Schistosoma mansoni, but the transcriptional changes that underlie this effect are poorly understood. To explore this, we sorted the two previously recognized hepatic macrophage populations (perivascular and Kupffer cells) from naïve and S. mansoni-infected male mice and performed microarray analysis as part of the Immunological Genome Project. The two hepatic macrophage populations exhibited remarkably different genomic profiles. However, this diversity was substantially reduced following infection with S. mansoni, and in fact, both populations demonstrated increases in transcripts of the monocyte lineage, suggesting that both populations may be replenished by monocytes following infection. Pathway analysis showed a profound alteration in global metabolic pathways, including changes to phospholipid and cholesterol metabolism, as well as amino acid biosynthesis and glucagon signaling. These changes suggest a possible mechanism for the previously reported athero-protective effects of S. mansoni infection. Indeed, we find that male ApoE null mice fed a high-fat diet in combination with S. mansoni infection have reduced plaque area and increased glucose tolerance as compared to control mice. Transcript analysis of infected and control high-fat diet fed ApoE−/− mice confirm that ApoC1, Psat1, and Gys1 are all altered by infection, suggesting that altered hepatic macrophage metabolism is associated with S. mansoni- induced protection from hyperlipidemia, atherosclerosis, and glucose intolerance. These results suggest a previously unknown and unreported role of hepatic macrophages in the modulation of whole body lipid and glucose metabolism during infection and provide a template for examining the role of immunomodulation on the long-term metabolism of the host.Hepatic macrophages play an essential role in the granulomatous response to infection with the parasitic helminth  Nos2) amongst other genes, and are pro-inflammatory, whereas M2 macrophages are induced by IL-4 and IL-13 and differentially express a range of genes including arginase-1 (Arg1), resistin like beta  and Ym1 (Chil3)  . M2 macr (Chil3) . Importanfection , 10 and nfection . Recent nfection , and ournfection . HoweverS. mansoni serve as a reproducible model for human schistosomiasis. In both humans and mice, the pathology progresses from an early Th1 response into a Th2 dominated one in response to egg deposition in organs such as liver, intestines, and lungs by worm pairs. Eggs are highly immunogenic and toxic, and the host response to this stimulus leads to fibrosis and portal hypertension  induced insulin resistance, and identify transcriptional changes to amino acid and glycogen metabolism that are likely mechanisms for this protection. Altogether, the data suggest an important and unexplored role of liver macrophages in the regulation of atherosclerosis and insulin sensitivity, a finding that has significant implications for diabetes, obesity and many other human diseases involving hyperlipidemia and insulin resistance.Here we describe microarray data of the transcriptional profiles of hepatic macrophages in naïve and This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocols were approved by the Institutional Animal Care and Use Committees of Washington University in St. Louis and Purdue University.ApoE−/− (B6.129P2-Apoetm1Unc/J) mice were bred at Purdue University. Mice were kept under specific pathogen–free conditions and male mice were infected at 6 weeks of age. Snails infected with S. mansoni  were provided by the Schistosome Research Reagent Resource Center for distribution by BEI Resources, NIAID NIH. Mice were exposed percutaneously to 75 S. mansoni cercariae. For experiments using Apoe−/− mice, animals were transitioned to HFD  10 days prior to S. mansoni infection. Control mice were fed standard rodent chow  and mice were sacrificed at 10-weeks post-infection.Male C57BL/6J mice were purchased from Jackson Laboratories, www.immgen.org). The following mAb  against mouse antigens were used as PE, PE-Cy5, PE-Cy7, allophycocyanin (APC), APC-Cy7, Pacific blue, or biotin conjugates: CD11c (N418), CD11b (M1/70), CD45 (30-F11), MHC-II (M5/114.15.2), F4/80 (BM8), and MERTK (BAF591). Cells were directly sorted from mouse tissues and were processed from tissue procurement to a second round of sorting into TRizol within 4 h using a Beckton-Dickinson Aria II instrument.Livers were removed from PBS-perfused animals, mashed, and incubated in RPMI (Mediatech) containing 250 μg/ml Collagenase D (Roche) at 37°C for 60 min. The resulting suspension was disrupted through a 100 μm metal cell strainer, washed, and then red blood cell lysed with ACK lysis buffer (BD) two times to remove hepatocytes followed by washing. The resulting pellet was washed and used for sorting. Surface staining with monoclonal antibodies, acquisition, and sorting were performed according to Immgen standard protocols (http://www.broadinstitute.org/cancer/software/genepattern/). Probesets were considered as differentially expressed with a coefficient of variation < 0.5 and a p < 0.05 (Student's T-test). Calculation and visualization of differential gene expression sizes  were performed using R-language for statistical computing.RNA was amplified and hybridized on the Affymetrix Mouse Gene 1.0 ST array by the Immgen consortium using double-sorted cell populations sorted directly into TRIzol. Data analysis utilized GenePattern analysis software. Raw data were normalized using the robust multi-array algorithm, returning linear values between 10 and 20,000. A common threshold for positive expression at 95% confidence across the dataset was determined to be 120. Differential gene expression signatures were identified and visualized using the “Multiplot” module of GenePattern (high (Kupffer cells) (foldchange > 0), but downregulated in F4/80int (perivascular macrophages) (foldchange < 0).Genes which are upregulated in F4/80high (foldchange < 0), but upregulated in F4/80int (foldchange > 0)Genes which are downregulated in F4/80Genes which are significantly (Benjamini-Hochberg adjusted p < 0.05) upregulated in both cell typesGenes which are significantly (Benjamini-Hochberg adjusted p < 0.05) downregulated in both cell typeGenes which differ substantially in the degree of regulation (regardless of the direction of regulation).To identify important genes  we used various Boolean selection strategies focusing on multiple scenarios:2A–C. The following set designations were used in the set notation:high cells (BH adjusted p < 0.05)q_hi = Significantly regulated genes in F4/80int cells (BH adjusted p < 0.05)q_nt = Significantly regulated genes in F4/80high cellshi_up = Genes upregulated in F4/80int cellsint_up = Genes upregulated genes in F4/80high cellshi_down = Genes downregulated in F4/80int cellsint_down = Genes downregulated in F4/80The described Boolean strategies are illustrated in the Venn diagrams in Figures A∩B–A intersect BA∪B–A union BA\B–A minus B, or A complement BThe employed set notation:http://www.advaitabio.com/ipathwayguide). Pathway analysis was performed on log2-transformed data using Bonferroni-corrected p-values. Data are deposited in GenBank under accession no. GSE37448 as part of the Immgen 2 dataset.The Data  were analyzed using Advaita Bio's iPathwayGuide .Livers were perfused with 1X PBS, mashed and digested in DMEM containing 250 μg/ml Collagenase (Sigma) at 37°C for 30 min. The digested livers were then mashed and filtered through a 100 μm metal strainer and digestion was repeated for 15 min. Total liver contents were strained and washed with DMEM. The pellet was lysed with 1X lysis buffer (BD PharmLyse), quenched, and washed. The resulting cell suspension was used in flow cytometry. Surface staining was performed using the following mAb against mouse antigens: CD45 , CD301(BioRad), CD206 , F4/80 , mouse Mer biotinylated (R&D), CD64. Samples were acquired using FACSCanto II flow cytometer (BD) and analyzed using Flowjo X 10.0.7r2 .At sacrifice, mice were perfused with PBS, and the heart was extracted for plaque assessment at the aortic sinus. Following fixation in 4% paraformaldehyde, tissue was cryoprotected in 30% sucrose, embedded in OCT compound (Fisher Scientific), and flash frozen. Fifteen-micron cryosections were made in the aortic sinus from initiation to termination of the aortic valve leaflets. Plaque assessment in the aortic sinus was performed as previously described , and is Total cholesterol was measured from plasma as previously described  using coCtΔΔ method.Sections of perfused livers were homogenized in Trizol, and RNA isolation was performed as previously described . RNA waApoC1 plasma concentrations were determined by ELISA using an anti-mouse ApoC1 ELISA kit as per the manufacturer's instructions (LSBio).P ≤ 0.05 were considered statistically significant. Graph generation and statistical analyses were performed using Prism (GraphPad v7.0) and R-language for statistical computing.Statistical analyses for non-microarray data were performed using one-way ANOVA, a non-parametric Mann-Whitney test, or unpaired Student's t-test depending on the distribution of the data. S. mansoni infection, the transcriptional changes that accompany the response to S. mansoni antigens are not entirely understood. We have previously established 10-weeks post-infection as a key time-point in immunomodulation where SEA (schistosome egg antigen) specific B cells are recruited to the liver, and immune-complex ligation of hepatic macrophages begins, leading to macrophage IL-10 production  and then sorted F4/80 high (Kupffer cells) and F4/80int (perivascular macrophages) populations from the livers of naïve and 10-week old S. mansoni-infected C57BL/6 mice according to Immgen standard protocols , 35.32 (naïve F4/80high cells vs. 10-week S. mansoni F4/80high cells). In both cases, the naïve vs. infected differences were statistically significant . Although distances for naïve F4/80high vs. naïve F4/80int cells and 10 -week infected F4/80high vs. 10-week-infected F4/80low cells were similar , the larger variance among naïve F4/80int cells made the former pair of expressions indistinguishable in the PC space . On the other hand, the dissimilarity between the gene expressions identified in the cells from the infected animals was significant (p = 0.004).We compared the gene expression profile of naïve Kupffer cells and perivascular macrophages and found very high transcriptional diversity between these two macrophage populations considering that they reside in the same tissue environment Figure . Subsequn Figure , suggestn Figure , 22. A rn Figure  indicates Figure . The ploS. mansoni infection. Among 57 genes in the Subset 3 (q_hi⋂hi_up⋂int_down)_(q_int), we located Cbr2 , which is significantly (p < 0.05) and highly (fold size = 20.7) upregulated in F4/80high, but does not demonstrate a statistically significant change in F4/80int, Alox , and F13a1 . Additionally, among 932 genes in Subset 4 (q_hi⋂q_int⋂int_down)_(hi_up), which is equivalent to (q_hi⋂q_int⋂int_down⋂hi_down) and contains the genes which are significantly downregulated (foldsize < 0) in both cell types we found Adh1, Alb, Aldh2, Apoa2, ApoB, ApoC1, ApoC3, Cd55, Clec4f, Creg1, Fabp7, Flt4, Gpr116, Hmcn1, Hpgd, Marco, Pde7b, Ptprb, Slc16a9, Slc40a1, Slco2a1, St3gal5, and Stab2. Surprisingly, this subset contains many genes involved in phospholipid and cholesterol metabolism, as well as solute transport. Further, we found that Prg4 and Spic belong to a Subset 5 of 795 genes that are downregulated in both macrophage types, but the downregulation in F4/80int has not been statistically significant. The set is designated as (q_hi⋂int_down)_(q_int∪hi_up). 