In the current study, we aimed at investigating if these Thai medicinal herb extracts played a molecular role in suppressing psoriasis via regulation of NF-κB signaling biomarkers. Using semi-quantitative RT-PCR and report gene assays, we analyzed the effects of these potential herbal extracts on 10 different genes of the NF-κB signaling network in HaCaT cells. In accordance with our hypothesis, we found that the extract derived from Alpinia galanga significantly increased the expression of TNFAIP3 and significantly reduced the expression of CSF-1 and NF-κB2. Curcuma longa extract significantly decreased the expression of CSF-1, IL-8, NF-κB2, NF-κB1 and RelA, while Annona squamosa extract significantly lowered the expression of CD40 and NF-κB1. Therefore, this in vitro study suggested that these herbal extracts capable of functioning against psoriasis, might exert their activity by controlling the expression of NF-κB signaling biomarkers.Psoriasis is a chronic inflammatory skin disorder characterized by rapid proliferation of keratinocytes and incomplete keratinization. Discovery of safer and more effective anti-psoriatic drugs remains an area of active research at the present time. Using a HaCaT keratinocyte cell line as an  Psoriasis, a chronic inflammatory skin disease affecting about 2–3% of the worldwide population, is characterized by hyperproliferation and abnormal differentiation of keratinocytes . At present, psoriasis is incurable, and its cause remains unelucidated. However, many studies have reported various factors contributing to the pathogenesis of psoriasis, including genetics, the immune system and environmental conditions, thus recognizing it as a multifactorial disease . Recently, a genome-wide scan study in 1,409 psoriasis cases and 1,436 controls revealed associations between three genes of the IL-23 pathway , two genes of nuclear factor-κB (NF-κB) pathway (TNIP1 and TNFAIP3) and psoriasis .in vitro screening of 11 Thai medicinal herbs used for treating skin diseases, we identified three herbal extracts, Alpinia galanga (rhizomes), Curcuma longa (rhizomes) and Annona squamosa (leaf), showing anti-psoriatic activity against a HaCaT cell line chosen as an in vitro model  were deposited at the Professor Kasin Suvatabhandhu Herbarium, Department of Botany, Faculty of Science, Chulalongkorn University, Thailand. All plants were extracted by maceration at room temperature with ethanol  using a 1:5 (w/v) ratio in a shaking incubator at 120 rpm for 48 h. The medicinal plant extracts were filtered, and the residues were subsequently extracted twice more. After the two filtrates were combined, the crude extracts were concentrated using the MiVac Quattro concentrator at 45 °C. Finally, the resulting crude extracts were dissolved in dimethyl sulphoxide  as stock solutions (100 mg/mL), stored at −20 °C and protected from light. Prior to incubation with HaCaT cells, the crude extracts were filtered through a 0.2 µm pore size filter .In brief, the Thai traditional medicinal plants, 2 for 24 h. HaCaT cells , the immortalized human epidermal keratinocyte cell line, were cultured in Dulbecco’s Modified Eagle Medium/High glucose (DMEM/HG)  with 10% fetal bovine serum, 100 U/mL penicillin and 100 µg/mL streptomycin (Hyclone). The cells were cultured at 37 °C in a humidified atmosphere at 5% CO50 value for each plant used was also addressed, particularly those Thai medicinal herbs having anti-psoriatic effect, Annona squamosa, Alpinia galanga, and Curcuma longa [6 cells. The cells were pretreated with pro-inflammatory cytokines, TNFα (10 ng/mL) and IFNγ (10 ng/mL) , for 12 h and 24 h or were left nonpretreated. After that, pretreated cells were washed with Phosphate Buffer Saline  and treated with various concentrations of each plant extract: 6.300, 3.150, 1.575 µg/mL (IC50 = 6.300) of Alpinia galanga rhizome extract, Annona squamosa leaf extract or 6.700, 3.350, 1.675 µg/mL (IC50 = 6.700) of Curcuma longa rhizome extract for the period of either 24 h or 48 h at 37 °C in a humidified atmosphere at 5% CO2. Control cells were either only pretreated cells or left nonpretreated cells depending on the experimental design. Plant materials and anti-psoriatic activity assays were previously described by our group, and ICma longa . HaCaT c® reagent  according to the manufacturer’s instructions. After extraction, total RNA was measured at 260 nm for quantification. Before performing the RT reaction, total RNA was adjusted to the final concentration of 1 μg/uL, and RNA was treated with 10−5 U deoxyribonuclease I (DNase I) (Invitrogen) for 30 min at 37 °C. DNase I-treated RNA was reverse transcribed by ImProm-IITM Reverse Transcription System  with oligo(dT)17 primer following the manufacturer’s protocol. For amplification of the cDNA, each desired DNA fragment was amplified for 35–40 cycles using each gene-specific primer pair as listed in Treated cells were washed with PBS. Total RNA was isolated from HaCaT cells by TrizolPCR reaction conditions consisted of 1 min denaturation at 94 °C, a 1 min annealing at 55 °C for TNFAIP3, IRF-1, RelB, IL-8 and NF-κB1, 60 °C for β-actin, 58.4 °C for NF-κB2, 55.9 °C for CD40, 58 °C for CSF1 and 67.7 °C for RelA, and a 1 min extension at 72 °C. The products of RT-PCR were performed on 2% agarose gel electrophoresis and visualized by ethidium bromide staining. All band densities were analyzed by the GeneTools program in a G:Box .Renilla luciferase) internal control plasmid  was used in this study.The promoterless luciferase expression vector (pSGG_prom vector) and NF-κB Biomarker Set consisting of eight luciferase reporter promoter gene constructs -CD40 , CSF-1 (914 bp), IL-8 , IRF-1 (958 bp), NF-κB2 , RelB , TNFAIP3 , VCAM1 - were kindly provided by SwitchGear Genomics . The pRL-CMV  for NF-κB1 and specific primers encompassing position −1061 to +165  for RelA. PCR conditions consisted of 30 s denaturation at 94 °C, a 30 min annealing at 58.1 °C for NF-κB1 promoter and 60.4 °C for RelA promoter, 58.4 °C for NF-κB2, and a 2 min extension at 72 °C. The PCR products were purified and cloned into pGEM-T Easy vector (Promega). Subsequently, target DNA fragments were digested from the clones with MluI/BglII and NheI/HindIII for NF-κB1 and RelA, respectively. Each corresponding promoter fragment was then purified and subcloned into restriction sites upstream of the luciferase gene of pSGG_prom empty vector. Finally, newly cloned reporter constructs were verified by DNA sequencing and used for reporter gene assays. In the present study, we made two additional reporter gene constructs (NF-κB1 and RelA) since they were also members of NF-κB family. Genomic DNA was extracted from HaCaT cells in order to use as a PCR template for amplifying promoters of both genes. For generating the promoter fragment, PCR was performed using specific primers encompassing position −979 to +15 (forward primer containing 50 = 6.300) of Alpinia galanga rhizome extract, Annona squamosa leaf extract, and 6.700, 3.350, 1.675 µg/mL (IC50 = 6.700) of Curcuma longa rhizome extract, for 24 h and 48 h or were left nonpretreated prior to lysis. The lysate was analyzed