoascus destructans responsible for white nose syndrome (WNS), is an extreme example of an extracellular pathogen that is capable of causing disease in North American microbats [In recent years, bats have been increasingly recognised for their role in maintaining numerous pathogens , with new pathogens being continually identified each year –18. In a of bats , 19–21.  of bats . Despite of bats , 22–29.  of bats . The devicrobats , 32.PSMB8 and 9), transporter associated with antigen processing 1 and 2 (TAP1 and 2) and Tapasin (TAPBP), are also found within the class II region, forming the AP gene cluster (DOB-TAP2-PSMB8-TAP1-PSMB9-DMB-DMA-BRD2 (bromodomain - containing protein 2)-DOA) [The MHC is the most gene-dense and polymorphic region of the genome and the majority of genes encoded within this region play a role in immune defence. The MHC region of eutherian mammals can be broadly divided into three regions, class I, class II and class III. The class I and II genes are highly polymorphic and evolve through gene duplication and conversion in response to strong selection pressure by pathogens , 34. The 2)-DOA) , 42. Num 2)-DOA) .DP, DQ and DR) are responsible for the presentation of extracellular antigens  to activate CD4+ (cluster of differentiation 4) T helper cells [+ T helper cells in turn coordinates the antibody- and cell-mediated immune responses. The non-polymorphic, non-classical class-II molecules (DM and DO) do not present antigens, but instead play an important role in antigen-processing [MHC-II molecules are heterodimers consisting of non-covalently linked α and β chains encoded by separate genes within the MHC-II region. They are expressed only on the surface of antigen presenting cells, such as B cells, monocytes, macrophages and dendritic cells, and accommodate antigens of 11 to 20 amino acid residues in length . MHC-II er cells . Activatocessing .Rousettus aegyptiacus) [DRB alleles, ectoparasite load and reproductive state were also identified in the insectivorous bat, Noctilio albiventris [Despite the importance of the MHC-II region in disease resistance, few studies have described MHC-II genes in bats. The limited work that has been reported to date has focused on the DRB locus of a variety of microbats and one species of megabat (ptiacus) . Extremeptiacus) –52. Corriventris , 53, 54.Pteropus alecto, revealing a relatively condensed, yet conserved MHC-I region [P. alecto, with the aid of its whole genome sequence [Recently we described the class I region of the Australian black flying fox, I region . Comparasequence . As batssequence , the batP. alecto genome was interrogated for MHC-II, AP and conserved class II flanking genes using the BLAST algorithm [The recently completed lgorithm . A singllgorithm  for genelgorithm  against Homo sapiens), horse  and pig (Sus scrofa) MHC-II regions were adapted from the Ensembl annotation  for comparative analysis with the bat  MHC-I region using EasyFig software [The human  were retrieved from Ensembl (version GRCh37.p11). The corresponding region was obtained for the bat MHC-II genes. The promoter regions of the bat class II genes were analysed by comparison to the human genes. All sequences upstream from the start codon were analysed using Clone Manager Professional Version 9 software to manually identify putative promoter S-X-Y motifs. Sequences were then collated and aligned, with sequence logos [http://www.geneious.com/).The region 600 bp upstream of human MHC-II genes (MEGA software version 5.2.1  was usedhttp://www.ncbi.nlm.nih.gov/Genbank) accession numbers and Ensembl (http://asia.ensembl.org/index.html) transcript ID for the genes and gene products discussed in this paper are P. alecto scaffold202 (KB030441.1); Homo sapiens HLA-DMA (NM_006120); H. sapiens HLA-DOA (M26039); H. sapiens HLA-DPw3 (M27487); H. sapiens HLA-DQA (M26041); H. sapiens HLA-DRA (NM_019111); H. sapiens HLA-DMB (U15085); H. sapiens HLA-DOB (L29472); H. sapiens HLA-DPB (M57466); H. sapiens HLA-DQB (M20432); H. sapiens HLA-DRB4 (NM_021983); H. sapiens PSMB8 (ENSP00000364016); H. sapiens PSMB (ENST00000374859.2); H. sapiens TAP1 (ENST00000354258.4); H. sapiens TAP2 (ENST00000374897.2); H. sapiens TAPBP (ENST00000434618.6); Equus caballus DMA (HQ890199); E. caballus DOA (XM_005603798.2); E. caballus DQA (L33909); E. caballus DRA (JQ254083); E. caballus DMB (XM_005603792.2); E. caballus DOB (XM_005603785.2); E. caballus DRB (JQ254095); E. caballus DQB (L33910); E. caballus PSMB8 (ENSECAP00000016548); E. caballus PSMB9 (ENSECAP00000014604); E. caballus TAP1 (ENSECAT00000024416.1); E. caballus TAP2 (ENSECAP00000005757); E. caballus TAPBP (ENSECAP00000010701); Sus scrofa SLA-DMA (NM_001004039); S. scrofa SLA-DOA (NM_001113705); S. scrofa SLA-DQA1 (NM_001130224); S. scrofa SLA-DRA (NM_001113706); S. scrofa SLA-DMB (DQ431246); S. scrofa SLA-DQB (AY102478); S. scrofa SLA-DRB (AY191776); Ovis aries DQB1 (L08792); Oryctolagus cuniculus DOB (M96942); O. cuniculus DPB (ENSOCUT00000008716.3); Mus musculus DOA (M95514); M. musculus DQA (M21931); M. musculus DRA (U13648); M. musculus DMB (U35332); M. musculus PSMB8 (ENSMUSP00000025196); M. musculus PSMB9 (ENSMUST00000174576.2); M. musculus TAP1 (ENSMUST00000170086.7); M. musculus TAP2 (ENSMUSP00000025197); M. musculus TAPBP (ENSMUSP00000025161); Rattus norvegicus DQB (X56596); Gallus gallus MHC-II α chain (AY357253); G. gallus MHC-II β chain (M29763) and G. gallus DMB (AB426148).The GenBank  and kinesin family member C1 (KIFC1), were highly contracted, spanning ~0.78 Mb compared to ~1.3 – 2.2 Mb in other mammals [Sus scrofa) is the only other mammalian species known to have a contracted MHC-II region (~0.5 Mb) [The bat classical and extended MHC-II sub-regions, bordered by butyrophilin-like protein 2  , 74. A s~0.5 Mb) . The con~0.5 Mb) , 75.BTNL2 and collagen, type XI, alpha 2 (COL11A2), is highly conserved in eutherians and contains all of the MHC class II genes [P. alecto, the gene order and content remain conserved, including the presence of the entire AP gene cluster (DOB-TAP2-PSMB8-TAP1-PSMB9-DMB-DMA-BRD2-DOA). Eleven MHC-II genes were found within the bat classical class II sub-region, compared to 16 in human [COL11A2 and KIFC1, is also highly conserved, with contraction observed in the bat (~200 kb) compared with human (~250 kb), horse (~250 kb) and pig (~400 kb). The essential AP gene, TAPBP, was also highly conserved in terms of location and orientation.The organisation of the bat MHC-II region was compared with corresponding regions from the human, horse and pig genomes. The human MHC region was used as a reference since it is the most well characterised, the horse was included due to its close phylogenetic relationship with bats, and pig was included due the similarity in size of its MHC to that of bats , 76. TheII genes , 37, 40.in human , 42, sevin human  and 19 iin human . The extDP, DQ and DR that are responsible for antigen presentation. The DM and DO genes, that encode non-classical class II molecules and play a vital role in peptide loading onto classical class II molecules, were also present in the bat MHC-II region. Bats appear to lack functional DP α and β chains, with only a single DPB1 locus encoding a partial DP β chain sequence, likely corresponding to a pseudogene . The baiversity . Alterna mammals . As illug horses , sheep [g horses  and pandg horses , but notg horses  speculathan meat .Fig. 3SiDRB2 was located ~355 kb upstream of SYNGAP1 which marks the end of the extended class II sub-region in other species and ~580 kb from the nearest class II gene (DPB)  in this location in the bat genome  appear to be functional based on the presence of intact ORFs. Two of the loci have been classified as pseudogenes, due to an incomplete ORF in the case of DPB1 or the presence of stop codons within the ORF for DRB2, which is within the range of two pseudogenes in pigs to eight in humans. Five MHC-IIA and four MHC-IIB transcripts previously identified in a P. alecto transcriptome dataset [Ten bat class II genes , which interacts with several transcription factors, particularly those that bind to the SXY motif [Ptal-DMA, −DOA, −DOB, −DQA2, −DQB1, −DQB2 and -DRA), with coordinates within the class II genomic region summarised in Table Ptal-DMB, −DPB1, −DQA1, −DRB1 or -DRB2. No CAAT or TATA boxes were identified in any of the bat MHC-II genes, similar to class II genes from the Tasmanian devil [DRB2 and DPB1.The transcription of MHC-II genes is regulated by conserved sequences in the proximal promoter