683 genes were significantly simultaneously upregulated in F4/80int and F4/80high forming a Subset 6 _(q_int∪int_down) contained upregulated genes, which changed in a statistically significant manner in F4/80high but not in F4/80int. Ccl12 with a foldchange of ~15 and Ccl8 with a very high foldchange of 76.6 in F4/80high as well as Mfsd6 were among these genes. Interestingly, there were only three genes in subset 1 (q_hi⋂q_int⋂hi_up⋂int_down): Atrnl1, Mef2c, Nav1, and only 2 genes in subset 2 (q_hi⋂q_int)_(hi_up∪int_down): Igf2bp2 and Ube2j2  Mgl2 , Retnla  as well as Ear11, Chi3l3, Ptprb, Ccr2, Gda, Gpr116, St3gal5, Stab2, Cd63.Some genes which were upregulated in both types of macrophages (or downregulated in both) nevertheless showed substantial differences in the level of regulation, exhibited by a large disparity in foldchanges. For instance, Rnase4 was upregulated in F4/80s Figure  expresseS. mansoni infection. Meta-analysis of transcriptional changes shared across both hepatic macrophage populations revealed striking alterations to multiple KEGG pathways, including multiple pathways of inflammation and cellular metabolism , others were not previously considered to be associated with either alternative activation, or S. mansoni infection . We conclude that S. mansoni infection strongly alters the metabolic potential of liver macrophages. Since the liver is responsible for regulating many aspects of whole body metabolism  or normal chow diet for 10 days before infection with S. mansoni, whereas controls were sham-infected. At 10-weeks post-infection we analyzed CD301  and CD206 (mannose receptor) expression as markers of alternative activation. Approximately 40–60% of macrophages from control chow uninfected were double positive, with the rest being CD206+  and ApoC3 . To confirm gene modulation on the ApoE−/− model, we assessed the expression of a list of genes of interest. Importantly, ApoC1 was significantly downregulated in the same manner in both the C57BL/6 and ApoE−/− model. Additionally, a previous report has indicated that ApoC1 production drives hyperlipidemia and pathogenesis in the ApoE−/− model  at 5-weeks post infection (pre-egg laying). We found that pre-patency, S. mansoni infection has no significant effect on glucose tolerance in normal chow fed mice as measured by area under the glucose excursion curve , normal chow fed infected mice have lower fasting glucose levels and significantly improved whole-body glucose tolerance , and one of the genes in that pathway, Psat1, stood out as a candidate for involvement in improving glucose tolerance as its transcript level is increased in infected C57BL/6 mice . The correlation between modulation of this transcript and infection was confirmed in our ApoE−/− model , in that pathway, S. mansoni infection decreases pygl expression while also increasing gys1 transcripts. Genetic disruption of PYGL has previously been shown to improve glucose tolerance in vivo  have a lower fold upregulation in perivascular macrophages, a finding that is matched by the frequency of these macrophages that are CD206+CD301+, suggesting that there may be degrees of alternative activation in the liver in the context of S. mansoni infection. The fact that only five genes (Subsets 1 and 2) are truly differentially regulated (opposite signs of significant fold changes) supports the hypothesis that after infection Kupffer cells and Perivascular macrophages act to a large degree in unison.Overall, our data provide the first complete transcriptional analysis of Kupffer cells and perivascular macrophages in steady state and chronic  healing , suggestS. mansoni infection, but our data also provide the first evidence that alterations to hepatic macrophage metabolism either reflect or influence infection-induced alterations to whole body metabolism. Macrophages are known to be critical sources of apolipoproteins like ApoC1  and glycogen phosphorylase (PYGL) acting to respectively increase and decrease stored glycogen to maintain blood sugar homeostasis. Our pathway analysis revealed key alterations to both macrophage amino acid and glucagon signaling/metabolic pathways. We identified key genes  from both pathways that have previously been demonstrated to play a role in glucose tolerance/ insulin sensitivity in models of obesity and diabetes , 41 and n humans , 45, 46 n humans , 16, 27 KF, EP, and GR conceived of the project. KF, AR, DC-S, and GR planned the experiments. DC-S, AE, AR, and KF performed the experiments. KF, BR, DC-S, and AE analyzed the data. DC-S, KF, and BR wrote the manuscript.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.


In this study, we investigated the effect of exogenous p-CA on plant growth, reactive oxygen species (ROS)-induced oxidative damage, photosynthetic metabolism, osmolyte content and changes in superoxide dismutase (SOD) enzymatic activity. Exogenous p-CA improved Salvia hispanica (chia) growth by significantly enhancing shoot length, fresh and dry weights coupled with augmented levels of total chlorophyll and carotenoid contents. Furthermore, p-CA also triggered an induction in proline, glycine betaine (GB) and superoxide (O2∙−) levels while no changes were observed for hydrogen peroxide (H2O2) and downstream malondialdehyde (MDA) content. Also, no change in SOD activity was observed in the p-CA treatment relative to the control. Therefore, the results suggest that exogenous p-CA improves chia seedling growth possibly via activation of a ROS-signalling pathway involving O2∙− under the control of proline accumulation. A few lines of research have shown that p-CA reduced the rate of seed germination, root length and biomass in different plant species [p-CA restricts plant growth and development in various plant species [p-CA) improved the growth of chia plants through differential regulation of photosynthetic metabolism, ROS content and antioxidant enzyme activities under salt stress conditions. This result is in contradiction to what was reported for legume plants [2∙−, H2O2 and hydroxyl radicals (OH∙) and if not controlled can lead to oxidative stress manifested as enhanced cellular death. However, increased ROS molecules (in moderation)—for example O2∙− and H2O2—have been shown to act as signalling molecules in various plant species [2∙− and H2O2 regulate physio-biochemical responses in Brassica napus plants via the modulation of SOD and APX activity, which in turn led to increased seedling growth. This evidence suggests that the mechanism controlling redox homeostasis of ROS molecules can be exploited to improve plant growth. To our knowledge, no study has investigated the effect of exogenous application of p-CA on the physio-biochemical responses (including ROS homeostasis) of pseudocereal plants.Phenolic acids are divided into two groups based on their chemical structure, namely hydroxybenzoic and hydroxycinnamic . Hydroxyactivity ,3,4. In activity ,6. Of th species ,9,10,11. species ,13. Jone species  showed te plants ,16. Anti species ,18. Furt species  showed tp-CA on the growth, photosynthetic pigments, osmolyte content, ROS-induce oxidative damage and changes in SOD activity in chia seedlings.Chia is an oilseed pseudocereal plant known for its nutritional and health promoting properties. The seed is a natural source of omega-3 fatty acids (α-linolenic acid), soluble and insoluble fibers, and proteins in addition to other important nutritional components, such as vitamins, minerals, and natural antioxidants ,21. Furtp-CA on plant growth and chlorophyll content. The results show that exogenous p-CA significantly increase shoot growth of chia seedlings  and carotenoid (25.1%) content in response to exogenous application of p-CA , when compared to the control. Furthermore, there was a significant increase in both proline (170%) and GB (22.4%) content  in the shoots of chia seedlings were investigated. Compared to the control, p-CA had no effect on H2O2 content . We observed that the final seed germination percentage was similar across all treatments except for 1000 μM at which no germination occurred  seedlings with exogenous p-CA and observed a reduction in chlorophyll content. Einhellig and colleagues [2∙−, which have been shown to interact directly with chlorophyll a ultimately leading to chlorophyll degradation [2∙− content in response to exogenous salicylic acid and sodium nitroprusside (nitric oxide donor) in wheat. In contrast, exogenous caffeic acid decreased O2∙− content in soybean plants [2∙− and chlorophyll in different plants, which has not been elucidated to date. In our study, higher levels of O2∙− were detected under exogenous p-CA and the scavenging mechanism of O2∙− did not involve a direct role of SOD enzymes. We observed that total SOD activity and individual SOD isoforms were unaltered in p-CA treated chia seedlings, which suggests that other mechanisms of O2∙− scavenging were possibly triggered by exogenous p-CA in the chia seedlings. This phenomenon was also observed by Yan et al. [2∙− did not result in an increase in SOD activity in response to exogenous sodium nitroprusside. Contrastingly, Klein et al. [2∙− content in soybean plants whereas a significant increase in SOD activity was observed. These studies show a complex interaction between O2∙− accumulation and scavenging via SOD, which is not well understood.In this study, we have dissected the role of exogenous  species ,9,13,27. species  performerred see . Our datp-CA see . Furtherr leaves . Similar biomass . In addipigments  in chia n et al.  that obsn et al. , which tlleagues  suggesteradation , howeverradation  observedn plants , which ln plants . These sn et al. , where tn et al.  showed t2O2 levels were similar to those of the control seedlings. This finding supports our result where no changes in SOD activity were observed. According to the Asada–Halliwell pathway, SOD scavenges O2∙− and produces downstream H2O2, which has the ability to oxidize polyunsaturated fatty acids (PUFA) ultimately producing secondary products such as MDA, which is an indicator of lipid peroxidation [p-CA treated seedlings when compared to the controls  in limiting membrane injury have been reported and studies have shown that proline can scavenge ROS molecules ,36. Furtained by , which saffected . In suppted  and allowed to grow on a 27/19 °C day/night temperature cycle under a 16/8 h dark cycle at a photosynthetic photon flux density of 300 μmol photons·m(R) solution diluted in water (1:300). For treatment with p-CA, plants were supplemented with Nitrosol(R) containing 100 µM p-CA  for a period of 14 days.Plants at the same developmental stage and of similar height were selected for all experiments. Control plants were supplemented with 50 mL of NitrosolChia seedlings were carefully removed from the growth medium to avoid damage. Subsequently, the roots were separated from the shoots to prevent erroneous data interpretation caused by possible root damage. The shoots from each treatment were scored for length (SL), fresh weight (FW) and dry weight (DW). The DW was determined by drying the seedlings in an oven at 55 °C for 48 h as described by .Total chlorophyll content in the shoots