for the firefly and Renilla luciferase activities by Dual-Luciferase® Reporter Assay System (Promega) following a procedure given by the manufacturer. VICTOR3 luminometer  was used for measuring chemiluminescence. Luciferase signals were normalized to Renilla luciferase within each sample.For transient transfection, HaCaT cells were cultured to reach about 70% confluence in six-well plates. Transfection of reporter plasmids into HaCaT cells was performed in DMEM without serum and antibiotics using FuGene HD reagent  following a procedure described by the manufacturer with little modifications. In particular, HaCaT cells were transfected with 2 µg of each reporter construct and cotransfected with 600 ng of pRL-CMV vector. Following 8 h of incubation with the transfection complex, the medium was changed to DMEM/HG with 10% fetal bovine serum, 100 U/mL penicillin and 100 µg/mL streptomycin, and cells were pretreated with proinflammatory cytokines, TNFα (10 ng/mL) and IFNγ (10 ng/mL) for 12 h and 24 h. Then, cells were treated with 6.300, 3.150, 1.575 µg/mL . Differences were considered to be significant at P < 0.05.Data are presented as the mean ± standard error of the mean (SEM) from three independent experiments for RT-PCR and nine independent experiments for reporter assays, and were analyzed by one-way analysis of variance (One-way ANOVA), followed by Alpinia galanga could regulate NF-κB networks via decreased expression of CSF-1 and NF-κB2, while increased expression of TNFAIP3. Curcuma longa could decrease the expression of CSF-1, IL-8, NF-κB2, NF-κB1 and RelA. Annona squamosa could reduce the expression of CD40 and NF-κB1. Therefore, we demonstrated the effects of Thai medicinal herb extracts on downregulation of NF-κB signaling molecules, reflecting their potential use in treating such disease with inflammation and hyperproliferation as psoriasis. In the future, we hope that many research groups will attempt to study and develop Thai medicinal herbs for therapeutic applications with improved efficacy, safe and reduced cost. The present data of treatment with Thai medicinal herb extracts suggest that ethanolic extracts of


Brassica have been widely cultivated due to the propagation of SI lines by spraying a salt solution. We demonstrated that suppression of Brassica napus SI from edible salt solution treatment was ascribed to sodium chloride and independent of S haplotypes, but it did not obviously change the expression of SI-related genes. Using the isobaric tags for relative and absolute quantitation (iTRAQ) technique, we identified 885 differentially accumulated proteins (DAPs) in Brassica napus stigmas of un-pollinated (UP), pollinated with compatible pollen (PC), pollinated with incompatible pollen (PI), and pollinated with incompatible pollen after edible salt solution treatment (NA). Of the 307 DAPs in NA/UP, 134 were unique and 94 were shared only with PC/UP. In PC and NA, some salt stress protein species, such as glyoxalase I, were induced, and these protein species were likely to participate in the self-compatibility (SC) pathway. Most of the identified protein species were related to metabolic pathways, biosynthesis of secondary metabolites, ribosome, and so on. A systematic analysis implied that salt treatment-overcoming SI in B.napus was likely conferred by at least five different physiological mechanisms: (i) the use of Ca2+ as signal molecule; (ii) loosening of the cell wall to allow pollen tube penetration; (iii) synthesis of compatibility factor protein species for pollen tube growth; (iv) depolymerization of microtubule networks to facilitate pollen tube movement; and (v) inhibition of protein degradation pathways to restrain the SI response.Self-incompatibility (SI) in plants genetically prevents self-fertilization to promote outcrossing and genetic diversity. Its hybrids in  Brassica napus), a plant from the Brassicaceae family, is one of the most important edible oil-producing crops, and its F1 hybrids have been widely cultivated worldwide for many years. However, self-incompatibility (SI) has been used in canola hybrid breeding for a long time. B. napus , a member of Brassica, was formed by natural allopolyploidization between Brassica oleracea  and Brassica rapa  [B. napus is a self-compatible species, SI lines can be generated by interspecific hybridization between B. oleracea and B. napus [B. oleracea and B. rapa [Brassica can be overcome by using physical and chemical methods, such as CO2 gas (3–5%) treatment [B. napus can efficiently overcome SI [B. oleracea and B. rapa for a long time, and recently in B. napus, which has sped up hybrid breeding by self-incompatibility in Brassica. Considering its significant economic contribution, it is necessary to understand the molecular mechanism of salt solution-overcoming SI. However, little is known, especially in B. napus.Canola  was first identified as an SI-related gene in stigma, and subsequently it was demonstrated that it was not the female S determinant that was required for full manifestation of the SI response [S-locus receptor kinase gene (SRK), and the male determinant S-locus protein 11 gene (SP11)/S-locus cysteine-rich protein gene (SCR)  are the two vital genes of the SI response [SRK was inhibited by the thioredoxin h proteins THL1 and THL2 [SRK is activated by SCR, accompanied by another S-locus cytoplasmic receptor kinase, M-locus protein kinase (MLPK) [SRK phosphorylates AMR-repeat containing 1 (ARC1), resulting in ubiquitination and proteasomal degradation of SC-related proteins, such as Exo70A1, which is a subunit of the exocyst consisting of eight subunits [SECRETORY3(SEC3), SEC5, SEC6, SEC8, SEC10, SEC15, and EXO84, were tested as compatible factors to promote compatible pollen grain acceptance [ARC1 linear ubiquitin–proteasome pathway, much evidence has suggested that the SI response is likely to be more complex, and to contain multiple parallel pathways rather than only the ARC1 linear ubiquitin–proteasome pathway [Self-incompatibility is one of the most important mechanisms for maintaining species diversity and offspring vigor . Self-inaplotype . S-locusresponse . The femresponse . Before and THL2 ,15. Oncee (MLPK) . Throughsubunits ,18. Receceptance . Althoug pathway . HoweverB. napus. Microarray analysis of B. napus SC/SI pollination stigmas revealed a rapid stigma senescence response following compatible pollination [Glyoxalase I (GLO1), one of the 19 proteins, was further detected by reverse genetics to be a stigmatic compatibility factor necessary for reproduction [B. napus self-incompatibility response have been limited to 2D gel electrophoresis analysis [B. napus.In recent years, omics technologies have played an increasingly important role in uncovering the self-incompatibility mechanism in lination . Time-colination . Nineteelination . Glyoxaloduction . So far,analysis . Isobarianalysis , and areanalysis ,28. TherIn this study, phenotypic observations of pollen tube elongation and pod seed-set were first performed, SI-related gene expression levels were checked by qPCR, and iTRAQ was carried out for self-incompatible pollination, salt-induced