regions  , 81. TheXY motif . To idenan devil . AlthougHLA-DOA, −DOB, −DPA1, −DPB1, −DQA1, −DQA2, −DQB1, −DQB2, −DRA, −DRB1 and -DRB5) for comparison, sequence logo diagrams of the S-X-Y motifs of the bat and human class II genes were generated  resides within the bat classical class II sub-region as discussed above  within the class II region revealed high genetic conservation compared with other mammals. Sequence alignments of AP genes indicated high nucleotide and amino acid similarity  appear to be pseudogenes, with the DPA locus remaining elusive. All identified bat class II loci have relatively conserved gene structures and were orthologous to other mammalian class II loci. Bats also possess an atypical MHC-II region, with at least one MHC-II gene (DRB2) located outside the class II region. The presence of a class II gene outside the MHC-II region is a first for a eutherian mammal and provides evidence for an ancient class II duplication block. This first resolution of the bat MHC-II region contributes valuable information on the comparative genomic evolution of the mammalian immune system. Detailed analysis of AP genes further suggests highly conserved and functional MHC-I and -II antigen presentation pathways, supporting the importance of this region in disease resistance in general. The characterisation of genes within the MHC-II region provides the first step towards understanding their roles in resistance to disease in response to the high diversity of viruses, bacteria and parasites identified in bats. Further studies to examine the presentation of peptides associated with extracellular pathogens by MHC-II molecules may contribute to our understanding of the response of bats to infection with extracellular pathogens. This study also provides the basis for further characterisation of the diversity of individual MHC-II genes in P. alecto and the role of selective pressures, including pathogens, in shaping their diversity. Detailed characterisation of MHC-II regions from different bat species will also be required to determine whether the architectural pattern observed in P. alecto applies across other bat species.The bat MHC-II region is condensed in size but highly conserved with that of other eutherian mammals. At least 12 MHC-II genes are present in the bat genome, two of which Previously Reported Additional file 2:Ptal) MHC-II A genes, (A) DMA, (B) DOA, (C) DQA and (D) DRA, against various mammals. Dashes indicate identical residues; Dots indicate gaps; P Peptide-Binding Sites; 4 CD4 Interaction Sites; C Cysteine residues in α2 domain likely to form intra-chain disulphide bonds. Percentage similarity of sequences within a particular α domain is reflected at the end of their respective sequences, with reference to the top sequence in the alignment. Red and blue represent percentage similarity of nucleotides and amino acid residues respectively within the stated α1 and α2 domains. Hosa – Homo sapiens; Eqca – Equus caballus; Susc – Sus scrofa; Mumu – Mus musculus. (DOC 412 kb)Amino acid alignment of bat  MHC-II B genes, (A) DMB, (B) DOB, (C) DPB, (D) DQB and (E) DRB, against various vertebrates. Dashes indicate identical residues; Dots indicate gaps; P Peptide-Binding Sites; 4 CD4 Interaction Sites; C Cysteine residues in β1 and β2 domains likely to form intra-chain disulphide bonds. Percentage similarity of sequences within a particular β domain is reflected at the end of their respective sequences, with reference to the top sequence in the alignment. Red and blue represent percentage similarity of nucleotides and amino acid residues respectively within the stated β1 and β2 domains. Hosa – Homo sapiens; Eqca – Equus caballus; Susc – Sus scrofa; Mumu – Mus musculus; Ovar – Ovis aries; Orcu – Oryctolagus cuniculus; Gaga – Gallus gallus. (DOC 364 kb)Amino acid alignment of bat  antigen-processing (AP) genes, (A) PSMB8, (B) PSMB9, (C) TAP1, (D) TAP2 and (E) TAPBP, against human (Hosa), mouse (Mumu) and horse (Eqca). Dashes indicate identical residues; Dots indicate gaps. Percentage similarity of sequences is reflected at the end of their respective sequences, with reference to the top sequence  in the alignment. Red and blue represent percentage similarity of nucleotides and amino acid residues respectively. (DOC 434 kb)Amino acid alignment of bat (


Antiretroviral therapy containing an integrase strand transfer inhibitor (INSTI) plus two NRTIs has become the recommended treatment for antiretroviral-naive HIV-1-infected patients in the updated guidelines. We aimed to determine the prevalence of INSTI-related mutations in Taiwan. Genotypic resistance assays were performed on plasma from ARV-naïve patients (N = 948), ARV-experienced but INSTI-naive patients (N = 359), and raltegravir-experienced patients (N = 63) from 2006 to 2015. Major INSTI mutations were defined according to the IAS-USA list and other substitutions with a Stanford HIVdb score ≧ 10 to at least one INSTI were defined as minor mutations. Of 1307 HIV-1 samples from patients never exposed to INSTIs, the overall prevalence of major resistance mutations to INSTIs was 0.9% (n = 12), with an increase to 1.2% in 2013. Of these 12 sequences, 11 harboured Q148H/K/R, one Y143R, and none N155H. Of 30 sequences (47.6%) with INSTI-resistant mutations from raltegravir-experienced patients, 17 harboured Q148H/K/R, 8 N155H, and 6 Y143C/R. Other than these major mutations, the prevalence of minor mutations were 5.3% and 38.1%, respectively, in ARV-naive and raltegravir-experienced patients. The overall prevalence of INSTI mutations remains low in Taiwan. Surveillance of INSTI resistance is warranted due to circulation of polymorphisms contributing to INSTI resistance and expected increasing use of INSTIs. Integrase strand transfer inhibitor (INSTI)-based regimens are recommended as first-line combination antiretroviral therapy (cART) in several updated treatment guidelines for HIV after the efficacy of the three INSTIs  has been demonstrated in randomized clinical trials1234567With the widespread use of INSTIs, surveillance of the prevalence of INSTI resistance among ART-naive patients is critical in optimizing ART efficacy, given the concerns that INSTI, particularly raltegravir and elvitegravir, have a relatively lower genetic barrier to emergence of resistance mutations when compared with boosted PIs101110 copies/mL , with 33.1% of the patients having PVL > 5 log10 copies/mL. The mean CD4 count was 299 cells/mm3 , with 31.7% having CD4 counts < 200 cells/mm3.From June 2006 to October 2015, a total of 1370 non-duplicated blood specimens were subjected to genotypic analysis of INSTI resistance at the National Taiwan University Hospital (NTUH). These specimens were from ART-naïve patients (N = 948), ART-experienced patients who had never been exposed to INSTIs (N = 359), and patients receiving raltegravir-containing regimens and experiencing virological failure (N = 63). Baseline characteristics of the patients included in this study are shown in P < 0.001), were younger  and were more likely to be MSM  and infected with subtype B virus , and to have higher mean CD4 count .Compared to ART-experienced/INSTI-naive patients, ART-naïve patients had a higher percentage of male gender . While the first case of INSTI-related resistance mutations was identified in a patient included in November 2006, the majority of the cases (n = 9) were identified in patients included in 2013. The prevalence of INSTI-related resistance mutations was <1% before 2012 , which increased to 1.2% (9/766) in 2013, and gradually declined to 0.5% (1/206) in 2014–15.Among the 1,307 INSTI-naive study subjects, 12 (0.9%) were infected with HIV-1 that exhibited major mutations shown to cause a marked reduction of viral susceptibility to raltegravir and elvitegravir . The preThe 12 INSTI-resistant HIV-1 sequences were all of subtype B; 6 of them were from ART-naive and 6 from ART-experienced/INSTI-naive patients. The amino acid mutations/polymorphisms detected in these individuals are listed in The presence of INSTI resistance mutations was also determined in 63 INSTI-experienced patients. Thirty patients (47.6%) harboured INSTI-related major mutations, which included 17 with Q148H/K/R mutations, eight with N155H mutations, and six with Y143C/H/R mutations. The concurrent major/minor INSTI-related mutations in these sequences are summarized in Beside major INSTI mutations, other integrase substitutions with a Stanford HIVdb score ≥ 10 to at least 1 INSTI, and