of chia seedlings was estimated using a method previously described by . Freshly2HPO4 at pH 7.4; 1 mM EDTA; 5% PVP MW = 40 000; 5% glycerol in distilled H2O) for the measurement and detection of SOD enzymatic activity. Protein concentrations were determined using the RC DC Protein Assay Kit 11 (Bio-Rad Laboratories).Shoots from all treatments were harvested and ground into a fine powder using liquid nitrogen. Shoots (0.1 g) was homogenized in 1 mL of PVP buffer (40 mM K2∙−) content in the shoots of chia seedlings was quantified using a method previously described in [2O2 (to inhibit Mn and Cu/Zn SODs), 2% (w/v) SDS (to inhibit Mn and Fe SODs), 80 mM Nitroblue tetrazolium chloride (NBT) and 50 mM potassium phosphate (pH 7.0) and incubated for 20 min. After incubation, shoots were homogenized and centrifuged at 10 000 × g for 5 min. The resulting supernatant was spectrophotometrically analysed at 600 nm using a FLUOstar Omega UV-visible spectrophotometer . The superoxide concentration was calculated using the NBT extinction coefficient of 12.8 mM·cm−1. Hydrogen peroxide (H2O2) content was measured in shoots of chia seedlings, using a method previously described by [w/v) TCA. The extracts were centrifuged at 12 000 × g for 30 min at 4 °C and 50 μL of the supernatant was used to initiate the reaction in a mixture  containing 5 mM K2HPO4 (pH 5.0) and 0.5 M potassium iodide (KI). The reaction mixture was incubated at 25 °C for 20 min and the absorbance readings were recorded at 390 nm using a FLUOstar Omega UV-visible spectrophotometer . H2O2 content was calculated using a standard curve based on the absorbance of H2O2 standards.Superoxide (Oribed in . Superoxribed by . Brieflyw/v) TCA. Aliquots of the supernatant (100 μL), were mixed with 400 μL of 0.5% thiobarbituric acid . The mixture was incubated at 95 °C for 30 min and the reaction was terminated on ice for 5 min. The mixture was centrifuged at 12 000 × g for 5 min at 4 °C. The absorbance of the supernatant was recorded at 532 nm and 600 nm using a FLUOstar Omega UV-visible spectrophotometer . After subtracting the non-specific absorbance, the MDA concentration was calculated using the extinction coefficient of 155 mM·cm−1.The extent of lipid peroxidation (MDA) in the shoots of chia seedlings were quantified as described by . Briefly2HPO4, pH 7.8, 0.1 mM EDTA, 10 mM methionine, 5 μM riboflavin, 0.1 mM NBT) and incubated at room temperature for 20 min under fluorescent light. Subsequently, the absorbance readings were recorded at 560 nm using a FLUOstar Omega UV-visible spectrophotometer . SOD activity was calculated based on the amount of enzyme that was required to cause a 50% reduction of NBT.Total SOD activity was measured using a method previously described by . The proIn-gel activity of individual SOD isoforms was detected in the shoots of chia seedlings according to a method described by . Proteinw/v) sulphosalicylic acid using a mortar and pestle. About 200 µL ml of each homogenate was mixed with 200 µL of glacial acetic acid to which 200 µL of ninhydrin was added. The reaction mixture was boiled in a water bath at 100 °C for 30 min and immediately cooled in an ice bath. After cooling, 400 µL of toluene was added to the reaction mixture. After thorough mixing, the chromophore containing toluene was separated and the absorbance of the red color developed was read at 520 nm against the toluene blank on FLUOstar Omega UV-visible spectrophotometer .Total free proline content in the shoots of chia plants was estimated using  and modi2SO4. An aliquot (250 µL) was transferred into a test tube and cooled in ice water for 1 h. Cold potassium iodide-iodine reagent (100 µL) was added, vortexed, and then centrifuged at 1000 × g for 30 min at 4 °C. The sample was incubated for 24 h at 4 °C. The formed periodite crystals were dissolved in 14 mL of 1,2-dichloroethane with gentle agitation at room temperature for 48 h. The absorbance was recorded at 365 nm using a FLUOstar Omega UV-visible spectrophotometer .Glycine betaine (GB) content in the shoots of chia seedlings was estimated using a method previously described in  with sliAll experiments described were performed six times independently. For superoxide content, shoot height, shoot fresh weight, shoot dry weight measurements, 30 individual chia seedlings per treatment were analyzed. For all other experiments, 50 chia seedling shoots were homogenized per treatment. For statistical analysis, the one-way analysis of variance (ANOVA) test was used for all data, and means (for six independent experiments) were compared according to the Tukey–Kramer test at 5% level of significance, using GraphPad Prism 5.03 software.p-CA on plants in a hypothetical model  specifically with regards to O2∙− plays a major role in synthesis and scavenging of the O2∙− in addition to the direct scavenging capacity of the p-CA. This carefully controls the O2∙− levels without triggering an increase in SOD activity. Furthermore, we observed no changes in H2O2 content and we attribute this to the unchanged SOD activity. We also conclude that the increase in GB by exogenous p-CA application led to improved chlorophyll and photosynthetic pigments. This result is supported by findings from [p-CA improves chia seedling growth via GB and proline activation. Future work should investigate whether the GB pathway occur separately or independently from the proline pathway in chia seedlings treated with p-CA.After summarizing studies on exogenous al model  and incoerved by  where intrast to , our res by p-CA ,52. Furt by p-CA  (after dngs from  which hi


Ligusticum chuanxiong hort, is known to exert a variety of antitumor effects including inhibition of tumor cell proliferation, metastasis, and drug resistance. In this research, we investigated antitumor effects of TMP combined with paclitaxel (PTX), a frontline chemotherapeutic drug, in vitro and in vivo. Our results indicate that TMP enhances the antitumor effects of PTX in ovarian cancer A2780 and SKOV3 cells. Furthermore, we found that combined treatment of TMP and PTX suppressed angiogenesis by inhibiting both ERK1/2 and Akt pathways and promoted apoptosis of tumor cells compared to TMP or PTX treatment alone. Moreover, TMP augmented the antitumor effects of PTX in ovarian cancer A2780 xenograft mouse models by significantly decreasing tumor burden and partially decreasing the toxicity of PTX, as evidenced by the decreased expression of proliferation and angiogenesis markers as well as the hematoxylin and eosin (H&E) staining and biochemical indexes assay. Overall, our findings provide novel mechanistic insight into the efficacy of combining of potent molecules present in traditional Chinese medicine with chemotherapeutic drugs for therapeutic intervention in cancer.Recent published findings have demonstrated the effectiveness of combining molecules from traditional Chinese medicine with chemotherapeutic drugs to treat cancer. Combined administration of these agents can overcome drug-mitigating responses as well as reduce adverse side effects, thereby enhancing the efficacy of the therapy. Tetramethylpyrazine (TMP), an alkaloid monomer from the medicinal herb  Cancer continues to pose a significant threat to human health worldwide . For decin vitro and in vivo  is an alkaloid monomer isolated from ong hort . A serieong hort . Reportsong hort . Additioong hort . TMP alsong hort .in vitro and in vivo for antitumor effects. Furthermore, we sought to dissect the molecular mechanisms driving the antitumor effects of this combination. Our results indicate that TMP enhances the antitumor efficacy of PTX by inhibiting angiogenesis, inducing apoptosis, decreasing tumor burden in A2780 xenograft mouse models, and partially reducing the toxicity of PTX.In this study, combined administration of TMP with PTX was investigated in vivo experiments including animal use and care protocols were carried out under the guidelines approved by the Institutional Animal Care and Use Committee of Chengdu University.TMP (purity ≥99%) was purchased from Energy Chemical Co., Ltd ; paclitaxel  was purchased from Dalian Meilun Biotech Co., Ltd ; vascular endothelial growth factor (VEGF) was bought from R&D systems . TMP and PTX were dissolved in dimethyl sulfoxide  as stock solutions, and diluted in relative culture medium with final DMSO concentrations no higher than 0.1% upon using. VEGFR tyrosine kinase inhibitor II (VRI) was obtained from CalBiochem . Antibodies recognizing phospho-p38 (Thr180/Tyr182), phospho-Akt (Ser 473), phospho-Erk1/2 (Thr202/Tyr204), Cleaved-Caspase 3, Cleaved-Caspase 9, Cleaved-PARP, and GAPDH were purchased from Cell Signaling Technology . Antibodies against Ki67 and CD31 were purchased from Abcam . Female BALB/c nude mice, aged 4–6 weeks (18–22 g), were obtained from Dashuo experimental animals Co., Ltd.  and maintained under specific pathogen-free conditions. All 2.Human umbilical vein endothelial cells (HUVEC) were obtained from American Type Culture Collection  and cultured in F-12K complete media with 100 µg/ml heparin, 30 µg/ml endothelial cell growth supplement (ECGS), 10% heat-inactivated fetal bovine serum (FBS), and 1% penicillin–streptomycin (P/S). Cells at early passage (3−8 passages) were used for the outlined experiments. A2780 ovarian cancer cells were obtained from ATCC and maintained in DMEM culture medium, supplemented with 10% heat-inactivated FBS and 1% P/S. SKOV3 ovarian cancer cells were obtained from ATCC and cultured in McCoy’s 5A medium, supplemented with 10% heat-inactivated FBS and 1% P/S. All the cells were incubated at 37°C in a humidified atmosphere with 5% CO3 cells/well. Cells were then treated with various concentrations of PTX (3.125−100 nM), TMP (6.25−200 µM), and the combination of the two compounds in low serum media (0.5%) for 48 h. Cells were treated with 0.5 mg/ml MTT. Four hours later, the formazan crystals were dissolved in 100 μl of DMSO and the absorbance was measured at 570 nm. Cell viability was calculated as the percentage of the controls. Each experiment was repeated at least three times.Cell viability was assessed using the MTT assay. Briefly, HUVEC cells, ovarian cancer A2780, and SKOV3 cells were seeded on 96-well plates at a density of 8 × 104 cells per well in F-12K complete media and cultured for 24 h for cell attachment. Cells were then starved with low serum media (0.5% FBS) overnight to achieve a quiescent state. After starvation, cells were treated with 100 nM PTX, 100 µM TMP, or the combination in low serum media containing VEGF (50 ng/ml) for 48 h. Cell proliferation was detected by MTT assay.HUVEC cells were seeded into a 48-well plate at a density of 3 × 104 cells) were resuspended in 200 ml of low serum (0.5% FBS) medium containing 20 ng/ml VEGF + 100 nM PTX, 20 ng/ml VEGF + 100 µM TMP, 20 ng/ml VEGF + 100 nM PTX + 100 µM TMP. Cells were then deposited into the 24-well companion plate with 500 μl of low serum (0.5% FBS) medium containing the same concentration of agents as were used to coat the upper sides. Cells were incubated at 37°C for 24 h. Cells on the upper surface of the membrane were removed using cotton swabs. Inserts were fixed with 4% paraformaldehyde for 15 min, and then stained with 10 μg/ml of Hoechst 33342 for 15 min.The effects of TMP combined with PTX on the migration and invasion of HUVEC cells were examined using the transwell migration assays (8-μm pores). In the migration assay, the upper side