self-compatible pollination, and self-compatible pollination. Our aims were (i) to elucidate some characteristics of salt-induced self-compatibility, (ii) to dissect any effect of salt solution on self-incompatibility-related gene expression, and (iii) to identify differentially accumulated proteins (DAPs) induced by salt solution and their functional annotations. All our results provide detailed information on the breakdown of SI by salt solution and abundant molecular data on compatible/incompatible pollen–stigma interactions, which provide new insights into networks of pollen–stigma interaction signal transduction.On the stigmas of both “W-3” and “Westar”, few pollen grain adhesions and no pollen tube growth were observed when pollinated with “W-3” pollen grains, and pollen tubes could traverse stigma when pollinated with “Westar” pollen grains A. Seed-sBnS-1300, while the dominant class I S haplotype BnS-1 controls the self-incompatibility of “W-3”. Like “W-3”, “S-1300” was observed to have self-pollen elongation, and set many seeds when pollinated with self-pollen grains after treatment with the salt solution  and BnGLO2 (GSBRNA2T00100627001) were glyoxalase I, with higher homology to AT1G11840 (ATGLYI3) and AT1G08110 (ATGLYI2), respectively, but the homology was very low among them (data not shown), which suggested that there existed more than one glyoxalase I gene participating in the SC response. As BnARC1 is a positive self-incompatibility factor and BnEXO70A1 and BnGLO1 are negative self-incompatibility factors, expression changes of SI-related genes could not explain the mechanism of edible salt solution-overcoming SI.To detect whether salt-induced self-compatibility was due to SI/SC-related gene expression changes, qPCR was carried out to analyze the quantitative expression of d BnGLO2 . The fou, and NA A,D. Both samples E,G. BnTH with UP F. NA hadd BnGLO2 B,C,H,I. SRK (GSBRNA2T00102188001) protein, but no quantitative data were acquired (SLG (GSBRNA2T00102191001), THL2 (GSBRNA2T00043000001) and GLO1 proteins were identified, their expression was not significantly changed based on screening criteria. Another glyoxalase I gene, GLO2, comparing with UP, was increased by ~1.55 and ~1.82 fold in PC and NA, respectively. However, other SI-related genes, ARC1 (GSBRNA2T00069618001), MLPK (GSBRNA2T00101663001), EXO70A1 (GSBRNA2T00136453001), and THL1 (GSBRNA2T00076902001) were not identified in our study. These results indicated that suppression of SI by salt solution was because of increasing SC-related protein species accumulation.In the following proteomics results, we identified acquired . AlthougiTRAQ analysis was conducted to detect any mechanism of salt-induced self-compatibility. The four samples for iTRAQ analysis were the same with those for qPCR analysis described above . After mFor the 5458 identified protein species, the protein mass from 10 to 100 kDa were approximately normally distributed, and the percentages of 10–50, 50–100, and above 100 kDa protein were 47%, 40%, and 13%, respectively . The pephttp://www.gene-ontology.org) against the non-redundant protein database . They possessed a wide range of biological processes, cellular components, and molecular functions, which could be grouped into 22, 15, and 16 categories, respectively  categories were conducted using the Blast2GO program  were considered as those displayed a fold change >1.3 or <0.77 in relative abundance and a ectively .A Venn diagram was generated to show the relationship of DAPs among PC/UP, PI/UP, and NA/UP . In totaOut of the 307 differentially accumulated proteins in NA/UP, 17 were shared only with PI/UP and 94 shared only with PC/UP, in addition to the 62 common and 134 unique DAPs. Eighty-nine of the 94 protein species displayed the same expression trend in PC/UP and NA/UP (45 up-accumulated and 44 down-accumulated) . We obsep = 2.39 × 10−6), “macromolecular complex assembly” (p = 1.41 × 10−5), “generation of precursor metabolites and energy” (p = 2.55 × 10−5), “photosynthesis, light reaction” (p = 6.31 × 10−5), “ribosome biogenesis” (p = 6.48 × 10−5), “ribonucleoprotein complex biogenesis” (p = 9.28 × 10−5), “photosystem II assembly” (p = 1.08 × 10−4), and “monosaccharide metabolic process” (p = 1.72 × 10−4) in biological processes  in PC/UP were classified into 41 functional groups C; the birocesses .p = 1.14 × 10−7), “translation” (p = 1.47 × 10−6), “RNA modification” (p = 2.61 × 10−5), “response to cold” (p = 1.01 × 10−4), “response to temperature stimulus” (p = 1.35 × 10−4), “plant-type cell wall organization” (p = 3.49 × 10−4), “defense response, incompatible interaction” (p = 3.52 × 10−4), “plant-type cell wall modification” (p = 3.70 × 10−4) in biological processes  in NA/UP were classified into 42 functional groups C, among rocesses .p = 4.59–5), “plant-type cell wall organization” (p = 8.51 × 10−4), “cell wall modification” (p = 9.30 × 10−4), “external encapsulating structure organization” (p = 0.0017), “oxidation–reduction process” (p = 0.0019), “fertilization” (p = 0.0032), “double fertilization forming a zygote and endosperm” (p = 0.0032), “pollen exine formation” (p = 0.0044) in biological processes  were classified into 44 functional groups C, of whirocesses .p = 1.05 × 10−7), “RNA methylation” (p = 3.38 × 10−6), “RNA modification” (p = 5.97 × 10−5), “outer mitochondrial membrane organization” (p = 3.27 × 10−4), “protein import into mitochondrial outer membrane” (p = 3.27 × 10−4), “macromolecule methylation” (p = 5.20 × 10−4), “ribosome biogenesis” (p = 8.89 × 10−4), and “ribonucleoprotein complex biogenesis” (p = 0.0010) in the biological processes (p = 0.16028) was not a significantly enriched term based on a cutoff value of a p < 0.05 threshold, 15 proteins were involved in “response to salt stress”, and all of these proteins were DAPs in PC/UP and NA/UP, rather than in PI/UP  in NA/UP, with 134 unique and 94 shared only with PC/UP. Functional classification showed that ribosome and phagosome were two significant enrichment pathways at the 94 DAPs. All the results indicated that SI destroyed by edible salt solution treatment was attributed to the protein species of self-compatible pollination response and to unique protein species, and the DAPs in the ribosome and phagosome pathways might be candidates induced by salt solution.Propagating SI lines is basic and necessary for the utilization of self-incompatibility in hybrid seed production. To obtain many self-incompatible seed lines, a double-cross model previously in Brassica  and a trB. napus  were proBnSP11-1 in self-compatible “Westar” pollen grains. We sampled stigmas of “Westar” rather than “W-3” because “Westar” produced many self-pollinated seeds, and did not have any gene transformation, most importantly, DAPs in NA/UP could accurately be compared with those in PC/UP. Out of the 19 proteins, glyoxalase I (GLO1) was confirmed to be a stigmatic compatibility factor [We identified 307 differentially accumulated proteins (DAPs) in NA/UP by iTRAQ technology, much more than the 19 DAPs by 2-D difference gel electrophoresis analysis . Furthery factor . Therefo+-ATPase, were enriched in