some polymorphisms that have been reported in previous studies or observed in this study were also determined. Their overall prevalence in INSTI-naive patients and INSTI-experienced patients was 5.2% (69/1307) and 38.1% (24/63), respectively. In INSTI-naive patients, the resistance mutations included L74M (n = 28), E92V (1), Q95K (4), T97A (4), E138AK (3), Y143S (1), V151AL (1), N155S (9), E157Q (11), G163K/R (5), S230R (1) and R263K (4) . The preP < 0.001), nucleos(t)ide reverse-transcriptase inhibitor (NRTIs) , and multi-drug resistance (MDR)  among patients with HIV-1 sequences harbouring INSTI mutations were all significantly higher than those without INSTI major mutations (Among the 1307 HIV-1 specimens from INSTI-naive (948 ART-naive and 359 ART-experienced) and 63 raltegravir-experienced patients, the PR and RT genes were successfully amplified and sequenced in 1292 specimens (94.3%). The baseline demographic and clinical characteristics of the patients with PR and RT genes successfully sequenced were similar to those without analyzable RT/PR sequences (data not shown). Among the 6 samples from ARV-naive patients with INSTI major mutations, only one harboured M184V mutation , known tutations .P = 0.02) and INSTI as the third agent . Accordingly, a significantly lower percentage of them used abacavir/lamivudine as the backbone  and PI as the third agent .When the current regimens used by the ART-experienced patients were analyzed, we found that zidovudine/lamivudine, abacavir/lamivudine, and tenofovir disoproxil fumarate (TDF)/emtricitabine or TDF and lamivudine were the major NRTI backbone antiretrovirals that were used in these patients . The thiPhylogenetic analysis was conducted to determine whether there were clusterings observed among these integrase sequences amplified from INSTI-resistant sequences . Three tIn this study conducted in Taiwan, where raltegravir was only available for clinical use since 2009, we demonstrate that the prevalence of major mutations to INSTI remains relatively low in INSTI-naive patients, 0.6% (6/948) among ART-naive and 1.7% (6/359) among ART-experienced/INSTI-naive patients, which is somewhat different from the absence of major mutations in ART-naive patients reported in the US and Europe1011et al.In patients experiencing virological failure to the first generation of INSTIs, raltegravir and elvitegravir, three genotypic mutation pathways have been defined: Q148H/R/K (±G140S or E138A/K), N155H (±E92Q), and Y143C/H/R (±T97A). Except E92Q, the amino acid substitutions in the parenthesis representing the compensatory mutations that can help restore fully or partly viral fitness1415in vitro studies19et al. in the UKP = 0.02). Whether decreased tolerance to zidovudine/lamivudine may contribute to the risk of accumulating secondary mutations to raltegravir remains to be investigated. Besides the Q148 mutation combined with one or more of G140A/C/S, L74I and E138A/K/T was identified to reduce viral susceptibility to dolutegravir, two amino acid mutations, G118R and R263K, have been reported to confer low-level resistance to dolutegravir22The susceptibility of these INSTI-resistant sequences to the second generation of INSTI, dolutegravir, was also evaluated based on the results from et al. who analyzed 56 specimens from the 278 specimens by 454 sequencing to detect the presence of scarce INSTI-related mutations found that the prevalence of mutations with Stanford HIVdb score ≧ 10 increased to 14.3%Although raltegravir and elvitegravir have a relatively lower genetic barrier than PIs and most NRTIs, the prevalence of primary INSTI resistance mutations is relatively rare based on a few published reports10111011in vitro passage experiment in the presence of raltegravir, and it has never been reported in sequences from patients receiving raltegravirIn INSTI-naïve patients, we observed that 0.7% of the sequences (N = 9) harboured the N155S/T mutation, which was not observed in any of the raltegravir-experienced patients in our study. N155S was first identified by Entamoeba histolytica have been observed in our MSM population in Taiwan25262728In this study, an increased prevalence of INSTI-related genetic mutations was noted in 2013. It could be related to transmission of resistant strains among patients with risky behaviors since increasing incidence of HDV, HCV, syphilis, and The findings of this study should be interpreted with the necessary caution. First, the study was based on patients included mainly in hospitals in northern Taiwan, where the majority of the patients were MSM. While MSM have become the leading risk behavior for HIV infections and TDR is more prevalent among MSM in Taiwan30In conclusion, the prevalence of primary INSTI resistance of the HIV-1 strains identified from ARV-naive patients in Taiwan remains relatively low, which is comparable to the previous reports in high-income countries. Nevertheless, given our previous observation that TDR to other antiretrovirals remains high among MSM in Taiwan, close and vigilant monitoring of INSTI resistance is important before its widespread use in Taiwan.In Taiwan, cART has been provided free of charge since its introduction in April 1997, but routine drug-resistance testing has not been available to clinicians. By the end of December 2015, there were a total of 31,036 people diagnosed as having HIV infection in Taiwan, and about 70% of those who survived were receiving cART. The prevalence of transmitted drug resistance (TDR) in Taiwan has increased from 1999 to 2006, with an overall prevalence of 9.4%TM Test, version 1.5,  and FACSFlow (Becton Dickinson), respectively. The study was approved by the Research Ethics Committee of the National Taiwan University Hospital, Taipei, Taiwan  and the experiment procedures were carried out in accordance with the approved guidelines. The patients who were linked to HIV care at the hospital gave written informed consent for determination of HIV-1 resistance mutations.HIV-infected Taiwanese patients receive HIV care according to the national treatment guidelines at designated hospitals around Taiwan by the Centers of Disease Control in Taiwan. Residual plasma samples for routine determination of plasma viral load (PVL) were obtained from HIV-1-infected patients seeking HIV care at the NTUH. A standardized case record form was used to collect information on demographics, antiretroviral medications, baseline CD4 lymphocyte count, and PVL. PVL and CD4 count were quantified with the use of Cobas Amplicor HIV-1 Monitorhttp://hivdb.stanford.edu; date last accessed, 10 November 2015), in accordance with the drug resistance mutation list of the International AIDS Society-USA Consensus GuidelinesFor those with confirmed HIV infection and linked to clinical care at NTUH, the genotypic resistance assays were performed retrospectively as described previously30integrase coding regions was PCR-amplified as described previouslypol2950A (5′-TCA KCA CCT GCC ATC TGT TTT CC-3′)/2064A (5′-AYA ARG GRA TTG GAG GAA ATG AAC A-3′). The amplification condition was 35 cycles of 94 °C for 15 s, 55 °C for 1 min, 72 °C for 2 min, and a final extension at 72 °C for 7 min. The major INSTI mutations, which have been determined having a marked reduction of viral susceptibility to raltegravir and elvitegravir, include Y143C/H/R, Q148H/K/R, and N155H. Besides these three major mutations, the integrase substitutions with a Stanford HIVdb score ≧ 10 to at least one INSTI were included, such as H51Y, T66A/I/K, L74M, E92G/Q/V, Q95K, T97A, F121Y, E138A/K, G140S/C/A, Y143G/K/S/A, P145S, Q146P, S147G, V151A/L, S153F/Y, N155S/T, E157Q, G163 K/R, S230R, and R263K. Some amino acid substitutions  that have been described in more than 2% of the study population in previous studies1113To determine the resistance mutations related to INSTIs, an 884-bp fragment of http://hiv-web.lanl.gov/seq-db.html). Sequences were aligned with the Clustal W listed in the MEGA  analytical package (version 3.0)Reference sequences of various subtypes and recombinants were retrieved from the Los Alamos database . Categorical data were analyzed using chi-square or Fisher’s exact tests, as appropriate, and continuous variables were compared using the Wilcoxon test. All tests were two-tailed and a P value < 0.05 was considered significant.How to cite this article: Chang, S.-Y. et al. Prevalence of Integrase Strand Transfer Inhibitors (INSTI) Resistance Mutations in Taiwan. Sci. Rep. 6, 35779; doi: 10.1038/srep35779 (2016).