of the membrane was pre-coated with collagen. HUVEC cells (5 × 104 cells) were then resuspended in 50 μl of medium containing 0.5% FBS and 20 ng/ml VEGF + 100 nM PTX, 20 ng/ml VEGF + 100 µM TMP, 20 ng/ml VEGF + 100 nM PTX + 100 µM TMP, respectively, and seeded onto the Matrigel-coated plate. After 4-h incubation, HUVEC cells in the control group formed tube-like structures, which were defined as endothelial cord formations connected at both ends. Tube formation was quantified by counting the number of branching points in three randomly selected fields of view.Matrigel matrix  was thawed at 4°C overnight and a 15-well u-slide was coated with 10 μl Matrigel/well and incubated at 37°C for 30 min for polymerization. HUVEC cells , harvested, re-suspended in 200 μl of binding buffer, and stained with 3 μl of Annexin-FITC for 10 min, followed by 3 μl of PI solutions for 15 min. Cells were analyzed immediately by flow cytometry. Ten thousand events were counted for each sample.An Annexin V-FITC assay kit was used to detect cell apoptosis. A2780 and SKOV3 cells were seeded at 2 × 105 per well for 24 h, then exposed to 100 nM PTX, 100 µM TMP, or 100 nM PTX + 100 μM TMP for 24-h incubation. After treatment, cells were collected and co-incubated with JC-1 dye (3 μg/ml) in incubation buffer for 20 min at 37°C. Cells were subsequently washed twice with PBS and examined by flow cytometry with 10,000 gated cells. The experiment was performed in triplicate.A2780 and SKOV3 cells were seeded at 2 × 10g for 20 min at 4°C, and the supernatant was collected. The total protein concentration was determined using a BCA Protein Assay kit. The lysates were subjected to Western blotting and incubated with phospho-Akt, phospho-Erk1/2, phospho-p38, Cleaved-PARP, Cleaved-Caspase 3, Cleaved-Caspase 9, and GAPDH antibodies. Images of protein bands were captured using a Molecular Imager ChemiDoc XRS . Densitometric measurements of band intensity were performed using Quantity One Software .Protein was extracted using lysis buffer containing 1% phenylmethylsulfonyl fluoride and 1% protease inhibitor. Lysates were centrifuged at 12,500 × in vivo model with potential clinical relevance, and has been widely used for preclinical evaluation of anticancer drugs. In vivo antitumor studies were performed on A2780-heterografted female BALB/c nude mice. When tumor volumes reached approximately 200 mm3, the mice were divided into four groups (eight mice per group): control, PTX-treated, TMP-treated, and PTX+TMP-treated. All components were dissolved in polyoxyethylene castor oil and ethanol mixture  and were diluted with normal saline to required concentrations before administration. According to the previous references, 5 mg/kg of PTX and 60 mg/kg of TMP were used for the in vivo study, and they were administered by tail vein injection once every 2 days  and were histologically analyzed  was considered to be significant.All data are expressed as mean ± SD for at least three independent experiments. Data were analyzed by GraphPad Prism 5.0. Statistical significance was assessed by one-way analysis of variance, and a in vitro cellular assays, we employed high pressure liquid chromatography with ultraviolet (HPLC-UV) methods to assess the purity of PTX and TMP as well as the potential interaction between them. We determined the purity of both compounds to be >99.5%  (We then used HUVEC cells as an P < 0.05), which indicates that the combination of TMP and PTX inhibits angiogenesis by reducing cell metastasis. On the other hand, tube formation assessment revealed that VEGF treatment increased the number of small tubes, while PTX and TMP decreased the number of small tubes. Moreover, co-treatment of PTX and TMP markedly decreased the number of small tubes , which strongly suggests that TMP can enhance the antitumor effect of PTX by promoting apoptosis.Next, we explored the mechanism of PTX+TMP treatment in promoting apoptosis from the molecular level. Specifically, we investigated the expression of apoptotic proteins caspase-3, caspase-9, and their downstream factor PARP. The results showed that PTX treatment partially activated caspase-3, caspase-9, and PARP in A2780 , Project of Sichuan Provincial Science and Technology Department (2019YJ0661), and Young Elite Scientiests Sponsorship Program by CAST (No. QNRC1-01).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.


Poly (ADP-ribose) polymerase 1 (PARP1) and sirtuin 1 (SIRT1) affect cellular NAD+ levels and play essential roles in regulating metabolism. However, there has been little research on the effects of PARP1 and SIRT1 crosstalk during senescence.Nicotinamide adenine dinucleotide  from human umbilical cord blood and treated them with a PARP1 inhibitor (PJ34).2O2, transfection with adenoviral vectors, and Western blot analysis, we observed that PJ34 treatment preserved intracellular NAD+ levels, increased SIRT1 activity, decreased p53 acetylation, and improved the function of stress-induced premature aging EPCs.Using a stress-induced premature aging model built by HOur results suggest that PJ34 improves the function of aging-induced EPCs and may contribute to cellular therapies for atherosclerosis.The online version of this article (10.1186/s13287-018-0961-7) contains supplementary material, which is available to authorized users. Atherosclerosis (AS) is a major cause of cardiovascular disease, and endothelial injury and dysfunction caused by aging are classical risk factors for AS –3. In re+) is a critical molecule involved in various biological functions such as energy generation, metabolism, and DNA repair. NAD+ also plays an important role during aging because it participates in oxidation-reduction (redox) reactions in the tricarboxylic acid (TCA) cycle [Nicotinamide adenine dinucleotide (NADA) cycle –14.+ to glutamate or aspartate residues within target proteins, resulting in protein-conjugated chains of poly ADP-ribose (PAR) polymers [+ consumption [Poly (ADP-ribose) polymerase 1 (PARP1) is a genome-stabilizing enzyme that catalyzes the covalent transfer of mono- or poly-adenosine diphosphate (ADP) units from NADpolymers . PARP1 isumption –18.+-dependent deacetylase that targets several transcription factors, including forkhead box O3, tumor protein p53, nuclear factor κB, and peroxisome proliferator-activated receptor γ coactivator 1 alpha. Therefore, its activity plays an important role in health maintenance [Sirtuin 1 (SIRT1) is a redox-sensitive protein involved in a wide range of cellular processes, including aging, oxidative stress responses, metabolism, circadian rhythm regulation, and proliferation –22. It intenance , 24.+ levels and play crucial roles in regulating cellular metabolism [PARP1 and SIRT1 affect NADtabolism . However+ levels, which improves mitochondrial function [PJ34, a PARP inhibitor, can inhibit the activation from PARP to PAR and thereby preserve intracellular NADfunction .2O2 to build a stress-induced premature aging model [2O2-activated PAR and the cellular states observed under a microscope, we selected an optimum concentration of H2O2. Furthermore, we used several concentrations of PJ34 to inhibit the activation of PARP and selected an optimum concentration on the basis of changes in PAR. Next, we established four treatments to study the effects of PJ34 on aging EPCs. Cells treated with PJ34 were used to measure the effects of PJ34 on young EPCs, cells treated with H2O2 were used to induce premature cellular senescence, and cells treated with H2O2 + PJ34 were compared with the cells treated with H2O2 to verify the effects of PJ34 on aging EPCs. Untreated cells were used as a negative control. Finally, we used SIRT1 short-hairpin RNA (Ad-sh-SIRT1) to silence SIRT1 expression in EPCs and measured the effects of PJ34 again. We measured and evaluated PARP1 activity by assaying the production of PAR [The overall objective of this study was to determine whether PJ34 is able to inhibit EPC senescence and, if so, the mechanism. Firstly, we used Hng model . Endogenng model . Accordin of PAR , 26. SIRn of PAR , 30. EPCn of PAR , 32, celn of PAR , transwen of PAR , 9, and n of PAR .The Ethics Committee of Xinhua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine approved this study. EPCs were isolated from human umbilical cord blood by density gradient centrifugation with Histopaque-1077 (Sigma). We gently added human blood to the separation solution and centrifuged at 2000 rpm for 20 min at 4 °C. After density gradient centrifugation, the uppermost layer contains the serum, while the lowermost layer is composed of red blood cells, and the middle layer of white blood cells is suspended in the separation liquid. We withdrew the white blood cells, resuspended these in complete endothelial cell growth medium (EGM)-2 (Lonza), and then seeded them in six-well plates. Cells from approximately 10 mL of cord blood were plated per well. The medium was changed every 3 days, and cells were subcultured at a 1:3 ratio. EPC endothelial markers were detected by immunofluorescence and flow cytometry. For immunofluorescence, antibodies against CD31, CD34, von Willebrand factor (vWF), vascular endothelial growth factor receptor 2 (VEGFR2), and CD133 were purchased from Cell Signaling Technology. For flow cytometry, antibodies against CD34, CD133, and VEGFR2 were purchased from Invitrogen.Cellular proteins were extracted using radioimmunoprecipitation assay buffer, and protein concentrations were measured using the bicinchoninic acid method. Approximately 30 μg of protein per well was loaded on 8 or 10% gels for sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Millipore), and sequentially detected by primary antibodies, secondary antibodies, and enhanced chemiluminescence (Millipore). Antibodies against PARP1, SIRT1, acetylated (ac)-p53, p53, and cyclin-dependent kinase inhibitor 1A (p21), as well as anti-mouse and anti-rabbit secondary antibodies, were purchased from Cell Signaling Technology. The anti-PAR antibody was purchased from Invitrogen. An anti-β-actin antibody  was used as an internal control.4/well) were suspended in 100 μL fresh medium containing the various treatments and then seeded. After treatment, CCK-8 (10 μL/well) was added for 3 h of additional incubation, and the absorbance was measured at 450 nm.CCK-8 (Dojindo) was used to analyze cell viability after various treatments. Cells  were used to observe cell migration. Complete fresh EGM-2 containing the various treatments (600 μL) was added to the bottom chambers, and 5 × 104 cells/well were seeded on the Matrigel and incubated at 37 °C. Images were acquired after 8 h.Matrigel™  was added to the wells of 96-well plates and incubated at 37 °C for 30 min. Then, 2 × 10An SA-β-gal staining kit  was used to identify cellular senescence. Cells were fixed for 20 min at room temperature and then incubated in staining solution overnight at 37 °C. Three random microscopic fields were selected, and the stained cells were counted.The reactive oxygen species assay kit (Sigma) was used to measure cellular ROS. Cells were seeded in a 96-well plate and incubated with 10 μM dichloro-dihydro-fluorescein diacetate (DCFH-DA) at 37 °C for 30 min. Cells were washed with phosphate-buffered saline three times, and then incubated in fresh medium containing the various treatments. After treatment, the absorbance was measured using a fluorescence enzyme-labeling device at excitation and emission wavelengths of 485 and 535 nm, respectively.