response to salt stress  was identified as a compatibility factor in B. napus [GLOI from Brassica juncea was considered to confer salinity tolerance in transgenic tobacco [BnGLO2 (GSBRNA2T00100627001) as glyoxalase I was identified and up-accumulated in PC/UP and NA/UP (GLOI and then led the SC response to occur. UDP-glucose pyrophosphorylase (UGPase) is a ubiquitous enzyme in plants, and produces UDP-glucose, which is essential for sucrose and polysaccharide synthesis. Studies by UGPase mutants revealed loss or drastically decreased activity of UGPase, resulting in a decreased number of seeds produced [UGPase in NA/UP indicated that salt solution treatment could promote seed production in our study. Salt stress can induce osmotic stress and ion stress [+-ATPaseH is a proton pump that uses the energy from ATP hydrolysis to produce a proton gradient, which can maintain ion balance. It has already been proven to be an essential component of key signal transduction [+-ATPaseH protein was significantly down-accumulated. By contrast, salt solution treatment induced +-ATPaseH up-accumulation, which was to maintain ion balance or promote SC response in stigmas. In conclusion, salt stress-related proteins might be helpful for salt-induced self-compatibility.A high salt concentration could depolymerize the polymerized MTs ,41, and sicaceae , and annB. napus ; overexpnd NA/UP , and qPCnd NA/UP E, which produced . The incn stress ; ion trasduction . After SB. napus. There were 389, 189, and 307 DAPs in PC/UP, PI/UP, and NA/UP, respectively  protein, consisting of 76 amino acids, is highly conserved in all eukaryotic species. It covalently marks target proteins for degradation by the 26S proteasome in different organisms, which has been well discussed in mediating different cellular processes [ARC1, as an E3 ligase, plays an important role in the SI of Brassica by targeting and transferring compatibility factors to the proteasome. In B. napus, 30 min after incompatible pollination, increased ubiquitination occurred, and the proteasome’s proteolytic pathway was essential for the SI response [Ubiquitination involves an intricate cascade process that is controlled by three enzyme families, including E1 (Ub-activating enzyme), E2 (Ub-conjugating enzyme), and E3 (Ub ligase), which are large extended gene families. There are 16 genes encoding E1s, 45 genes encoding E2s, and more than 1400 genes encoding E3s in rocesses ,49,50. Aresponse ,23. Howeresponse . Six DAPARC1 linear ubiquitin–proteasome pathway, and edible salt solution-overcoming SI might affect these pathways. The two pathways of endocytosis and phagosomes, which are essential processes in cells to control the dynamics and turnover of plasma membrane proteins, were detected in the current study (FLS2) and brassinosteroid-insensitive 1 (BRI1) were well studied and associated with endocytosis in Arabidopsis thaliana. Inactive FLS2 localizes to the PM, whereas it is rapidly internalized and degraded upon binding to the flg22 protein, which is a 22-amino acid peptide containing a conserved epitope from bacterial flagellin [BRI1 localizes both to the PM and cytoplasmic compartments, and its localization and trafficking are independent of its ligand [SCR–SRK interaction took place at the plasma membrane, and that SRK moved into endosomes where the SRK negative regulator THL1 was enriched, which was different from the BRI1 signaling pathway, and was more similar to FLS2 [ATPase HSC70 (heat shock cognate 70) to initiate the disassembly of the clathrin coat fusing with endosomes [Hsc70-1 was identified as significantly down-accumulated in PC/UP and PI/UP, and conversely, Hsc70-G8 was upregulated in NA/UP; these results suggested that salt-induced self-compatibility was likely to inhibit or degrade SRK by promoting fusion with endosomes , which are a family of subtilisin-like serine proteases [SBT was detected and up-accumulated in PI/UP , another important plant cell wall component, are a diverse family of hydroxyproline-rich glycoproteins (HRGPs), and are characterized by the repeated occurrence of serine (Ser) followed by several consecutive prolines (Pro) [Many studies have suggested that the recognition response between pollen and stigma is similar to that of plant–pathogen interactions ,30,31, bractions . In our ractions . Extensies (Pro) ,66. The es (Pro) ,68. In oLRR) family protein, and peptidoglycan-binding LysM domain-containing protein. Ca2+ plays important signaling roles in the response to biotic and abiotic stress, including plant–pathogen interaction and salt stress. The Ca2+ signal is transduced through Ca2+ sensors, which include calmodulins (CaMs), CaM-like proteins (CMLs), Ca2+-dependent protein kinase (CDPKs), and calcineurin B-like proteins (CBLs). Ca2+ as a signaling molecule also participates in pollen–stigma interactions [2+, were significantly up-accumulated in PC/UP, PI/UP and NA/UP, indicating that Ca2+ plays an important role in pollen–stigma interactions and salt-induced self-compatibility. Many resistance (R) proteins have a conserved LRR domain, which functions in some developmental and immune signaling pathways, and participates in receptor/co-receptor complex formation [LRR proteins, two were down-accumulated in NA/UP and PC/UP, and one was down-accumulated in PI/UP  as the ratio of the number of seeds to the number of flowers was calculated. At least twenty siliques were sampled for every treatment [Stigmas of fresh flowers of “Westar”, “W-3”, and “S-1300” were treated with edible salt (NaCl ≥ 98.5%) solution. The five concentrations of salt solution were 650 mM, 850 mM, 1050 mM, 1250 mM, and 1450 mM. The stigmas were smeared with salt solution by Chinese writing brushes, and then were artificially pollinated. Pollination with “Westar” pollen grains was taken as one self-compatible control. For “W-3” and “S-1300”, self-incompatible controls were self-pollination and self-pollination after water treatment. Since “Westar” is self-compatible, resulting from the non-functional pollen self-incompatibility determinant gene reatment . Pure NaB. napus pistils were fixed in 3:1 ethanol, glacial acetic acid for 2 h, and softened in 1 N NaOH at 60 °C for 1.5 h. Following softening, the pistils were subjected to washing three times with distilled water, and then stained with basic aniline blue (0.1% aniline blue in 0.1 M K3PO4) for 2.5 h. The stained pistils were gently put onto a microscopic slide glass and gently squashed by the cover glass. Samples were observed under a fluorescence microscope . At least six pistils were observed for every pollination treatment.Aniline blue assay was performed as previously described  with min“Westar” flowers were emasculated one day before anthesis. Stigmas were unpollinated (UP), pollinated with “Westar” pollen (PC), pollinated with “W-3” pollen (PI), and pollinated with “W-3” pollen after edible salt solution (1050 mM) treatment (NA). Thirty minutes after pollination, stigmas of each sample were harvested by cutting the pistil just below the base of the stigma, immediately frozen in liquid nitrogen, and stored at −80 °C. Each sample had three biological