Caenorhabditis elegans epidermal stem cells, known as seam cells, as a readout. We employ genetics, cell lineage tracing, and single molecule imaging to show that mutations in lin-22, a Hes-related basic helix-loop-helix (bHLH) transcription factor, increase seam cell number variability. We show that the increase in phenotypic variability is due to stochastic conversion of normally symmetric cell divisions to asymmetric and vice versa during development, which affect the terminal seam cell number in opposing directions. We demonstrate that LIN-22 acts within the epidermal gene network to antagonise the Wnt signalling pathway. However, lin-22 mutants exhibit cell-to-cell variability in Wnt pathway activation, which correlates with and may drive phenotypic variability. Our study demonstrates the feasibility to study phenotypic trait variance in tractable model organisms using unbiased mutagenesis screens.Biological systems are subject to inherent stochasticity. Nevertheless, development is remarkably robust, ensuring the consistency of key phenotypic traits such as correct cell numbers in a certain tissue. It is currently unclear which genes modulate phenotypic variability, what their relationship is to core components of developmental gene networks, and what is the developmental basis of variable phenotypes. Here, we start addressing these questions using the robust number of  C. elegans is notable for its highly reproducible development, showing an almost invariant pattern of cell division and differentiation during development; it is thus an ideal model organism in which to search for genes that regulate phenotypic consistency among genetically identical individuals. We focus on a group of lateral epidermal cells—the seam cells—which undergo stem cell-like divisions during postembryonic development. These divisions can either be symmetric towards the seam cell fate, acting to increase the total number of cells, or asymmetric, giving rise to one daughter cell that differentiates into its final fate and another one that serves to keep the number of seam cells constant. We show here that mutations in the transcription factor lin-22 increase seam cell number variability due to stochastic conversion of symmetric divisions into asymmetric ones and vice versa during development, thereby altering the number of terminal seam cell number in opposing directions. We also show that the observed phenotypic variability correlates with the stochastic activation of the conserved Wnt signaling pathway. Our work suggests that core components of developmental gene networks modulate phenotypic variability in multicellular animals.Organisms are exposed to both internal and external perturbations in every molecular process they go through, and robustness—the ability to maintain their systems unchanged—is crucial for their development and survival. However, the processes that keep the variability of cells as low as possible are barely known. The nematode  It is remarkable how biological systems manage to operate consistently despite facing several types of variation, including the intrinsic stochasticity in every molecular process. This ability of a given system to produce an invariable output in the presence of internal and external perturbations is called robustness , 2. DeveHsp90 impairment are not understood. Furthermore, recent evidence has suggested a more complex picture as perturbations of Hsp90 can also reduce phenotypic variance, indicating a dual role for this chaperone as either a potentiator of variability or buffer  was injected at 30 ng/μl with myo-2∷dsRed as co-injection marker.To construct a vector to allow seam cell transgene expression, we used the last intron of All constructs used in this study were verified by sequencing and at least 2 independent transgenic lines were obtained and compared. A list of all oligos used in this study is presented in lin-22 RNAi feeding vector, lin-22 was amplified using the TOPO cloning compatible lin22-15F primer and lin22-14R from the fosmid WRM0627dG07 . The amplicon was inserted by TOPO cloning in pDONR/D-TOPO vector, creating the entry vector pENTR lin-22. The pENTR lin-22 plasmid was used to insert lin-22 in a gateway compatible L4440 vector via an LR reaction. To construct a vrp-1 RNAi feeding vector, vrp-1 was amplified from N2 genomic DNA using primers Y54G2A.3a F1 and Y54G2A.3a R1, first cloned in a pDNR/D-TOPO vector  and then inserted into a gateway compatible L4440 vector. Both feeding vectors were transformed into Escherichia coli HT115 to be used for nematode feeding. The elt-1 RNAi feeding vector is pAW565 as described in [Animals were fed with dsRNA expressing bacteria as a food source. Bacteria were grown overnight and then seeded directly onto NGM plates containing 1 μM IPTG, 25 μg/ml ampicillin and 6.25 μg/ml tetracycline. To construct a ribed in . All othS1 TextCR1, conserved region 1; CR2, conserved region 2.(DOCX)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.S2 Data(XLSX)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S1 FigHsp90/daf-21 leads to marginal seam cell number variability in the seam (n ≥ 40). Red star depicts change in variance with a Levene’s median test (* P < 0.05). Error bar shows mean ± SD. (B) Graph showing the selected recombinant lines with CB4856 based on quantitative phenotyping of seam cell number standard deviation (SD), percentage of animals with extreme seam cell counts, and percentage of animals with 16 seam cells. Each circle represents 1 line. The parental vsc1 mutant strain is depicted in red (SD = 1.9) and the wild-type JR667 in blue (SD = 0.3). (C) Mapping the causative mutation in vsc1 by whole genome sequencing of recombinant lines with CB4856. Graphs show the ratio of mapping strain (CB4856) alleles to the total number of reads for 2 different chromosomes. Arrow points to the left arm on chromosome IV that lacks mapping strain polymorphisms. Another chromosome (III) is shown for comparison. Numerical data used for S1 Fig A, B can be found in (A) Down-regulation of (TIF)Click here for additional data file.S2 Fign = 43) and lin-22 mutants (n = 43). (C-D) Phenotypic comparison between lin-22 RNAi treated animals (n = 30) and control (empty vector) treatment (n = 29). RNAi-treated animals show multiple PDE neurons (C) and seam cell number variance (D). (E-F) Phenotypic comparison between vrp-1 RNAi treated animals (n = 35) and control (n = 40). No defect was found with regard to number of PDE neurons (E) or seam cell number (F). (G) Quantification of seam cell number in lin-22(icb38) mutants based on the bro-1CNE∷GFP marker (n ≥ 32). (H-I) Phenotypic characterisation of lin-22(icb49) in the CB4856 background, showing multiple PDE neurons (n ≥ 31) (H) and seam cell number variance (n ≥ 30) (I). (J) Quantification of seam cell number in males carrying the lin-22(icb-38) mutation (n = 31). Note that terminal seam cell number in wild-type males is 18 per lateral side. (K) Heatmap illustrating the relationship between seam cell number counts on 1 lateral side and those on the other lateral side in wild-type and lin-22(icb38) animals. The majority of animals show 16 seam cells on both sides in wild-type and moderate correlation of errors (R = 0.37). In lin-22(icb-38) mutants, there is even less correlation between the seam cell number deviations on one side and the other (R = 0.23). Black stars show statistically significant changes in the mean with a t test or one-way ANOVA and Dunnett’s test; red stars depict changes in variance with a Levene’s median test as follows: *** P < 0.001, **** P < 0.0001. For PDE scorings, error bars show mean ± SEM and for seam cell number counts error bars show mean ± SD. Numerical data used for S2 Fig A, B, C, D, E, F, G, H, I, J, K can be found in (A-B) PDE neuron number (A) and seam cell number (B) comparison between wild-type animals ((TIF)Click here for additional data file.S3 Figlin-22 promoters that are conserved between the following Caenorhabditis species: C. elegans, C. briggsae, C. remanei, and C. angaria. The position of these elements is shown with the C. elegans lin-22 promoter as a reference. Note that CR1 overlaps with Y54G2A.67 that is annotated on Wormbase as a putative noncoding RNA. Part of the CR1 sequence with 2 putative GATA sites and the position of the icb38 and ot269 mutations are also shown. (B) lin-22 smFISH in late L1 wild-type and lin-22(icb38) animals. In wild-type lin-22, spots were observed in anterior seam cells and not posterior (dashed line marks the seam cell boundary). (C) Quantification of lin-22 spots in the 4 V1-V4 daughter cells early (n ≥ 11) and late (n ≥ 22) after the asymmetric division. (D) lin-22 smFISH in wild-type and lin-22(icb38) L4 animals. Note expression in intestinal cells in