+ concentration was calculated according to the formula NAD+ = total NADH − NAD.The NAD/NADH assay kit (Abcam) was used to measure NAD/NADH levels. The standard solution was prepared according to the manufacturer’s protocol, and the NAD/NADH concentration was calculated using the standard curve. The NADAdenoviral vectors containing green fluorescent protein (Ad-GFP) and Ad-sh-SIRT1 were purchased from Hanheng Biotechnology. Ad-GFP was used as a control. Cells were transfected for 6 h and then incubated with fresh medium for 48 h, after which protein expression levels were analyzed by Western blot, or additional treatments were performed.t test. P values < 0.05 were considered statistically significant. All experiments were performed independently in triplicate at a minimum.The results are expressed as the mean ± standard error. Comparisons between two groups were performed using the independent samples The isolated cells exhibited monolayer growth and cobblestone morphology, like typical EPCs Fig. . Most ofTo investigate the differences between young and senescent EPCs, we repeatedly subcultured EPCs for up to 21 passages (to P21). Senescence was confirmed through morphology and SA-β-gal assays. Cells at P1 were small and displayed typical cobblestone-like morphology. With repeated subculture, P21 cells became larger and more irregular in shape, and some were branch-like and polygonal or long and spindle-shaped Fig. . SA-β-gaWe next examined the levels of SIRT1, PARP1, PAR, ac-p53, p53, and p21. Our results showed that with the repeated subculture SIRT1 decreased, while PAR, ac-p53, p53, and p21 increased. There was no significant difference in PARP1 levels between young and replicative aging EPCs Fig. .2O2, and then used various concentrations of PJ34 to inhibit PARP1 activation. By Western blot, we found that H2O2 decreased SIRT1 levels and increased PAR synthesis by activating PARP1  with HRP1 Fig. , b. PJ34nce Fig. , d.Fig. 2O2 and PJ34. EPCs (P3–P5) were incubated with normal fresh culture medium, 2 μM PJ34, 100 μM H2O2, or 2 μM PJ34 + 100 μM H2O2 for 6 h. After incubation, the culture medium was removed. Cells treated with PJ34 were incubated in fresh culture medium containing 2 μM PJ34 for 18 h, and cells subjected to other treatments were incubated in normal fresh culture medium for 18 h.To further verify the effects of crosstalk between PARP1 and SIRT1 on senescence, we first determined the proper concentrations of H2O2 treatment , we speculated that increased NAD+ levels, as a cofactor of SIRT1, were unable to promote SIRT1 expression but activated the biological activity of SIRT1, which could be observed by the improvement of deacetylation functionality.Our results suggested that SIRT1 levels decreased and P53 levels increased with replicative senescence, which is consistent with the results of previous studies , 36. H2O studies , 30. MorThere are some advantages to our study. First, we used a novel approach to understand the effects and mechanism by treating EPCs with PJ34, which will aid further research on senescent EPCs Fig. . Improve2O2 for 6 h and then with fresh culture medium for 18 h exhibited the same state as P20 EPCs, which were identified as being in stress-induced premature cellular senescence [2O2 to establish a senescent model for further and easier research. However, there are many differences between induced senescence and natural senescence. Whether our results can be applied to naturally senescent cells in vitro, and even in vivo, remains to be discussed. Secondly, compared with knockdown, shRNA attenuated protein expression enormously. Hence, our results were not perfect. In addition, NAD+ is widespread in cells and is associated with various functions, and senescence is a slow and complex process involving many regulators and signaling pathways. Therefore, other signaling pathways may play roles in preventing senescence after the elevation of intracellular NAD+ levels. Finally, we found that excessive PJ34 was harmful to EPCs, but the most appropriate concentration remains to be determined.There are several limitations to our study. Firstly, EPCs cultured from human umbilical cord blood entered a senescent stage after a limited number of cell divisions, which was identified as replicative senescence in our study , 38. In nescence . Therefo+ levels, and increased SIRT1 activity.PJ34 can improve the function of aging EPCs through PARP1 inhibition, preservation of intracellular NADAdditional file 1:Figure S1. Identification of EPCs from human umbilical cord blood. Cells were characterized by flow cytometry detection of CD34, VEGFR2, and CD133. (TIF 138 kb)


Camellia sinensis) is enriched with bioactive secondary metabolites, and is one of the most popular nonalcoholic beverages globally. Two tea reference genomes have been reported; however, the functional analysis of tea genes has lagged, mainly due to tea’s recalcitrance to genetic transformation and the absence of alternative high throughput heterologous expression systems. A full-length cDNA collection with a streamlined cloning system is needed in this economically important woody crop species. RNAs were isolated from nine different vegetative tea tissues, pooled, then used to construct a normalized full-length cDNA library. The titer of unamplified and amplified cDNA library was 6.89 × 106 and 1.8 × 1010 cfu/mL, respectively; the library recombinant rate was 87.2%. Preliminary characterization demonstrated that this collection can complement existing tea reference genomes and facilitate rare gene discovery. In addition, to streamline tea cDNA cloning and functional analysis, a binary vector (pBIG2113SF) was reengineered, seven tea cDNAs isolated from this library were successfully cloned into this vector, then transformed into Arabidopsis. One FL-cDNA, which encodes a putative P1B-type ATPase 5 (CsHMA5), was characterized further as a proof of concept. We demonstrated that overexpression of CsHMA5 in Arabidopsis resulted in copper hyposensitivity. Thus, our data demonstrated that this represents an efficient system for rare gene discovery and functional characterization of tea genes. The integration of a tea FL-cDNA collection with efficient cloning and a heterologous expression system would facilitate functional annotation and characterization of tea genes.Tea ( Arabidopsis [Full-length cDNA (FL-cDNA) is the DNA complement to an mRNA sequence that covers the region near the 5′ cap structure to the poly(A) tail . It is ebidopsis , rice [5bidopsis , corn [7bidopsis , citrus bidopsis , cotton bidopsis ,10, and bidopsis . Howeverbidopsis . Thus, abidopsis . A normaCamellia sinensis) is rich in bioactive secondary metabolites, and one of the most popular nonalcoholic beverages globally. In 2017, 6 million tons of tea was produced by 20 top production countries, according to the Food and Agriculture Organization of the United Nations (http://faostat.fao.org/). To this day, tea plants have been cultivated in more than fifty countries, and provide massive job opportunities and social wealth [Camellia sinensis var. assamica  and Camellia sinensis var. sinensis  have been reported, with 36,951 and 33,932 annotated protein-coding genes, respectively [Tea , was cloned and expressed in Arabidopsis. The overexpression of CsHMA5 resulted in copper hyposensitivity, and the phenotypes are consistent with a role of CsHMA5 as a bona fide tea orthologs of the Arabidopsis HMA5 gene. The protocols described in this study have thus been proven to be useful for functional characterization of tea genes. Molecular biology and functional genetic studies have been limited in tea tree, mainly due to two factors: (1) Tea tree is recalcitrant to genetic transformation. The first transgenic tea was reported more than a decade back , since tCamellia sinensis cv. ‘Fuding Dabaicha’ was selected as material for several considerations: (1) it is a national elite germplasm and is widely cultivated in China for the production of green tea, white tea, oolong tea, and black tea; (2) it has been widely used as a maternal line in multiple tea breeding programs in China and produced dozens of new elite germplasms; (3) it is diploid. To maximize the cDNAs included in this library, total RNA was isolated from nine different tissue types, including buds, tender stems, tender leaves, mature leaves, flower buds, flowers, fruit pericarps, roots, and bark. Equal amounts of total RNAs from these tissues were pooled for cDNA library construction. A λ phage (λTriplEx2) was selected as cloning vector since it is not biased toward short insert size. In total, 580 µL of unamplified cDNA library was obtained with a titer of 6.89 × 106 cfu/mL and recombination rate of 87.2%; the titer reached 1.8 × 1010 cfu/mL after library amplification. In this study, a new normalized EST collection with enrichment of full-length and rare transcripts of tea was successfully generated by applying an integrated method. The insert size was evaluated by colony PCR from randomly selected white plaques, which were in the range of 0.8–2.5 kb with medium insertion size of 1.5 kb, as seen in Yunkang 10 ) and Shuchazao ) have been published with 36,951 and 33,932 annotated protein-coding genes, respectively [Two tea reference genomes from ectively ,21. The ectively . Based oectively , were noThese 44 FL-cDNA sequences were used to query the CSS and CSA database, respectively. Thirty-three cDNAs were commonly detected in both databases; seven cDNAs  were found from CSS database but not from CSA database; two cDNAs (MN102719 and MK795766) were found from CSA only. The other two cDNAs, one encoding a natural antisense transcript (clone 34), and the other one encoding a putative long chain noncoding RNA (clone number 43), were not found from CSS and CSA databases, as seen in At the whole-genome level, UTRs provide functional specificity to genes in a length-dependent manner . For theFor MN027184, the annotated CDS from the tea genome database (CSA015215 and TEA028910) has an additional 900 nt before our identified 5′-UTR sequence, as seen in Arabidopsis [Arabidopsis than rice. This is unexpected, since the tea genome (3.02 Gb) is 24-fold larger than that of Arabidopsis (129 Mb); In addition, as a perennial species tea tree may require more complicated gene regulations compared to annual plant species such as Arabidopsis or rice. Thus, it is anticipated that the average length of 5′-UTR and 3′-UTR of tea should be longer than that of Arabidopsis or rice. Arabidopsis tea tree is a dicot, which may suggest that the 5′-UTR and 3′-UTR from dicot and monocot have been independently evolved after their evolutionary divergence. Since only 41 randomly selected tea FL-cDNAs were used here for UTR length calculation, this tentative estimation requires further validation. The extensive sequencing of this cDNA library in the future would provide more conclusive evidence.Based on the validation results above, these three clones were excluded from the calculation of the average length of 5′-UTR, but they are included for the calculation of the average length of 3′-UTR. Our data indicated that the average lengths for the 5′-UTR and 3′-UTR are 128 and 296 bp, respectively. In rice, the average lengths of 5′- and 3′-UTRs are 259 and 469 bp, respectively, but only 155 and 242 bp in bidopsis . Thus, UArabidopsis has several advantages for functional analysis of heterologous