replicates, and the stigmas of every biological replicate came from at least three individual plants.g for 15 min. The supernatant was mixed well with a 5× volume of chilled acetone containing 10% (v/v) TCA, and incubated at −20 °C overnight. After centrifugation at 4 °C, 30,000g, the supernatant was discarded. The precipitate was washed three times with chilled acetone. The pellet was air-dried and dissolved in lysis buffer . The suspension was sonicated at 200 W for 15 min and centrifuged at 4 °C, 30,000g for 15 min. The supernatant was transferred to a new centrifuge tube, and 10 mM DTT was added , followed by incubation at 56 °C for 1 h to reduce disulfide bonds of protein species. After that, 55 mM IAM  was added  to inhibit cysteines, and incubated in a darkroom for 1 h. To precipitate protein species, a 5× volume of chilled acetone was mixed well with the supernatant, and incubated for 2 h at −20 °C. The mixture was centrifuged at 4 °C for 3000× g, the supernatant was discarded, and the pellet was dried in air for 5 min. The pellet was dissolved in 500 µL, 0.5 M TEAB  and sonicated for 15 min at 200 W. Finally, centrifuged the mixture at 4 °C, 3000× g for 15 min. The supernatant was transferred to a new tube, and the protein concentration was quantified with a Bio-Rad protein assay kit  based on the Bradford method using BSA as a standard. The proteins in the supernatant were kept at −80 °C until further analysis.Proteomics analysis was carried out at BGI . Two independent biological replicates were performed. For each biological replicate, approximately 1500 stigmas were ground into powder in liquid nitrogen, extracted with lysis buffer  containing 1 mM PMSF and 2 mM EDTA . After 5 min, 10 mM DTT  was added to the samples. The suspension was sonicated at 200 W for 15 min, and then centrifuged at 4 °C, 30,0002PO4 in 25% ACN, pH 2.7) was added, and loaded on a 4.6 × 250 mm Ultremex SCX column containing 5 µm particles . The peptide mixtures were eluted in a gradient of buffer A for 10 min, 5–60% buffer B  for 27 min, 60–100% buffer B for 1 min, at a flow rate of 1 mL/min. Before equilibrating with buffer A for 10 min prior to the next injection, the system was maintained at 100% buffer B for 1 min. By measuring the absorbance at 214 nm to monitor the elution results, and fractions were collected every 1 min. Finally, the eluted peptides pooled into 20 fractions, then the fractions were desalted with a Strata X C18 column  and vacuum-dried.Total protein (100 µg) was collected from each sample solution, and the protein was then digested with Trypsin Gold  at a 30:1 ratio of protein/trypsin at 37 °C for 16 h. After trypsin digestion, peptides were dried by vacuum centrifugation. The peptides were reconstituted in 0.5 M TEAB, and processed according to the manufacturer’s protocol for the 8-plex iTRAQ reagent (Applied Biosystems). Briefly, one unit of iTRAQ reagent was thawed and reconstituted in 24 µL isopropanol. Samples were labeled with the iTRAQ tags, and the tag for each sample is shown in g; the final average concentration of peptide was approximately 0.5 µg/µL. Supernatant (10 µL) was loaded on an LC-20AD nano HPLC  by autosampler onto a 2 cm C18 trap column. The fractions were eluted on a 10 cm analytical C18 column (inner diameter 75 µm) that was packed in-house. The peptides were loaded at 8µL/min for 4 min, the 35 min gradient was run at 300 nL/min from 2% to 35% B , then a 5 min linear gradient to 60%, a 2 min linear gradient to 80%, with maintenance at 80% B for 4 min, and finally, a return to 5% in 1 min. Data were acquired using a Triple TOF 5600 System fitted with a Nanospray III source  and a pulled quartz tip as the emitter . Data acquisition was operated with an ion spray voltage of 2.5 kV, curtain gas of 30 PSI, nebulizer gas of 15 PSI, and an interface heater temperature of 150 °C. The MS was performed with an resolving power (RP) greater than or equal to 30,000 FWHM for TOF MS scans. For information dependent acquisition (IDA), survey scan acquisitions were operated for 250 ms with a 2+ to 5+ charge-state, and if exceeding a threshold of 120 counts per second (counts/s), as many as 30 product ion scans were collected. The total cycle time was set to 3.3 s. The Q2 transmission window was 100 Da for 100%. Each scan summed 4 time bins at a pulser frequency value of 11 kHz by monitoring of the 40 GHz multichannel TDC detector with a four-anode channel detection ion. All precursor ions for collision-induced dissociation applied a sweeping collision energy setting of 35 ± 5 eV coupled with iTRAQ adjust rolling collision energy. Dynamic exclusion was set as 1/2 of peak width (15 s), and then the precursor refreshed from the exclusion list. Raw data files were acquired from Orbitrap, which were converted into MGF files with Proteome Discoverer 1.2 ,  and the files were searched. Protein identification was operated using a Mascot search engine  against a database containing Brassica napus sequences (http://www.genoscope.cns.fr/brassicanapus/). For protein identification, a mass tolerance of 0.05 Da was the allowance for intact peptide masses, and ±0.1 Da for fragmented ions, permitting only one missed cleavage in the trypsin digests. Set Gln->pyro-Glu (N-term Q), Oxidation (M), and Deamidated (NQ) were set as the potential variable modifications, and Carbamidomethyl (C), iTRAQ 8plex (N-term), and iTRAQ 8plex (K) were set as fixed modifications. The charge states of peptides were set to +2 and +3. In particularly, an automatic decoy database search was performed in Mascot, where the decoy checkbox was chosen with a random sequence of database generated and tested for raw spectra, as well as the real database. To reduce the probability of false peptide identification, only these peptides were counted as identified, which were greater than “identity” with the 95% confidence interval by a Mascot probability analysis. Each confident protein identification used at least one unique peptide. A protein containing at least two unique spectra was required for protein quantitation. Then, protein ratios were weighted and normalized by the median ratio in Mascot. Protein ratios with p-values < 0.05, and fold changes of >1.3 or <0.77 were considered significant. Functional classifications of the protein species were conducted with the Blast2GO program against the non-redundant protein database . All identified protein species were classified and grouped by using KEGG database (http://www.genome.jp/kegg/) and COG database (http://www.ncbi.nlm.nih.gov/COG/).Each fraction was resuspended in buffer A  and centrifuged for 10 min at 20,000http://www.ambion.com), and first-strand cDNA synthesis and qPCR were carried out as previously described [B. napus cDNA sequences . After RNA quality and quantity measurement, five micrograms of RNA was DNase-treated using a DNA-free kit (Ambion, escribed . Gene-spequences . Actin (B. napus. A large number of differentially accumulated proteins were identified, including 307 proteins in UP/NA. Further bioinformatics analysis indicated that the DAPs were involved in ribosome, endocytosis, proteasome, plant–pathogen interaction, phagosome, and ubiquitin-mediated proteolysis pathway. Upregulated expression of SC-related proteins may be required for salt-solution destruction of SI, and may help plants facilitate SC responses. Knockout or overexpression of the DAPs in NA/UP would identify genes responsible for salt-induced self-compatibility. In summary, our results showed comprehensive extractome coverage of salt-induced self-compatibility, and the potential candidate proteins may provide a starting point in the elucidation of the molecular mechanisms and provide potential genetic resources for the breeding of self-incompatibility lines.Our study showed that iTRAQ was a powerful tool for quantitative proteomics analysis of salt-induced self-compatibility in