the mutant (arrows). Nuclei DAPI staining is shown in magenta. (E) Quantification of lin-22 spots in pooled posterior V1–V4 daughter cells at the L2 asymmetric division stage in wild-type animals treated with control bacteria (n = 93), and elt-1 (n≥57) or egl-18 RNAi (n = 90). Black stars show statistically significant changes in the mean with one-way ANOVA and Dunnett’s test as follows: *** P < 0.001, **** P < 0.0001. Scale bar in B, D is 10 μm and black spots correspond to mRNAs. Error bars in C, E show mean ± SEM. Numerical data used for S3 Fig C, E can be found in (A) Vista analysis (70% identity and 100 base-sliding window) depicting 2 regions (CR1 and CR2) in (TIF)Click here for additional data file.S4 Figlin-22(icb38) animals. Solid black lines indicate seam cell fate, gray lines indicate daughters that differentiate into hypodermal cells, and blue dots depict lineages that give rise to PDE neurons. Errors in the lineages that result in terminal seam cell number reduction derive from loss of the L2 symmetric division of V1–V4 cells and adoption of a V5-like pattern (highlighted in red boxes). Hybrid lineages where ectopic neurogenesis co-occurs with seam cell fate maintenance (thus do not change seam cell number) are shown in yellow boxes. Errors that increase the terminal seam cell number, such as V1–V4 symmetric divisions at the L3/L4 stage or H2 symmetric divisions at the L2 stage, are shown in blue and green boxes, respectively. Errors that do not change the terminal seam cell number, such as V cell polarity defects mostly at the L4, V1–V4 cells skipping an asymmetric division, or V1–V4 cells showing an extra asymmetric division are highlighted in gray. Note that timing of divisions is not generally affected to suggest broad developmental timing defects. However, within single lineages we observed a rare manifestation at the L3 stage of a repeat of the L2 pattern of V1–V4 symmetric division followed by an asymmetric division (see lineage right underneath panel A). Asterisks depict lineages that show errors that both decrease and increase the total seam cell number for that lineage. Numerical data used for S4 Fig A can be found in (A) Heat map illustrating the increase (in blue) or decrease (in red) in cell number output per cell lineage (H0–V6) compared to the wild-type. Each line is an independent lateral side of one animal, while white colour indicates a wild-type cell number output. Note the presence of lineages producing extra and fewer cells within the same lateral side. (B) Seven representative postembryonic lineages of H0–V6 seam cells from (TIF)Click here for additional data file.S5 Figmab-5 expression by smFISH. Note that posterior V(1–4).pp cells express mab-5 at similar levels to that of wild-type V5. Error bars show mean ± SEM. (B) Quantification of the frequency of symmetrization of elt-1 expression in the H2 daughters (n = 13) and loss of expression in anterior V cell daughters (n ≥ 30) in lin-22(icb38) mutants assessed by smFISH. (C) Quantification of the frequency of detection of mab-5 expression in posterior daughters of the H2 (n = 10) and V1–V4 (n ≥ 20) cells in lin-22(icb38) mutants assessed by smFISH. (D) Representative smFISH image of lin-22(icb38) mutants using an elt-1 or mab-5 probe during the symmetric division of L2 stage and an egl-18 probe at the asymmetric L2 stage. Arrowheads mark the pattern changes described in this work. Note cell-to-cell variability as V3 in these particular images does not show the described pattern change while adjacent cells do. (E) Quantification of cases in which the V.paa daughter cell in lin-22(icb-38) mutants expresses egl-18 (assessed by smFISH) outside the WT range. Red box marks such cases, grey depicts expression within the WT range, and white when expression was nondetermined due to lack of expression data for this cell. Lines correspond to different animals. Note cell-to-cell variability in the vast majority of animals. (F) Fluorescent images of the wild-type and lin-22(icb38) head region with seam cells marked by scm∷GFP. Note the presence of H cell clusters in lin-22(icb38) mutants in which H cells appear to be in duplicates (arrowheads). (G) The occurrence of the above phenotype was scored in early adult animals selected for presence  or absence  of POPHHOP marker expression in the head during the L2 stage. A random population (n = 38) was also scored in parallel for the phenotype of interest. Note the significant increase in the frequency of the phenotype in animals selected for the presence of POPHHOP signal (positive) in comparison to both the negative and random populations. (H) lin-17 smFISH images at the L3 stage showing the entire anterior-posterior axis of a wild-type (same with lin-22(icb38) mutant animal. Black stars show statistically significant changes with a Fisher’s test: * P < 0.05, ** P < 0.01. Scale bar in D, F, H is 10 μm, and black spots in D, H correspond to mRNAs. Black bars in B, C, G show frequency ± standard error of the proportion. Numerical data used for S5 Fig A, B, C, G, E can be found in (A) Quantification of (TIF)Click here for additional data file.S6 FigP-value of < 0.1 in lin-22(icb38) mutants. (B) Heat map showing the expression of selected genes. Changes in ceh-16, lin-14, nhr-25, egl-18 in lin-22(icb38), and lin-22(ot269) are statistically significant. In both panels, the color of each cell in the heat map indicates the difference in the mean expression level in lin-22 mutants relative to the wild-type JR667 strain as per the color key.(A) Heat map showing the expression of the top 50 upregulated genes that are statistically significant at FDR-adjusted (TIF)Click here for additional data file.S7 Figlin-22(icb38) animals at the early L3 stage . (B) Quantification of the number of π cells in wild-type (n = 29) and lin-22(icb38) (n = 34) animals at the L3 stage. (C) Quantification of the number of intestinal nuclei in wild-type and lin-22(icb38) animals at the early L1 stage (n = 39). Black stars show statistically significant changes in the mean with a t test as follows: ** P < 0.01. Error bars show mean ± SD. Numerical data used for S7 Fig A, B, C can be found in (A) Quantification of the average number of induced vulval cells in wild-type and (TIF)Click here for additional data file.


Here we describe derivatives of the HEK293T cell line that are defective in their ability to generate mucin-type O-linked glycosylation. Using CRISPR/Cas9 and a single-cell GFP-sorting procedure, the UDP-galactose-4-epimerase (GALE), galactokinase 1 (GALK1), and galactokinase 2 (GALK2) genes were knocked out individually and in combinations with greater than 90% of recovered clones having the desired mutations. Although HEK293T cells are tetraploid, we found this approach to be an efficient method to target and disrupt all 4 copies of the target gene. Deficient glycosylation in the GALE knockout cell line could be rescued by the addition of galactose and N-acetylgalactosamine  to the cell culture media. However, when key enzymes of the galactose/GalNAc salvage pathways were disrupted in tandem (GALE+GALK1 or GALE+GALK2), O-glycosylation was eliminated and could not be rescued by the addition of either galactose plus GalNAc or UDP-galactose plus UDP-GalNAc. GALK1 and GALK2 are key enzymes of the galactose/GalNAc salvage pathways. Mass spectrometry was performed on whole cell lysate of the knockout cell lines to verify the glycosylation phenotype. As expected, the GALE knockout was almost completely devoid of all O-glycosylation, with minimal glycosylation as a result of functional salvage pathways. However, the GALE+GALK1 and GALE+GALK2 knockout lines were devoid of all O-glycans. Mass spectrometry analysis revealed that the disruption of GALE, GALK1, and GALE+GALK2 had little effect on the N-glycome. But when GALE was knocked out in tandem with GALK1, N-glycans were exclusively of the high mannose type. Due to the well-characterized nature of these five knockout cell lines, they will likely prove useful for a wide variety of applications. Cell lines that are deficient in their ability to form particular protein-linked carbohydrate have been a critical tool for the study of N- and O-linked glycans across many fields of research –5. Such The Leloir pathway of galactose metabolism is highly conserved from bacteria to humans . The enzPreviously, using non-specific methods, a derivative of the Chinese hamster Ovary (CHO) cell line was developed that is devoid of GALE activity –23. ThisGALE, GALK1, and GALK2, we developed glycosylation-defective mammalian cell lines in a HEK293T background. These cell lines are not only phenotypically defined but also genetically defined. These cell lines are now made available to the scientific community for a broad range of possible