genes, such as its small size, short generation time, and its high transformation efficiency [Sfi I cloning sites identical to those of the λ phage vector [ficiency . Thus, ie vector , thus teSfi I digestion, ligated into pBIG2113SF–M vector, and transformed into Arabidopsis. RT-PCR analysis of transgenic plants demonstrated that all seven tea genes were successfully transformed and expressed in Arabidopsis, as seen in To validate this cloning and expression system, seven tea FL-cDNAs were selected , as seen in Arabidopsis HMA5 protein, as seen in AtHMA5. Thus, we renamed the clone 8 cDNA as CsHMA5. CsHMA5 was transformed into Arabidopsis. Several hygromycin-resistant homozygous lines were identified, and two overexpression lines (OX-5-1 and OX-6-2) were characterized further. When germinated on standard 0.5× MS solid medium, transgenic plants showed shorter primary and lateral roots than that of WT control, as seen in CsHMA5 was highly and similarly expressed from both overexpressors, but was undetected in the WT control, as seen in Arabidopsis. The standard 1× MS medium contains 0.1 µM copper. To test if the short root phenotype of the overexpressors was a result of their copper hypersensitivity, WT and two overexpressors were germinated on synthetic 1× MS medium without copper or low copper (0.01 µM). Under both growth conditions, the root lengths of OX-5-1 and OX-6-2 were still shorter than that of control, as seen in CsHMA5 in Arabidopsis did not result in copper hypersensitivity. To test if CsHMA5 overexpressors could show altered response to higher concentrations of copper, seeds of WT and two CsHMA5 overexpressors were germinated on standard 0.5× MS medium supplemented with 12.5–50 µM copper. The exogenously supplementation of copper into standard MS medium would increase its free copper concentration accordingly. After plant growth for 10 days, WT root length from control plate was almost two-fold longer as that of CsHMA5 overexpressors. With the increasing concentration of copper, WT root length became gradually shorter, and this growth inhibition became more evident when exogenously supplied copper reached above 12.5 µM.; in contrast, the root growth from CsHMA5 overexpressors was not affected by as high as 37.5 µM copper, as seen in CsHMA5 in Arabidopsis led to copper hyposensitivity.Clone 8 corresponds to a 1183 nt transcript with a 209-nt 5′-UTR and 248-nt 3′-UTR, and encodes a deduced 25.6 kDa protein of 241 amino acids. Its amino acid sequence showed high similarity to Arabidopsis, HMA5 is primarily expressed in roots, and strongly and specifically induced by copper in whole plants [CsHMA5 overexpression rendered copper sensitivity of transgenic plants, 16 genes from copper signaling pathways were selected, and their expression levels in the root tissues were compared between WT and CsHMA5 overexpressors. We found that the expression levels of the endogenous AtHMA5 from CsHMA5 overexpressors were not affected, as seen in COPT3 and COX17-1 in transgenic lines were significantly higher than that of WT, while COPT5, RAN1, and PAA2 showed significantly lower expression levels than that of WT, as seen in HMA1, PPA1, CPOT1, CPOT2, CPOT4, COX17-2, ZIP2, ZIP4, ATX1, and CCS1, were not significantly affected, as seen in COX17-1 is a soluble protein located in the mitochondrial intermembrane space and participates in the transfer of copper for cytochrome oxidase assembly [COPT5 is a tonoplast copper exporter responsible for the transport of copper ions from root to reproductive organs [RAN1 plays an essential role in the biogenesis of the ethylene receptors and copper homeostasis [PAA2 is a chloroplast copper transporter of the thylakoid membrane [HMA5 in Arabidopsis results in hypersensitivity to copper. In contrast, expression of SvHMA5II in Arabidopsis increases copper tolerance [CsHMA5 in Arabidopsis resulted in hyposensitivity to copper, as seen in Arabidopsis can be used as a heterologous host to study the functions of tea genes.In e plants . To furtassembly , the expassembly . COPT5 ie organs . RAN1 pleostasis . PAA2 ismembrane . These oolerance . In thisCamellia sinensis (L.) O. Kuntze cv. ‘Fuding Dabaicha’) were grown in the tea garden of Fujian Agriculture and Forestry University. Nine different tissues, including roots, tender stems, tender leaves, mature leaves, flower buds, flowers, fruit pericarps, and bark, were collected on September 2015.Tea plants  , mixed well, incubated on ice water bath for 10 min, then centrifuged at 12,000× g for 10 min at 4 °C. The above extraction steps were repeated, and the supernatant was then transferred into fresh tube containing equal volume of prechilled chloroform:isopropanol  , mixed well, and centrifuged at 12,000× g for 15 min at 4 °C. The supernatant was once again transferred into a fresh tube containing half volume of prechilled 8 M LiCl  and 1% β-mercaptoethanol, and then stored at −20 °C overnight. The next day, the mixture was spun at 12,000× g for 30 min at 4 °C, the supernatant was discarded, and the pellet was rinsed with prechilled 75% ethanol, air dried for 10 min, and dissolved in 200–500 µL DEPC-treated water, as seen in Total RNA was isolated by using modified CTAB method . A totalA 500 µg RNA aliquot from each tissue type was pooled together, then used to isolate mRNA by applying PolyATract mRNA Isolation System . The isolated mRNA was eluted into 1 mL of RNase-free water, as seen in The first cDNA strand was synthesized from 1 µg of purified mRNA by using SMART cDNA library construction kit , SMART IV Oligonucleotide and CDS-4M adapter were used for amplification, as seen in DSN  isolated from Kamchatka crab was used for normalization. A total of 0.6–1.2 µg purified ds cDNA, 4 µL of 4× hybridization buffer, and an appropriate volume of sterile RNase-free water was combined to make total volume of 16 µL, mixed well, and spun briefly. A 4 µL aliquot of the reaction mixture was taken into each of the four appropriately labeled sterile PCR tubes, the reaction mixture was overlaid with a drop of mineral oil, and the tubes were centrifuged for 2 min at maximum speed in a microcentrifuge. The tubes were incubated in a thermal cycler at 98 °C for 2 min, followed by incubation at 68 °C for 5 h. Shortly before the end of the hybridization procedure, two DSN dilutions were prepared in two sterile tubes to final DSN concentrations of 0.5 and 0.25 U/µL, then placed on ice. The DSN master buffer was preheated at 68 °C for 3–5 min, 5 µL of the hot DSN master buffer was added to each tube containing hybridized cDNA, and the tube was briefly spun in a microcentrifuge and returned quickly to the thermal cycler, and incubated at 68 °C for 10 min. Then 0, 0.25, 0.5, and 1 U DSN were added into each tube, then immediately returned to the thermal cycler, incubated at 68 °C for 25 min. A total of 5 µL of DSN stop solution was mixed in the tubes and spun briefly, incubated in the thermal cycler at 68 °C for 5 min, and then the tubes were placed on ice. A total of 25 µL of sterile RNase-free water was mixed in each tube and spun briefly, then placed on ice, as seen in Well-normalized cDNA was amplified by Advantage 2 PCR Kit . PCR Primer M1 was used for amplification, as seen in Sfi I  at 50 °C for 2 h, then size fractioned by using CHROMA SPIN TM-400 column. The first four fractions (lanes 5–8) were collected and pooled into a clean tube, as seen in 2O. The Sfi I-digested cDNA is then ready to be ligated into the Sfi I-digested, dephosphorylated λTriplEx2 vector .Purified cDNA was digested by Sfi I digested cDNA to λTriplEx2 vector (500 ng), three different ligations with varying insert to vector ratio were performed, as seen in To ligate To package into the λTriplEx2 vector, 25 µL of MaxPlax Lambda Packaging Extracts  was added into above ligation mixture, the detailed packaging procedure followed manufacturer’s instructions. −2–10−3 with phage dilution buffer, then a 100 µL aliquot was mixed with 100 µL E. coli XL1-blue cell, incubated at 37 °C for 15 min, and then plated onto LB/MgSO4 medium. To determine the recombinant rate, before plating onto LB/MgSO4 medium, 3.0 mL of melted LB/MgSO4 top agar (45 °C) containing 75 µL IPTG Fand 75 µL X-gal was added and mixed well. The plates were incubated at 37 °C for 16–18 h, then plaque numbers were counted. The recombinant rate was expressed as the ratio of white (recombinant) to blue (nonrecombinant) plaques.To titer the unamplified library, the library was diluted to 10The library was amplified and titered according to product manual. E. coli BM25.8 at 31 °C for 30 min without shaking, and 1–10 µL of infected cell suspension was spread onto LB/carbenicillin plate to obtain isolated colonies. A well-isolated positive plaque from secondary or tertiary screening plate was picked, coincubated with 200 µL of Plasmid DNAs were prepared from randomly selected 44 white clones and sequenced by using standard ds-DNA sequencing protocols. The forward and reverse sequencing primers were: 5′-TAATACGACTCACTATAGGGC-3′; and 5′-CTCGGGAAGCGCGCCATTGTG-3′, respectively. The 5′-UTR and 3′-tail sequences were determined from individual cDNA. The CDS sequence was used to query the CSA and CSS genomes using BLAST .Sfi IA and Sfi IB cutting sites of pBIG2113SF vector are different from λTriplEx2 vector and were re-engineered to make them same as λTriplEx2 vector. As a first step, the Sfi IA and Sfi IB cutting sites from vector U12445 were first modified by using Fast Site-Directed Mutagenesis Kit . The primer sequences for Sfi IA mutagenesis were: 5′-ATCTGGAATTCGGCCATTACGGCCAGAAGGAGATA-3′; and 5′-TATCTCCTTCTGGCCGTAATGGCCGAATTCCAGAT-3′. The primer sequences for Sfi IB mutagenesis were: 5′-AAAGTGCCTAAGGCCGCCTCGGCCGTCGACTAGAA-3′; and 5′-TTCTAGTCGACGGCCCGGAGGGCCTTAGGCACTTT-3′. The new U12445 vector with altered Sfi IA and Sfi IB recognition sequence was renamed as U12445M. Binary vector pBIG2113SF was selected to express tea FL-cDNAs in planta . The SfiSfi IA and Sfi IB from U12445M vector was amplified by using following pair of primers: 5′-TACAACTACATCTAGAGTTATCTGGAATTCGGCCATTA-3′; and 5′-CCGGGGATCCTCTAGAAATTCTAGTCGACGGCCGAG-3′. The PCR products were separated by agarose gel electrophoresis and purified by using E.Z.N.A.