Zika virus (ZIKV) causes congenital neurologic birth defects, notably microcephaly, and has been associated with other serious complications in adults. The virus has been detected in human breast milk and possible transmissions via breastfeeding have been reported. Breast milk is rich in nutrients and bio-active substances that might directly affect viral infectivity. Thus, here, we analyzed the effect of human breast milk on ZIKV infection. We observed that fresh human breast milk had no effect on ZIKV, but found that upon storage, milk effectively suppressed infection. The antiviral activity is present in the fat-containing cream fraction of milk and results in the destruction of the structural integrity of viral particles, thereby abrogating infectivity. The release of the factor is time dependent but varies with donors and incubation temperatures. The viral titer of milk that was spiked with ZIKV decreased considerably upon storage at 37 °C for 8 h, was lost entirely after 2 days of 4 °C storage, but was not affected at −20 °C. This suggests that cold storage of milk inactivates ZIKV and that the antiviral factor in milk may also be generated upon breastfeeding and limit this transmission route of ZIKV.  Zika virus (ZIKV) is a (re-)emerging virus that was originally identified in 1947 in Uganda , the SouAedes species mosquito [Ae. aegypti and Ae. albopictus mosquitoes are endemic [ZIKV is mainly transmitted to people through the bite of an infected mosquito . This ve endemic . ZIKV tr endemic ,15,16,17 endemic , or from endemic , intrapa endemic  or possi endemic ,23,24,25 endemic ,22,23,25 endemic ,24,25. A endemic . Neverth endemic  includin endemic ,29 and i endemic ,29. Howe endemic ,20.Breast milk is a body fluid that nurtures and protects the infant. It is rich in nutrients and vitamins and provides the child with carbohydrates, proteins, fat, minerals, hormones, growth factors and antibodies ,31. BreaCercopithecus aethiops derived epithelial kidney) cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 2.5% heat-inactivated fetal calf serum (FCS), 2 mM l-glutamine, 100 units/mL penicillin, 100 μg/mL streptomycin, 1 mM sodium pyruvate, and non-essential amino acids . For experiments in the presence of breast milk, the medium was supplemented with 100 µg/mL gentamicin. Cells were grown at 37 °C in a 5% CO2 humidified incubator.Vero E6  titration and the genome copy number of the stocks was assessed by RT-qPCR.The African ZIKV strain MR766 was isolated in 1947 from a sentinel rhesus macaque  or from Human breast milk was donated by healthy volunteering nursing mothers after informed consent had been signed. None of the mothers reported stays in ZIKV endemic regions or previous infections with flaviviruses. Samples were collected into sterile 50 mL Falcon tubes immediately before breastfeeding and either used for analysis within 30–60 min after donation or after long-term (>6 months) storage at −20 °C. All procedures were approved by the local ethics committee of Ulm University . ® Luminescent Cell Viability Assay . It was performed according to the manufacturer’s instructions under conditions corresponding to the respective infection assays. Briefly, the medium was removed from the culture after 2 days of incubation and 50 µL phosphate-buffered saline (PBS) and 50 µL substrate reagent were added. After 10 min, the luminescence of the samples was measured in an Orion II Microplate Luminometer . Untreated controls were set to 100% viability.The effect of human breast milk on the metabolic activity of the cells was analyzed using the CellTiter-Glo50), ZIKV stocks were serially diluted 10-fold and used to inoculate Vero E6 cells. To this end, 6000 Vero E6 cells were seeded per well in 96 flat-bottom well plates in 100 µL medium and incubated over night before 80 µL fresh medium was added. Next, 20 µL titrated ZIKV of each dilution was used for inoculation, resulting in final ZIKV dilutions of 1:101 to 1:109 on the cells in triplicates. Cells were then incubated for at least 6 days and monitored for cytopathic effect. TCID50/mL was calculated according to Reed and Muench.To determine the tissue culture infectious dose 50 (TCIDv/v) FCS and 0.3% (v/v) Tween 20) at 37 °C. After 1 h, the cells were washed three times with washing buffer (0.3% (v/v) Tween 20 in PBS) before a secondary anti-mouse antibody conjugated with horseradish peroxidase (HRP)  was added and incubated for 1 h at 37 °C. Following four times of washing, the TMB peroxidase substrate  was added and the reaction was stopped with 0.5 M H2SO4 after 5 min of light-protected room temperature incubation. The optical density (OD) was recorded at 450 nm and baseline corrected for 650 nm using the VMax Kinetic ELISA microplate reader . For infection, 6000 Vero E6 cells were seeded in flat-bottom 96-well plates in 100 µL medium, incubated overnight, and then topped to 180 µL medium. ZIKV stocks were diluted to the desired multiplicity of infection (MOI). In the “virion treatment” setting, virus was then mixed (1:1) with serially titrated breast milk samples and incubated for 10 min at room temperature. Then, cells were inoculated with 20 µL of the mixture in triplicates (resulting in a 10-fold lower concentration of the milk samples) and incubated for 2 h before the medium was replaced by fresh medium. Alternatively, cells were first incubated with the breast milk samples for 10 min, followed by ZIKV inoculation (“cell treatment”). Two days post infection, the flavivirus E protein content in the cells was quantified by cell-based immunodetection assay as described . To thisv/v) FCS in PBS) and permeabilized for 5 min with permeabilization solution . Cells were washed with 0.1% (v/v) Tween 20 in PBS and incubated with mouse anti-flavivirus protein E antibody 4G2 (in PBS containing 0.1% (v/v) Tween 20) for 30 min at room temperature. After a next washing step, cells were incubated with a secondary anti-mouse antibody conjugated with Alexa Fluor 488  for 30 min at room temperature protected from light. After further washing steps, cells were resuspended in FACS buffer and analyzed in a BD FACS Canto™ II Cell Analyzer and evaluated with BD FACS Diva™ and FlowJo 7 software .For infection, 100,000 Vero E6 cells were seeded in 24 well plates in 500 µL medium, incubated overnight, and then replaced by 180 µL fresh medium. Cells were then inoculated with 20 µL ZIKV that was