projects.In this study, we set out to develop a set of human cell lines for the study of O-linked glycosylation. By using CRISPR/Cas9 to make targeted mutations in GALE (Gene ID: 2582), GALK1 (Gene ID: 2584), and GALK2 (Gene ID: 2585). Target sequences were determined using the GeCKO v2 human library. Three gRNAs per target gene were used, targeting both strands of DNA, to ensure full knockout (KO) of the gene of interest. gRNAs were cloned into an expression vector with a GFP tag to allow for single cell GFP sort.Guide RNAs (gRNAs) (SantaCruz Biotechnology) were generated to our genes of interest: CCGGGATTACATCCATGTCGTCAGCTCCTGGACCCGCCGCCAGGCTGGGGTTGGCGTAACCACAATAGCTGCCCGCGCCCACGCGCTGCTCATTGACTGCTCGGTGGGCCAACTATGTCAGAAAACGTACGCTCTCCAACGCCAAGAGTGAGCGTTACATGCCAATGTAACGCTCACTCTHEK293T cells (ATCC) were transfected using jetPrime transfection reagent (Polyplus-Transfection) with three gRNAs for the particular target gene. Cells were examined by GFP fluorescent microscopy at 24 hours post-transfection using a Zeiss Axio Observer A1 Microscope to gauge sufficient levels of expression necessary for downstream flow cytometric analysis and cell sorting. Axiovision software was used for image acquisition. Following microscopy, cells were harvested, washed in PBS, and resuspended in DMEM with 1mM EDTA to prevent the formation of cell aggregates. Cells were sorted on a 5 laser 17-color BD FACS SORP Aria-IIu with an Automatic Cell Deposition Unit (ACDU). The top 10% GFP-expressing cells were individually sorted into a 96-well plate. FSC-W by FSC-A and SSC-W by SSC-A were used to reduce the rate of duplets. Four hours post-sort, cells were inspected to ensure that all wells contained only one cell. Any wells that contained duplets were excluded from further processing. Once clones reached confluency in a 6-well plate, cells were lysed in RIPA buffer (Life Technologies) and used for western blot analysis.Upon acceptance of this manuscript, GALE, GALE+GALK1, GALK1, GALE+GALK2, and GALK2 knockout cell lines will be submitted to the ATCC for use as research reagents.Cell lysate was harvested by incubating cells in RIPA buffer. Clarified lysate was loaded onto a 4–12% SDS-PAGE gel (Life Technologies). Wild-type HEK293T cell lysate was loaded in the first lane as a control. Protein was transferred to a PVDF membrane using the iBolt Dry Blotting System (Life Technologies). Membranes were probed with anti-GALE (C-4), anti-GALK1 or anti-GALK2 antibodies followed by a secondary HRP-conjugated antibody staining using the iBind western system (Life Technologies). Membranes were developed using the SuperSignal Pico Substrate (ThermoFisher) and images captured on a ImageQuant LAS 4000 mini Luminescent Image Analyzer .® 2.0 Protein Staining Device (Genscript). The second gel was probed for gp120 using 500ng/ml rhesus anti-gp120 monoclonal antibody 3.11H (purified in-house). The third gel was probed for O-glycosylation using 4μg/ml HRP-labeled Jacalin lectin (BioWorld). For protein production, cells were grown in 10% FBS, 3% lipo-depleted fetal bovine serum (LDFBS), or 3% LDFBS with galactose and GalNAc (+sugars) for 4 days until 12 hours post-transfection, at which point cells were washed and changed to serum free media. In a separate experiment, galactose and GalNAc were added to the cell culture media separately to identify whether galactose or GalNAc was the limiting precursor for O-glycosylation.GALE enzymatic activity was analyzed on the protein level. GALE knockout cells (GALE KO) were transfected with an expression vector for SIVmac239 gp120 made as a truncated secreted product with a C-terminal polyhistidine tag. Secreted gp120 was purified from supernatant 48 hours post transfection using HisPur nickel-NTA columns (Thermo Fisher). 3μg of purified gp120 was run on three 4–12% SDS-PAGE gels in triplicate. The first was analyzed by Coomassie staining using the eStainGALK1 enzymatic activity was analyzed on the protein level. HEK293T, GALK1 KO cells, and GALE+GALK1 double KO cells were transfected with expression vectors for SIVmac239 gp120 made as a truncated secreted product with a C-terminal polyhistidine tag. Secreted gp120 was purified from supernatant 48 hours post transfection using nickel-NTA columns. 3μg of purified gp120 was run on two 4–12% SDS-PAGE gels in duplicate. The first gel was probed for gp120 using 500ng/ml rhesus anti-gp120 monoclonal antibody 3.11H. The second gel was probed for O-glycosylation using an HRP labeled Jacalin lectin. For protein production, cells were grown in serum free media, serum free media +galactose, or serum free media +galactose +GalNAc as indicated.GALK2 enzymatic activity was analyzed at the protein level. HEK293T, GALK2 KO cells, and GALE+GALK2 double KO cells were transfected with expression vectors for SIVmac239 gp120 made as a truncated secreted product with a C-terminal polyhistidine tag. Secreted gp120 was purified from supernatant 48 hours post transfection using nickel-NTA columns. 3μg of purified gp120 was run on three 4–12% SDS-PAGE gels in triplicate. The first was analyzed by Coomassie staining. The second gel was probed for gp120 using 500ng/ml rhesus anti-gp120 monoclonal antibody 3.11H. The third gel was probed for O-glycosylation using an HRP-labeled Jacalin lectin. For protein production, cells were grown in serum free media, serum free media +galactose +GalNAc (+sugars), or serum free media +UDP-galactose +UDP-GalNAc (+UDP) as indicated.RNA was purified from mutant cell lines using the Quick-RNA Miniprep Plus Kit (Zymo Research). First-strand complementary DNA (cDNA) was synthesized by priming with oligo-DT and reverse transcribed using the Super Script IV Reverse Transcriptase (Thermo Fisher) according to manufacturer’s specified protocol.E. coli (Thermo Fisher) by heat shock transformation techniques. 15 colonies were grown up for each gene of interest and purified using the ZR Plasmid Miniprep kit (Zymo Research). Sequencing was performed by Genewiz on miniprep DNA using the T7 and M13R sequencing primer sites of the pCR-Blunt sequencing vector.Genes of interested were PCR amplified from cDNA using gene specific primers designed to flank areas just outside target gRNA sites and the Phusion Hot Start II High-Fidelity PCR Master Mix (Thermo Fisher) according to manufacturer’s specified protocol. Bands of the correct length were gel purified using the Zymo Gel DNA Recovery Kit (Zymo Research) and blunt-end ligated into a sequencing vector using the Zero Blunt TOPO PCR Cloning Kit (Thermo Fisher). Ligation was transformed into One Shot TOP10 Chemically Competent Upon acceptance of this manuscript, sequencing results from the GALE, GALE+GALK1, and GALE+GALK2 cell lines will be submitted to the Genbank sequence database.Glycoprotein derived N-linked and O-linked glycans were extracted from the various HEK293T cells as previously described . Glycans4 cells were used to seed wells in triplicate. Every 24 hours cells were trypsinized and counted using a Beckman Coulter Z1 Coulter Particle Counter. Measurements were taken for 4 days at which time cells reached relative confluence.HEK293T, GALE KO, GALK1 KO, GALE+GALK1 KO, GALK2 KO, and GALE+GALK2 KO cells were plated in 6 well plates on day 0. 4x10GALE, GALK1, and GALK2 genes were cloned into an expression vector containing a green fluorescent protein (GFP) tag. HEK293T cells were transiently transfected with three gRNA expression vectors all targeting the same gene. Cells were examined by GFP fluorescent microscopy at 24 hours post-transfection to gauge sufficient levels of expression necessary for downstream flow cytometric analysis and cell sorting (DNA sequences encoding Guide RNAs (gRNAs) that target human  sorting . Cells w sorting . Cells wGALE gene. Of the 15 cDNA clones used for sequencing, none had an intact copy of the GALE gene, supporting the claim that all copies of the GALE gene were successfully knocked out in GALE KO clone 4  were analyzed by western blot, GALE was undetectable for all 8 clones . GALE KO clone 4 .GALE KO clone 4 was chosen for further validation at the enzymatic level. To analyze if GALE KO clone 4 was indeed defective for GALE enzymatic activity, wildtype HEK293T and GALE KO cells were transiently transfected with an expression vector encoding truncated secreted SIVmac239 gp120 with a polyhistidine tag. SIVmac239 gp120 is known to contain 4 sites of O-glycosylation and to react strongly to Jacalin lectin, , 27 makiGALE enzymatic activity is only one way that UDP-GalNAc and UDP-galactose is generated inside a cell. Cells can also salvage exogenous GalNAc and galactose from the cell culture media and convert these salvaged sugars into usable UDP-sugar precursors for glycosylation . Due to 3μg of purified SIV gp120 generated in parental