® Gel Extraction Kit , then cloned into pBIG2113SF vector by gene fusion method. The engineered pBIG2113SF vector with new Sfi IA and Sfi IB sites was renamed as pBIG2113SF-M. The DNA fragment spanning Sfi I digestion at 50 °C for 3 h, purified by gel extraction kit , and then ligated into Sfi I sites of pBIG2113SF-M vector by T4 DNA ligase  at 16 °C overnight. In this report, seven tea FL-cDNAs were cloned into the pBIG2113SF-M vector. Individual clones from the tea cDNA library were converted into pTriplEx2 plasmid, the tea FL-cDNA insert was recovered from the plasmid by Agrobacterium tumefaciens GV3101 and transformed into Arabidopsis thaliana Columbia by the floral dip method. The T1 seeds were harvested and screened in 0.5× MS medium containing 50 μg mL−1 hygromycin. Resistant seedlings were transferred into soil pots, and T2 seeds were harvested from individual plants. T2 seeds were screened in 0.5× MS medium containing 50 μg mL−1 hygromycin. Six resistant seedlings per line were transferred into soil pot, and T3 seeds were harvested individually. Homozygote transgenic T3 seeds were identified and used for further characterization. Tea gene-specific primers were used to confirm transgene expression by RT-PCR, as seen in Individual recombinant pBIG2113SF-M vectors containing tea FL-cDNA insert were introduced into 2O, the pH adjusted to 5.7, and 8 g of agar powder  was added, then copper sulfate was supplemented to specified concentrations. The composition of the synthetic copper deficiency 1× MS medium was (per liter of medium): 1650 mg NH4NO3, 1900 mg KNO3, 440 mg CaCl2.2H2O, 370 mg MgSO4.7H2O, 170 mg KH2PO4, 6.2 mg H3BO3, 16.9 mg MnSO4.H2O, 8.6 mg ZnSO4.H2O, 0.83 mg KI, 0.25 mg NaMoO4.2H2O, 0.025 mg CoCl2.6H2O, 27.8 mg FeSO4.7H2O, 37.3 mg Na2EDTA.2H2O, 103 mg B5 vitamin mix, 15,000 mg sucrose, 8000 mg agar. All the macrosalts and sucrose used in this study were analytical grade. Homozygote transgenic T3 seeds were germinated vertically on growth medium under continuous light (100 µmol photon m−2 s−1). After plant growth for 10 days, root lengths were measured by using Image J software . Data were expressed as mean ± SE (n = 28). After root length measurement, root tissues were harvested to isolate total RNA. One microgram of total RNA was reverse transcribed using PrimeScript™ RT Reagent Kit   and a mixture of oligo (dT)12–18 primer and random hexamers were added. The RT products were diluted three-fold before use as qPCR templates. The primer pairs for copper signaling genes are listed in Arabidopsis ADAPTOR PROTEIN-2 MU-ADAPTIN  was used as loading control. Each reaction was performed in triplicate along with an internal control reaction. Relative gene expression levels were calculated according to the 2Ct−ΔΔ comparative CT method [The standard 0.5× MS medium was prepared from Murashige & Skoog basal medium vitamins . A total of 2.2 g of MS basal medium and 0.5 g of MES were dissolved in 1 L of ddHT method .Arobidopsis P1B-type ATPases and CsHMA5 were done by using DNAMAN software .Protein sequence comparison and the phylogenetic tree construction of Fuding Dabaicha’ were used to make the RNA pool. As a result, our cDNA library also excludes those genes that are specifically expressed in tea seeds.Tea is an obligate out-breeding plant species, which makes its genome highly heterozygous. Its seeds contain genome from two different tea germplasms. To avoid RNA contamination from other tea germplasms, in this study, only vegetative tissues from a single cultivar ‘There are several procedures to make a FL-cDNA library, mostly based on the mRNA cap structure ,41. ThesArabidopsis transformation will obviate the technical hurdle of direct tea transformation, thus facilitating functional analysis of tea genes. This system has the potential to be scaled up by using pooled inserts for cloning and transformation, and a large transgenic population can be easily generated for phenotypic observation. Once phenotypes were scored and corresponding tea transgenes identified, the functions of corresponding tea genes can be elucidated. Such strategies have been applied to study gene functions of model plant species, such as Arabidopsis and rice [Here we performed a small-scale trial and demonstrated that this FL-cDNA library is a valuable resource for new tea gene discovery and functional characterization. A more depth sequencing of this library would be simply a matter of cost. The establishment of easy tea gene cloning in combination with highly efficient and rice ,46, and


Plasmodium sporozoites traverse cells searching for a “suitable” hepatocyte, invading these cells through a process that results in the formation of a parasitophorous vacuole (PV), within which the parasite undergoes intracellular replication as a liver stage. It was previously established that two members of the Plasmodium s48/45 protein family, P36 and P52, are essential for productive invasion of host hepatocytes by sporozoites as their simultaneous deletion results in growth-arrested parasites that lack a PV. Recent studies point toward a pathway of entry possibly involving the interaction of P36 with hepatocyte receptors EphA2, CD81, and SR-B1. However, the relationship between P36 and P52 during sporozoite invasion remains unknown. Here we show that parasites with a single P52 or P36 gene deletion each lack a PV after hepatocyte invasion, thereby pheno-copying the lack of a PV observed for the P52/P36 dual gene deletion parasite line. This indicates that both proteins are equally important in the establishment of a PV and act in the same pathway. We created a Plasmodium yoelii P36mCherry tagged parasite line that allowed us to visualize the subcellular localization of P36 and found that it partially co-localizes with P52 in the sporozoite secretory microneme organelles. Furthermore, through co-immunoprecipitation studies in vivo, we determined that P36 and P52 form a protein complex in sporozoites, indicating a concerted function for both proteins within the PV formation pathway. However, upon sporozoite stimulation, only P36 was released as a secreted protein while P52 was not. Our results support a model in which the putatively glycosylphosphatidylinositol (GPI)-anchored P52 may serve as a scaffold to facilitate the interaction of secreted P36 with the host cell during sporozoite invasion of hepatocytes.Within the liver,  Plasmodium parasites are responsible for taking nearly half a million lives worldwide every year , searching for a suitable host hepatocyte. Upon encountering such a cell, sporozoites switch to “invasion mode” and enter by creating the replication-permissive parasitophorous vacuole (PV)  strain , and ΔP52ΔP36 knockout parasite lines were created by gene replacement through double crossover homologous recombination  relied upon double crossover homologous recombination using a modified plasmid pDEF , transferred onto a PVDF membrane, blocked for 1 h at room temperature in 5% milk in TBST , incubated overnight with α-mCherry (16D7) rat mAb in 2% milk/TBST at 4°C, washed, and incubated with donkey α-rat horseradish peroxidase (HRP)-conjugated IgG in 2% milk/TBST for 1 h at room temperature. The membrane was stripped with Restore (Thermo Fisher Scientific) twice and re-incubated with α-PyP52 (13G10) mouse mAb  containing 10% FBS (Sigma-Aldrich), 200 U/mL penicillin with 200 μg/mL streptomycin (Thermo Fisher Scientific), and 2.5 μg/mL Fungizone (GE Life Sciences) were plated in 8-well chamber slides (1 × 105 cells per well) allowing them to rest overnight. Sporozoites were extracted, activated by incubating with 20% FBS in DMEM for 15 min at room temperature, diluted in complete DMEM at 37°C, and added to the Hepa 1–6 cells to a final concentration of 1 × 105 sporozoites per well in 150 μl total volume. Chamber slides were centrifuged at 500 g for 3 min and invasion was allowed for 90 min for P36mCherry sporozoites or 2 h for RFP-labeled strains at 37°C followed by one quick wash with 1 × PBS. Samples were then fixed with 10% neutral buffered formalin (VWR) for 20 min at room temperature and washed twice with PBS. The slides were kept in a wet chamber at room temperature. Blocking and permeabilization was done simultaneously with 2% (wt/vol) BSA , 0.2% (vol/vol) Triton X-100 (Thermo Fisher Scientific) in PBS for 3 h. After washing, samples were incubated with primary antibody (Table For dy Table  diluted dy Table  in blockFor sporozoite IFAs: sporozoites (3 μl) were spotted onto a glass slide and allowed to dry overnight; fixing, blocking, permeabilization, staining, and mounting were performed as described above. All slides were analyzed by fluorescence microscopy acquiring images with Olympus 1 × 71 DeltaVision Elite deconvolution microscopy. Imaris software was used for 3D image visualization.Py P36mCherry sporozoites were dissected, collected in cold PBS, and fixed in 1% paraformaldehyde (PFA)/0.2% glutaraldehyde (Electron Microscopy Sciences) in PBS for 20 min on ice with frequent agitation. The glands were washed with PBS, stored in 4% sucrose in PBS at 4°C and then embedded in LR White resin (Polysciences). Sections (70 nm thickness) were blocked for 30 min in blocking buffer , incubated overnight at 4°C in blocking buffer containing mouse and/or rabbit primary antibodies  was thoroughly resuspended in 200 μl of ice-cold lysis buffer  containing 2 × protease inhibitor cocktail , placed on ice, and vortexed every 10 min for 1 h. Sporozoite lysates were centrifuged at 13 krpm for 20 min at 4°C and the supernatants were collected and mixed with 200 ul of dilution buffer . P36mCherry lysate, and wild-type lysate as a control for non-specific binding, were incubated with RFP-Trap®_MA beads (Chromotek) to immunoprecipitate mCherry following the manufacturer's instructions with modifications. Briefly, the sporozoite lysates were pre-cleared by incubating with 50 μl of unconjugated control beads at 4°C for 1 h with rotation. After magnetically separating the beads, the lysates were transferred to a new tube containing 50 μl of previously washed RFP-Trap®_MA beads and incubated at 4°C for 1 h with rotation. Control and RFP-Trap®_MA beads were washed four times with dilution buffer, magnetized, and stored at −80°C. Frozen bead pellets were resuspended in 2 × SDS buffer, boiled and magnetized, subjecting supernatants to SDS-PAGE and western blot analysis following the procedure described above; mass spectrometry analysis was also performed in parallel. For the crosslinking experiment, freshly extracted sporozoites were purified on an Accudenz  discontinuous gradient as previously described , spun at 6 krpm for 6 min and stored at −80°C. To create lysates, a frozen sporozoite pellet (3–4 × 102). The following steps were performed on a thermomixer at 800 RPM and 37°C. Gel pieces were de-stained with 50 mM ABC in 50% acetonitrile (ACN) and dehydrated with ACN. Gel pieces were incubated for 30 min in 10 mM dithiothreitol in 100 mM ABC and 20 min in 50 mM iodoacetamide in 100 mM ABC, washed with 50 mM ABC in 50% ACN, dehydrated with 100% ACN, and rehydrated with 6.25 ng/μL sequencing grade trypsin (Promega) in 50 mM ABC. After incubating overnight at 37°C, the supernatant was recovered, and peptides were extracted by incubating the gel pieces 20 min each with 2% v/v ACN and 1% v/v formic acid (FA) in water, then 60% v/v ACN and 1% v/v FA in water, then 100% ACN. The extractions were combined with the digest supernatant, evaporated to dryness in a rotary vacuum, and reconstituted in liquid chromatrography (LC) loading buffer consisting of 2% v/v ACN and 0.2% v/v trifluoroacetic acid (TFA).With the exception of the cross-linked sample, proteins isolated from the RFP-Trap® IP were subjected to SDS-PAGE pre-fractionation and in-gel tryptic digestion essentially as described in Lindner et al. . BrieflyThe BS3-crosslinked sample was immunoprecipitated with RFP-Trap® and eluted from beads by adding 50 μL of 5%SDS/50 mM Tris pH 8.5 to which 0.5 mM Bond-Breaker TCEP solution (Thermo Fisher Scientific) was added to 19 mM per 100 μL of beads and incubating 10 min at 95°C. A total of 300 μL eluate was collected, to which 8 μL of 1 M iodoacetamide was added and incubated at room temperature in darkness for 20 min. The eluted protein was digested using an S-Trap Micro (Protifi) following the manufacturer's protocol. Briefly, 30 μL of 12% v/v phosphoric acid was added to the eluate, followed by 1.980 mL S-Trap buffer (90% v/v methanol/ 50 mM Tris pH 8.5). The sample was loaded onto the S-trap and washed with S-Trap buffer by centrifugation. Sixty microliters of 50 mM Tris pH 8.5 containing 0.6 μg of trypsin (Promega