treated for 10 min with thawed human milk. After 2 h, the inoculum was removed, cells were washed with PBS and fresh medium was added. Three days post infection, cells were detached (0.05% trypsin in PBS) and flow cytometric analysis was performed to determine infection rates. Specifically, cells were washed with PBS and stained with fixable viability stain 450  for 15 min at room temperature, followed by a washing step. Next, cells were fixed with 4% PFA for 10 min. Cells were then washed with fluorescence-activated cell sorting (FACS) buffer (1% (g at 4 °C to skim the fat (cream) from milk. Next, cream was resuspended in 10 mL PBS and the skim milk was ultracentrifuged at 100,000× g for 90 min at 4 °C to separate caseins from serum proteins. The casein-containing pellet was resuspended in 10 mL PBS and the supernatant (whey) was collected.The fractionation of human breast milk was performed as previously described . Briefly10 genomic RNA copies/mL was incubated with 90% PBS, 0.5% SDS, 1% Triton X-100, or 90% thawed breast milk for 1 h at 37 °C. Next, the free (and not virion protected) viral RNA of one aliquot of each sample was degraded by incubation with 10 U RNaseA  for 1 h at 37 °C. As control , one aliquot was in parallel incubated with PBS. After treatment, the samples were analyzed for remaining RNA copy numbers. Quantitative real-time PCR was performed using qScript™ XLT One-Step-RT-qPCR ToughMix® . The final 20 µL RT-qPCR reaction mixture contained 5 µL RNA template, 10 pmol forward primer ZIKV-RKI-F , 10 pmol reverse primer ZIKV-RKI-R  and 3 pmol probe ZIKV-RKI-P . The amplification was done under the following conditions, 10 min at 50 °C for RT reaction, 10 min at 95 °C for polymerase activation, followed by 40 cycles for 15 s at 95 °C, 60 s at 55 °C and 10 s at 72 °C.To determine the amount of intact ZIKV virions and their destruction by breast milk or detergents, the total and virion-associated viral RNA was determined by qPCR, as previously described for HCV . To this50) by four-parametric nonlinear regression was performed using GraphPad Prism version 8.1.0 for Windows, GraphPad Software, San Diego, California USA, www.graphpad.com.The determination of the inhibitory concentration 50 , showing that the antiviral activity in milk is directed against the virus. We next analyzed whether milk may directly disrupt virion integrity, applying a protocol established for HCV [8 to 109) were detected in all control samples, demonstrating that detergents or milk components do not interfere with RT-qPCR-based detection of viral genomes. RNase treatment, however, resulted in an almost entire loss of viral RNA when virions were exposed to breast milk  and then added to cells, resulting in a 10-fold dilution of the inoculum, and final cell culture concentrations of up to 2% of milk. Simultaneously, Vero E6 cells were first incubated with breast milk at concentrations of up to 2% for 10 min, before ZIKV was added (“cell treatment”). Thus, in both treatments the final milk concentrations in cell culture are the same, however, due to virion pre-exposure with 10-fold higher concentrations different outcomes could arise if the antiviral factors target the virion. After 2 h, the supernatants were exchanged with fresh medium, and viral infection was quantified 2 days later. Under both conditions, milk inhibited ZIKV infection a. Howeve for HCV . In brieast milk b. Similaast milk c. Howeveast milk c. These v/v) (50 of 0.85 ± 0.15% during virion treatment) that was comparable to whole milk (IC50 of 0.41 ± 0.03%)  whereas the casein fraction had no effect  a. We tes± 0.03%) b. Skim mo effect b.Our finding that milk has potent anti-ZIKV activity  was obta50 analysis showing that ZIKV that was incubated in PBS remained fully infectious, even after incubation for 8 days  of ZIKV-contaminated milk effectively destroys viral infectivity . AlthougRecent reviews investigated transmission from ZIKV-infected mothers to their children and did not find evidence for efficient ZIKV transmission via breastfeeding, despite the presence of virus in breast milk ,24,25 an


This study aimed to develop a new oral paclitaxel formulation (DHP23002) and to evaluate its absorption and antitumor effects in a pancreatic tumor mouse model.® injections at 10 mg/kg once per week. After 3 weeks of administration, tumor growth in mice belonging to each group was further monitored for 4 weeks after discontinuing medication. Moreover, to examine paclitaxel (DHP23002) accumulation in the tumor tissue, the amount of paclitaxel in tumor/blood was quantified using liquid chromatography with quadruple-TOF mass spectrometry.To investigate the oral absorption of DHP23002, a newly developed lipid-based orally active paclitaxel formulation, a pharmacokinetic study of DHP23002, was conducted in mice (62.5 and 125 mg/kg). Moreover, to evaluate the antitumor effect of DHP23002 in pancreatic cancer treatment, the drug was administered to female athymic nude mice at 0 (vehicle), 25, 62.5, and 125 mg/kg on alternate days; the efficacy of the agent was compared with the efficacy of intravenous Taxol® showed a negligible absorption, whereas DHP23002 showed a high absorption rate dependent on dosage, with a bioavailability of approximately 40% at a dose of 62.5 mg/kg. In efficacy-related studies, DHP23002 administration at a dose of 25, 62.5, or 125 mg/kg on alternate days for 3 weeks showed a superior tumor inhibitory effect of 80%, 92%, and 97% in a xenograft mouse model, respectively, after 7 weeks. Paclitaxel accumulation in tumors persisted for >24 h in mice, when orally administered once at doses of 25, 62.5, and 125 mg/kg DHP23002.In the mouse pharmacokinetic study, oral TaxolOral chemotherapy with DHP23002 showed excellent absorption in animals owing to a strong antitumor activity in a pancreatic cancer mouse model. This demonstrates that paclitaxel is largely distributed and persists for a prolonged period at the tumor site owing to oral DHP23002 administration. Globally, pancreatic cancer has been reported as the twelfth most common cancer and is the seventh leading cause of cancer-associated mortality. More than 330,000 people die from pancreatic cancer, and almost the same number of new cases are reported annually –3. BecauOver the past few decades, small chemotherapeutic agents such as 5-FU or gemcitabine and drug combinations have been tested for treating progressive pancreatic cancer . AlthougCre-paclitaxel; Taxol®) [re-paclitaxel formulation. Moreover, The Cremophor EL solvent also altered the pharmacology of paclitaxel, thereby affecting its toxicity profile and anticancer efficacy [®, and gemcitabine, revealed an increased survival rate of patients compared with the survival rate of patients who received FOLFIRINOX [Because taxane can enhance microtubule assembly and inhibit tubulin depolymerization and consequently block cell proliferation, use of docetaxel or paclitaxel as a single agent or in combination with other chemotherapeutics has been proposed for pancreatic cancer. Poor solubility problem of taxane could be resolved by developing a polyoxyethylated castor oil solvent (Cremophor EL) formulation, which can be intravenously administered ( Taxol®) . Howeverefficacy –10. ThusLFIRINOX –12. AddiBecause the oral paclitaxel formulation can be conveniently administered and provides timing and location flexibility for patients with pancreatic cancer, the development of an oral formulation would prevent unnecessary hospitalization and hypersensitivity reactions. Moreover, as opposed to intermittent iv exposure, oral administration of paclitaxel reportedly allows continuous exposure at low and effective concentrations during the treatment period and thereby facilitates a more flexible drug administration procedure. It has been reported that continuous exposure to low paclitaxel concentrations exerts a superior anti-tumor effect by inhibiting tumor-associated angiogenesis .