HEK293T cells and in the GALE KO cell line were loaded onto an SDS-PAGE gel and confirmed by Coomassie Blue staining and with the rhesus anti-gp120 monoclonal antibody (3.11H) probe . When prSince FBS is known to contain high levels of galactose and glycoproteins, it was somewhat surprising that no O-glycosylation was observed in GALE KO cells grown in 10% FBS. Previously, it was found that the GALE-deficient ldlD cells can salvage sugar precursors from serum containing media to restore O-glycosylation . To furtGALK1 gene and sorted for high GFP expression as outlined above. Since the levels of GALK1 protein were not easily detectable by western blot, potential clones were sequenced to identify clones with a disrupted GALK1 gene . Both HEK293T and GALE KO cells were transfected with gRNAs for the LK1 gene . To furtGALE gene. However, when GALK1 was knocked out in combination with GALE, no O-glycosylation of SIV gp120 was observed in all culture conditions. Since no O-glycosylation was observed when GALE+GALK1 KO cells were cultured with the addition of GalNAc and galactose, we can conclude that all copies of the GALK1 gene were appropriately disrupted.Truncated secreted SIV gp120 was generated in both our HEK293T-GALK1 KO and HEK293T-GALE+GALK1 KO cell lines. Since GALK1 is a critical enzyme in the salvage of galactose, we cultured each line with either galactose alone or GalNAc and galactose. As expected, GALK1 KO cells produced fully glycosylated SIV gp120 in all culture conditions . This isThe addition of GalNAc to a threonine or serine is the first step in all forms of mucin-type O-glycosylation. Since it remains possible that the GALE KO cell line could salvage a small amount of GalNAc from glycoproteins present in FBS, we decided that a HEK293T-GALE+GALK2 double knockout cell line (GALE+GALK2 KO) would be a useful tool for experiments when it is imperative to use a cell line devoid of all O-glycosylation. With this cell line, there would be no concern of salvage of GalNAc in all media conditions.Following transfection, GFP sort, and expansion, cell lysate from 7 GALE+GALK2 knockout clones was analyzed for the presence of full-length GALK2 protein by western blot. GALK2 protein was detectable by western blot at a size of 61kDa in wildtype HEK293T cells. When cell lysate from our GALE+GALK2 knockout cell lines was analyzed by western blot, no presence of GALK2 was detectable in lanes 2, 3, 5, 6, and 8 . GALE+GAGALK2 gene. Of the 15 cDNA clones sent for each cell line, none had an intact copy of the GALK2 gene, further supporting the claim that the GALK2 was successfully knocked out (Clones for both HEK293T-GALK2 (clone 7) and HEK293T-GALE+GALK2 (clone 7) were sequenced for mutations in the cked out .GALE gene. However, when GALK2 was knocked out in combination with GALE, no O-glycosylation was observed in all culture conditions. Since no O-glycosylation was observed with GALE+GALK2 KO clone 7 cultured with the addition of GalNAc and galactose, we can conclude that all copies of the GALK2 gene were correctly knocked out.Monomeric secreted SIV gp120 was generated in both the GALK2 (clone 7) and GALE+GALK2 (clone 7) knockout cell lines. Since GALK2 is a critical enzyme in the salvage of GalNAc, we cultured each line with galactose and GalNAc . This isGALK2 and GALE+GALK2 KO cell lines were also cultured in the presence of 100uM UDP-GalNAc and 10uM UDP-galactose. Since both GalNAc and galactose must be converted into their respective UDP-precursors before being utilized for glycosylation, we were interested in whether the UDP forms of the sugars could be salvaged and the knockout phenotype could thus be rescued. However, even at high concentrations of UDP-sugar precursors, no restoration of O-glycosylation was observed by lectin western blot  Lane 6. Due to the established importance of O-glycosylation to support life and the known medical disorders associated with defects in GALE and galactokinases, it was somewhat surprising to find that our knockout cell lines devoid of all O-glycosylation grew equally well as the wildtype HEK293T cells . Even afMatrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectra of permethylated O-glycans isolated from total cell lysate were recorded. As expected, HEK293T and GALK1 KO cells displayed a normal range of O-glycans . ConsistMALDI-TOF MS profiles of the permethylated N-linked glycans from total cell lysate of HEK293T cells, HEK293T-GALE KO, HEK293T-GALK1 KO, HEK293T-GALE+GALK1 KO, HEK293T-GALE+GALK2 KO, and HEK293T-GALE+GALK1 KO cells +galactose were also generated. Major peaks are annotated with relevant N-glycan structures shown in symbol form . WildtypAlthough glycosylation-deficient cell lines have been used for decades, to our knowledge, this is the first report using CRISPR/Cas9 technology to make human cell lines with drastic alterations to the O-linked glycosylation machinery. Compared to traditional methods of lectin based selection or radioactive suicide, CRISPR/Cas9 proved to be a far superior method for the generation of glycosylation deficient cell lines . With thOne somewhat surprising finding from our data was that even the most extreme double knockout (GALE+GALK2), which was shown to be devoid of all O-glycosylation Figs  and 7A, et al. (TIF)Click here for additional data file.


Bidens pilosa effectively suppresses tumor metastasis and inhibits tumor-induced accumulation of granulocytic (g) MDSCs, but does not result in body weight loss in a mouse mammary tumor-resection model. BP-E-F1 is further demonstrated to exert its anti-metastasis activity through inhibiting the differentiation and function of gMDSCs. Pharmacokinetic and mechanistic studies reveal that BP-E-F1 suppresses the differentiation of gMDSCs via the inhibition of a tumor-derived, G-CSF-induced signaling pathway in bone marrow cells of test mice. Taken together, our findings suggest that specific plant polyacetylenic glycosides that target gMDSC differentiation by communicating with bone marrow cells may hence be seriously considered for potential application as botanical drugs against metastatic cancers.Myeloid-derived suppressor cells (MDSCs) are implicated in the promotion of tumor metastasis by protecting metastatic cancerous cells from immune surveillance and have thus been suggested as novel targets for cancer therapy. We demonstrate here that oral feeding with polyacetylenic glycosides (BP-E-F1) from the medicinal plant  Owing to the recent advancement in precision surgeries, early diagnosis of cancer, and adjuvant therapies used alongside chemotherapeutic drugs, cancer death rate now mainly reflects the degree and pattern of residual or circulating tumor cells metastasizing from the primary tumor site to secondary tissue target sites1245791112Myeloid-derived suppressor cells (MDSCs) and T-regulatory cells (Tregs) are the two main immunosuppressive cell types that negatively regulate immune responses against cancers1415161620212324262728Bidens pilosa L., an Asteraceae plant, has been traditionally used as a folk medicine and an herbal tea constituent in many Asian cultures. Plant extracts of B. pilosa have been reported to confer a spectrum of immunomodulatory and anti-inflammatory activities. Previously, we reported that a phytoextract and phytochemicals isolated from B. pilosa exhibit immunomodulatory activities that regulate the functions of dendritic cells and T cells3132B. pilosa (BP-E) was found to exhibit a strong immunomodulatory effect that effectively suppressed the G-CSF-induced differentiation of gMDSCs from test bone marrow cells ex vivo. In vivo studies further demonstrated that simple oral delivery of BP-E can suppress 4T1 tumor metastasis with high potency in a tumor-resection model. Mechanistically, we show that this effect of BP-E is mediated via inhibiting the differentiation and function of gMDSCs in test mice. Bio-organic chemical analysis showed that a specific group of polyacetylenic glycosides, which made up the great majority (>89%) of the constituents of one fraction of BP-E (BP-E-F1), act as the active phytochemicals responsible for the detected MDSC activities ex vivo and in vivo, and the resultant anti-metastatic activities in vivo. Taken together, our results strongly suggest that specific polyacetylene phytochemicals in BP plant extracts and the derived ethanol fraction may have therapeutic or other clinical applications against metastasis of mammary tumor. Therefore, we would like to consider that gMDSC activities/populations have potential to serve as an immune checkpoint modifier. Our findings on BP-E-F1 phytochemicals may also have applications as botanical drugs against metastatic cancers.9 photons/sec), in accordance with the time course pattern observed for expression of G-CSF in test