sequencing grade) was added and the trap was incubated 3 h at 47°C. Peptides were eluted with 40 μL each of 50 mM Tris pH 8.5, 0.2% v/v formic acid, and 50% v/v ACN/0.2% v/v formic acid, dried down in a speed vac, and reconstituted in 20 μL 5% ACN/0.5% TFA.Except for the crosslinked sample, LC was performed using an Agilent 1100 nano pump with electronically controlled split flow. Peptides were separated on a column with an integrated fritted tip  packed in-house with a 20 cm bed of C18 . Prior to each run, sample was loaded onto a trap column consisting of a fritted capillary  packed with a 1 cm bed of the same stationary phase and washed with loading buffer. The trap was then placed in-line with the separation column for the separation gradient. The LC mobile phases consisted of buffer A (0.1% v/v FA in water) and buffer B (0.1% v/v FA in ACN). The separation gradient was as follows: 5–35% B over 60 min, 35–80% B over 10 min, 10 min at 80% B, 80–5% B over 1 min, then 29 min at 5% B. Tandem MS (MS/MS) was performed with an LTQ Velos Pro-Orbitrap Elite (Thermo Fisher Scientific). A top-20 data-dependent acquisition method was employed to select the top precursors for collision-induced dissociation (CID) and analysis in the ion trap. Dynamic exclusion was enabled to exclude a precursor mass with a ± 5 ppm mass tolerance for 30 s after observing it once. Precursor charge state selection was employed to selecting only doubly- and triply-charged precursors. Two nanoLC MS technical replicates were performed for each fraction, with roughly half the available sample injected for each replicate.The BS3-crosslinked peptides were analyzed using an EASY nLC coupled to a Q-Exactive HF (Thermo Fisher Scientific). Peptides were separated on a column with an integrated fritted tip  packed in-house with a 50 cm bed of C18 . Prior to each run, sample was loaded onto a trap column  and washed with buffer A. The separation gradient was as follows: 5–35% B over 120 min, 35–80% B over 1 min, 15 min at 80% B, 80% B to 2% B over 1 min, then 20 min at 2% B. A top-15 data-dependent acquisition method was employed to select the top precursors for higher-energy collision-induced dissociation (HCD). Twenty seconds of dynamic exclusion was enabled. Precursor charge state selection was employed to exclude singly-charged precursors and those with charge state 6 and higher. Half the available sample was injected.P. yoelii 17 × . Decoy proteins with the residues between tryptic residues randomly shuffled were generated using a tool included in the Trans-Proteomic Pipeline (TPP) and interleaved among the real entries. The precursor mass tolerance was set to ±10 ppm. Fragment ion bin and offset were set to 1.0005 and 0.4, respectively, for ion trap spectra and 0.02 and 0.0, respectively, for HCD spectra. Semi-tryptic peptides and up to 2 missed cleavages were allowed. The search parameters included a static modification of + 57.021464 Da at Cys for carbamidomethylation by iodoacetamide and potential modifications of +15.994915 Da at Met for oxidation, + 17.026549 Da at peptide N–terminal Gln for deamidation from formation of pyro–Glu, + 18.010565 Da at peptide N–terminal Glu for loss of water from formation of pyro–Glu, + 17.026549 Da at peptide N–terminal carbamidomethylated Cys for deamidation from formation of S–carbamoylmethylcysteine, and + 42.010565 at the N–terminus of the protein  for acetylation. An additional variable modification of + 156.0786 at Lys and peptide N-terminus for dead-end BS3. The MS/MS data were analyzed using the TPP  were kept on ice, washed once with ice-cold PBS, and split into two equal aliquots. After centrifuging the sporozoites for 4 min at 4 krpm, the pellets were resuspended in 30 ul of ice-cold PBS, incubating the control aliquot on ice and the activated aliquot in a 37°C water bath for 40 min. The aliquots were then centrifuged at 6 krpm for 6 min at 4°C, collecting the supernatants; pellets and supernatants were stored at −80°C. Supernatants were mixed with 4 × SDS buffer, and pellets were fully resuspended in 2 × SDS buffer to create lysates, followed by SDS-PAGE and western blot analysis as previously described.Isolated sporozoites  and is an AAALAC accredited facility. The facilities and programs of the vivarium are operated in compliance with state law, federal statute and NIH policy. All protocols involving research animals were reviewed and approved by the Center for Infectious Disease Research IACUC . Ketamine/Xylazine was used as an anesthetic. Euthanasia methods followed the recommendations of the American Veterinary Medical Association's Panel on Euthanasia. Mice were euthanized by COPlasmodium yoelii parasites were genetically modified by double crossover recombination to create ΔP36 and ΔP52 single gene deletion mutants and a ΔP52ΔP36 dual gene deletion mutant, all expressing red fluorescent protein (RFP) in their cytoplasm  and salivary gland-derived sporozoites by live fluorescence microscopy  approximately at the predicted molecular weights (MW) of 66 and 53 kDa, respectively , both proteins previously reported to be localized in the micronemes  labeled with P36 were also labeled with P52, while more than two thirds (F = 0.68) of all P52-labeled micronemes were simultaneously labeled with P36. For P36 and TRAP, out of all micronemes labeled with TRAP, 42% also contained P36; and about 58% of all P36-labeled micronemes were also labeled with TRAP. For P52 and TRAP, there was a similar percentage of single and double labeled micronemes containing TRAP; however, the majority of all P52-labeled micronemes (nearly 88%) were also labeled with TRAP. Rather than reflecting a random distribution, these findings are indicative of an intrinsic higher probability for P52 to be localized in micronemes containing either TRAP or P36, instead of being found individually. Therefore, a mathematical correlation should exist between the probabilities of finding P52 co-localized [(P52 + P36) and (P52 + TRAP)] and the probability of finding TRAP and P36 in a given microneme. Accordingly, and taking the observed frequencies (F) as probabilities (p), p[P52 + P36]* p[P52 + TRAP] = p[P36 + TRAP] resulting in (0.68*0.875) = 0.595, which is remarkably close to the experimentally observed frequency (F = 0.58) for finding P36 and TRAP together, suggesting a real tendency for P52 to be found co-localized. Triple labeling experiments were attempted, however none of the rat α-mCherry antibodies tested worked well for this purpose.To determine the precise subcellular localization of P36, and to confirm the localization of P52 with higher resolution than previously reported  from salivary gland sporozoite lysates using RFP-Trap®, technology based on alpaca nanobodies with remarkable affinity and specificity to the mCherry tag . Wild-type sporozoite lysates were used in parallel as a control for non-specific binding. Analysis of the immunoprecipitated samples by western blot  and in the RFP-Trap® precipitated sample (IP). The unconjugated bead control (C) did not show a signal, as expected, and only a faint band was seen in the flow through (FT), indicating most of P36mCherry was successfully immunoprecipitated. Probing with α-P52 antibody after stripping revealed the presence of P52 in the input (IN) of both wild-type and P36mCherry sporozoites near the expected MW (53 kDa), and an equal intensity band for the wild-type flow through was observed as there was no depletion from incubating with RFP-Trap®. In the P36mCherry sporozoite sample, P52 was observed with the same pattern as P36mCherry with a strong band in the immunoprecipitated sample (IP), only a faint band left in the flow through (FT) sample, and no signal for the unconjugated bead control (C), indicating a specific affinity of P52 for P36mCherry. Probing with α-TRAP antibodies after stripping, as an additional control, did not show a signal for any of the immunoprecipitated (IP) samples (data not shown).To explore the possible interacting partners of P36 within the sporozoite, P36t Figure  probed wmCherry sporozoite lysates were chemically crosslinked using 2.5mM bis[sulfosuccinimidyl] suberate (BS3)  was observed for the crosslinked lysates when probing with α-mCherry indicating that P36mCherry is part of a protein complex with an apparent MW of ~120–150 kDa when crosslinked. The detected signal was within range of the expected MW of the P36mCherry-P52 complex (119 kDa) considering that a more native-like conformation should be preserved by the crosslinker. Furthermore, stripping and re-incubating the blot with α-P52 confirmed the presence of P52 within the same crosslinked complex. An equivalent shift in MW was also observed for P52 using crosslinked lysates from wild-type sporozoites (data not shown).To further investigate the P52-P36 interaction, P36) Figure  and analmCherry and co-precipitated protein partners were analyzed in more detail by mass spectrometry comparing against the IP of the wild-type lysate made with the same number of sporozoites; both lysates were previously incubated with unconjugated beads to assess non-specific binding. The relative abundance of proteins detected from test and control samples was assessed by spectral counting. The Plasmodium proteins exhibiting the highest enrichment relative to control were P36mCherry, P52, and HSP70-2/BiP  but was high-abundance in the test IP (78 PSMs from 25 unique peptides). Other Plasmodium proteins in the test IP were less abundant relative to the control  displayed a bulk center-concentrated signal for P36mCherry and an outer rim-like signal for TRAP  lacked circumferential filipin staining, further showing the inability of single gene knockouts to form a PVM. Sporozoites also traverse host cells but in transient vacuoles (TV) formed without rhoptry secretion or membrane remodeling before committing to productive invasion, which could confound the detection of true PVMs in which the parasites are ensconced. However, our analysis of UIS4, which is only inserted into the PVM but not the TV, as well the time of analysis of 2 h post sporozoite infection, when cell traversal has mostly ceased, make this unlikely  and Maurer's clefts in blood stages  (Carruthers and Sibley, Pb P52 was originally described as being secreted and retained onto the sporozoite surface upon gliding (Ishino et al., At the time of invasion, proteins stored in the micronemes are secreted to establish contact with the corresponding receptors. Microneme secretion is a temperature dependent process and it is regulated by the mobilization of parasite intracellular CaDespite both P36 and P52 being essential for productive hepatocyte invasion, recent studies suggest that only P36 might interact with the host cell. Previous indirect evidence was reported for the interaction of P36 with the host cell receptors EphA2, CD81 and SR-B1. All three host cell receptors are required for productive invasion to various degrees and, in the case of EphA2 and CD81, for PVM formation (Kaushansky et al., Conceptualization: SA, SM, AV, and SK; formal analysis: KS; funding acquisition: KS, NS, RM, and SK; investigation: SA, KS, TM, RS, DD, AH, SM, and MT; methodology: SA, KS, TM, RS, NC, SM, AV, and MT; resources: BO, VV, NK, TI, NS, and WB; supervision: NS, RM, and SK; visualization: SA, KS, and MT; writing-original draft: SA, KS, RS, VV, and AV; writing-review and editing: SK.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.