mice. The population of gMDSCs expressing CD11b+Ly6G+ in white blood cells (WBCs) of peripheral blood reached 66.7% at day 7, and maintained a high level  from day 14 to day 42  from day 6 to day 19 (The MDSC population has been shown to increase in cancer patients3435o day 42 . A high o day 42 . Monocytthe WBCs . In addithe WBCs . The popo day 19 .5 cells) and gMDSCs (1 × 107 cells) into the mammary fat pad of test mice. At 18 days post tumor implantation, the primary tumor mass was gently removed surgically. Experimental results showed that the co-transplanted gMDSCs could indeed promote tumor growth and metastasis (Expression levels of gMDSCs and G-CSF have previously been shown to be closely associated with the progression of tumor growth in mouse modelstastasis . It is itastasis . We therB. pilosa (BP-E) plant extract significantly suppressed the G-CSF-induced differentiation of gMDSCs from bone marrow cells ex vivo (P < 0.05). The MDSC populations in the spleen tissue of each test group were then investigated. As shown in To investigate potential therapeutic agents against tumor metastasis, a number of phytoextracts or the derived phytochemicals were evaluated in our laboratory for inhibition of the function and differentiation of MDSCs. The ethanol partitioned fraction of the  ex vivo . MTT ass ex vivo . In addi ex vivo . In orde ex vivo . We next ex vivo . This is ex vivo . These rex vivo culture conditions. To identify candidate active components or phytochemicals from the BP-E phytoextract that can confer anti-metastasis activity, BP-E was further fractioned into four sub-fractions (F1 to F4) using high performance liquid chromatography (HPLC) analysis with an absorbance of UV at 235 nm . These f+, and [M + Na]+ peaks were detected at m/z 365.16 and m/z 387.14, respectively  were isolated , and theectively , Peak A rent ion , Peak A.ectively , Peak B,rent ion , Peak B.Since we were able to separate the phytochemicals of the BP-E extract into four major fractions, we then investigated the possible inhibitory effect of the BP-E-F1 on tumor growth in our orthotopic mammary tumor growth/tumor resection mouse model. At 7 days post tumor implantation, test mice were randomly divided into untreated and BP-E-F1 groups . Like oral administration of BP-E, BP-E-F1 treatment had little or no significant effect on primary tumor growth, as measured by tumor volume . We nextOur current results suggest that BP-E as well as its derived F1 fraction can effectively suppress tumor metastasis via inhibition of the differentiation of MDSCs from bone marrow cells and the accumulation of MDSCs in the tumor microenvironment. For subsequent experiments, we injected 4T1 cells alone or co-injected with granulocytic MDSCs into the mammary fat pad of test mice. Starting at 7 days post tumor implantation, mice were fed orally with F1 (5 mg/kg) every day. At 18 days post tumor implantation, the tumor masses of test mice were gently removed surgically and measured. F1 treatment significantly inhibited the effect of MDSCs on tumor growth as measured weekly by tumor volume and tumor mass . In addiIn our current study, we established a luminescent murine mammary 4T1-luc2 orthotopic tumor model, with which tumor resection and subsequent monitoring of tumor metastasis were conducted. We systemically investigated the role of MDSCs in tumor growth and metastasis in this system. Our findings suggest that an immunotherapeutic strategy that involves MDSCs may be usefully employed to treat against metastatic cancers. Our results showed that granulocytic MDSCs (gMDSCs) are the major MDSC population accumulated in the peripheral blood and spleen tissue of 4T1 tumor-bearing mice, and are present from the early stage to the later stage of tumor growth . In addi38404243B. pilosa (BP-E) or the phytochemicals from a derived fraction (BP-E-F1) can significantly suppress 4T1 mammary metastasis. Importantly, the efficacy of the F1 fraction in inhibition of metastasis and MDSC accumulation was at a level comparable with docetaxel treatment  of BP-E-F1 in the blood of test mice. Oral administration was also used to investigate the effect of BP-E-F1 on suppression of 4T1 metastasis. The bioavailability of the three BP-E-F1 compounds (A-C) was assessed in BALB/c mice (n = 12) via administration of BP-E-F1 by either intravenous (iv) or oral delivery, both at 10 mg/kg. As seen in in vitro and in vivo. Tumor-derived G-CSF has been demonstrated to play a key role in promotion of gMDSC developmentin vivo  plants were grown on farms in Sanxia District, New Taipei City, Taiwan, in 2013. Air-dried shoot, leaf and root tissues of whole plants, weighing 228.2 g were imbibed, stirred and extracted in 2.28 liters of 95% ethanol (EtOH) at room temperature for three days. This total crude extract was evaporated in a vacuum to yield a dried residue (6.3955 g) that was then resuspended in methanol (MeOH) and eluted with a water-MeOH mixture of decreasing polarity, using a PR-18 preparative HPLC column  with a flow rate of 0.5 ml/min, and detected at UV 235 nm to give a total of four sub-fractions (BP-E-F1 − BP-E-F4). The BP-E-F1 (eluent of 73.5% MeOH/water from the PR-18 column) was identified as the bioactive fraction. Therefore, BP-E-F1 was repeatedly separated by the same eluent with 70% to 72% MeOH in water for further use in in vitro and in vivo studies.nd sub-fractions ). These 2nd sub-fractions were then further separated from BP-E-F1 (40 mg) by the same PR-18 preparative HPLC column as mentioned above and eluted with 31.2% ACN with 0.05% FA to yield compounds A , B , and C . LC-MS/MS was performed to identify the chemical formulas of compounds A, B and C. The LC-ESI-Orbitrap Elite MS data were processed using the Xcalibur version 2.7.0 SP1 (Thermo Scientific). For the LC-MS analysis, data were generated by intensities of the peaks, with the retention time (RT) and the m/z data pairs were used to identify each peak.BP-E-F1 was subsequently separated via chromatography by a RP-18 UPLC column , eluted with 30% to 32% acetonitrile (ACN) with 0.2% formic acid (FA), to give a total of three 22 and 95% humidity.293T cells were transfected with pMD.G, pCMVΔR8.91, and pIF4g.As2.luc.bla to construct a lentivirus vector expressing luc2 gene. Twenty-four hours later, cell culture media were collected and employed to transfect 4T1 cells with the constructed viral vector. Blasticidin S (10 μg/mL) was applied to select the single clones of transfected 4T1-luc2 cells. Transgenic 4T1-luc2 cells were cultured and maintained in RPMI-1640 medium supplemented with 10 μg/mL blasticidin S, 10% fetal bovine serum, 1 mM penicillin/streptomycin and 1 mM sodium pyruvate in an incubator at 37 °C in 5% CO5 cells/100 μl PBS)in vivo imaging system (IVIS). All experiments were performed in accordance with relevant guidelines, and experimental protocols involving animals were approved by the Institutional Animal Care and Utilization Committee (IACUC) of Academia Sinica, Taipei (IACUC ID: 10-12-105).Mouse mammary carcinoma 4T1-luc2 cells .To purify Ly-6G2 incubator at 37 °CBALB/c mouse bone marrow cells from femora and tibiae were depleted of RBCs with ACK lysis buffer and cultured in RPMI 1640 medium supplemented with 20 ng/ml GM-CSF, 10% fetal bovine serum, 50 μM 2-mercaptoethanol, 100 unit/ml penicillin and 100 μg/ml streptomycin in a humidified 5% COCell lysates were prepared using M-PER Mammalian Protein Extraction Reagent . Lysates were run on 5% to 20% gradient polyacrylamide-sodium dodecyl sulphate (SDS) gels (20 μg protein per lane), proteins were then transferred onto Hybond-ECL membranes  and immunoblotted with anti-G-CSFR antibody, anti-stat3 antibody, or anti-phosphorylated stat3 antibody. Protein bands were detected by enhanced chemiluminescence  and developed by autoradiography.Serum from test mice and conditional medium of test cell cultures were collected and stored at −80 °C until use. Samples were checked for expression levels of G-CSF (R&D Systems) and quantified at a wavelength of 450 nm using a Biotek PowerWave HT spectrophotometer.Anti-stat3 antibody and anti-phosphorylated stat3 antibody were purchased from Cell Signaling Technology. Anti-G-CSFR antibody was purchased from Abcam.Data are presented as fold-changes or percentages with mean ± SEM as indicated in the corresponding figure legends. All statistical analyses were determined using GraphPad software. For comparison between multiple data sets, a one-way ANOVA analysis with Tukey–Kramer method was performed.How to cite this article: Wei, W.-C. et al. Inhibiting MDSC differentiation from bone marrow with phytochemical polyacetylenes drastically impairs tumor metastasis. Sci. Rep.6, 36663; doi: 10.1038/srep36663 (2016).Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.