®in vivo using a xenograft mouse model of chemotherapy-sensitive BxPC-3 pancreatic cancer.Recently, a clinically usable, mucoadhesive, lipid-based oral paclitaxel formulation (DHP107) has been developed –14. The All animals including ICR and BALB/c nude mice were maintained and used in accordance with the Guidelines for the Care and Use of Laboratory Animals of the Institute of Laboratory Animal Center, Daegu-Gyeongbuk Medical Innovation Foundation. The animal studies were conducted after approval by the Institutional Reviewer Board (IRB) on the Ethics of Animal Experiments of the Daegu-Gyeongbuk Medical Innovation Foundation . All efforts were made to minimize and euthanize the pain of mice according to the end points of an IACUC-approved protocol.® was purchased from Bristol-Myers Squibb , and paclitaxel powder for preparing DHP23002 was purchased from Samyang Bio Pharmaceutical® . Oral paclitaxel (DHP23002) and vehicle were prepared by Dae Hwa Pharmaceutical Co. Ltd. , and their preparation method has been described in the patent [Taxole patent . All sol® (Cre-paclitaxel) was diluted six times with saline solution and administered via a bolus tail-vein injection at a dose of 12.5 mg/kg as a positive control. Taxol® (Cre-paclitaxel) for oral administration was prepared by dissolving paclitaxel at a concentration of 36 mg/mL in an equivolume mixture of Cremophor EL & ethanol and diluted with saline solution; this mixture was orally administered at a dose of 62.5 mg/kg and used as another control. To determine paclitaxel concentration in plasma, a 200-μL plasma sample was mixed with 800 μL acetonitrile after centrifuging at 10,000×g for 10 min to recover the supernatant. The reconstituted solutions were centrifuged for 10 min at 10,000×g at 4°C to eliminate the proteins in plasma samples. The supernatant was transferred to a total recovery vial for analysis. LC-MS/MS analysis was performed using Xevo G2-XS  coupled to a Q-TOF mass spectrometer (Waters Corporation) equipped with an electrospray ionization source. The two mobile phase gradient composition was solvent A (water with 0.1% formic acid) and solvent B (acetonitrile with 0.1% formic acid). A 3-min gradient was developed from 50% to 10% A at a flow rate of 0.3 mL/min. The analytical column was a Waters BEH C18 UPLC Column . Ion detection was performed in MS mode, and the parent ions were monitored at 854.3 m/z. Data acquisition was performed using TargetLinx software (Waters Corporation). The mean and standard error of the mean at each timepoint were calculated, and PK parameters were determined from the mean concentration data via a non-compartmental analysis conducted using the WinNonlin 6.4 program .Six female ICR mice were administered with an oral formulation of paclitaxel (DHP23002) at doses of 62.5, and 125 mg/kg. Taxol® Brite cell line), was purchased from Perkin Elmer. The BxPC3 cell line was maintained in RPMI-1640 supplemented with 10% fetal bovine serum  and 1% PS (Penicillin/Streptomycin). Six-week-old female, athymic nude mice (NCr-nu) were adapted for 1 week. Before cell line injection, pancreatic cancer cells were washed twice with phosphate-buffered saline (PBS), detached using 0.5% Trypsin-EDTA, harvested by centrifugation, and resuspended in Hank’s balanced salt solution . Cell viabilities were determined by trypan blue exclusion method. For injection into nude mice, mice were anesthetized by continuous inhalation of 3% isoflurane gas for 5–10 min using an anesthesia system . A suspension of 2.0×106 cells/100 μL was subcutaneously injected on the right lower back of the mouse. The skin around the tumor was swabbed using a combination of betadine and alcohol for minimizing the risk of bacterial infection. Tumor cell-implanted animals were placed in a separate individual cage and then returned to IVIS cages  when they regained consciousness after anesthesia.A luciferase-expressing human pancreatic adenocarcinoma cell line, BxPC3-Red-Fluc (Bioware® (BMS) and DHP23002 . To evaluate the efficacy of DHP23002, eight female athymic nude mice received 0, 25, 62.5, and 125 mg/kg DHP23002 orally on alternate days for 3 weeks. Oral administration of DHP23002 vehicle (DHP system) was used as negative control; Taxol® intravenous injections at 10 mg/kg were administered weekly as positive control. Mice (8 per treatment group) were randomly assigned to receive one of five treatments: (1) 5 mL/kg of vehicle administered once every other day (control group); (2) 10 mL/kg of Taxol® administered intravenously once weekly; (3) 1 mL/kg of DHP23002 administered once on alternate days (4) 2.5 mL/kg of DHP23002 administered once on alternate days (5) 5 mL/kg of DHP23002 administered once on alternate days. Treatments were continued for 3 weeks, and the tumor size of each group was observed for 48 days . Tumor volume was measured every week after inoculation and calculated as follows: tumor volume = short length×short length×long length×0.5. After observation, all mice were imaged using IVIS-spectrum-CT  after injection of luciferin for luciferase activation  and oral administration of DHP23002  and to monitor the amount of paclitaxel in blood after administration. Taxol® or DHP23002 was administered at the same dose as the abovementioned tumor growth inhibition experiment. At 6, 24, and 48 h after the administration of each formulation, mice were euthanized by CO2 asphyxiation and tumor homogenates and blood plasma were collected for further analysis. Tumor tissues were homogenized in four volumes of PBS and centrifuged for 10 min at 10,000×g. Two hundred microliters of supernatant fraction was mixed with 800 μL acetonitrile and filtered through a 0.2-μm PTFE syringe filter (Whatman). Acetonitrile extracts of tumor tissue and blood were analyzed via LC-MS/MS analysis . The composition of the two mobile phase gradients was solvent A (water with 0.1% formic acid) and solvent B (acetonitrile with 0.1% formic acid). Ion detection was performed in MS mode, and parent ions were monitored at m/z 854.3. Data acquisition was performed using TargetLinx software (Waters Corporation).To evaluate the amount of paclitaxel in tumor or blood after the first administration of paclitaxel formulation via i.v. or oral routes, tumor-bearing mice were prepared. BxPC-3 cells at a concentration of 2.0×10®-, or DHP23002-treated cells. Briefly, 2×104 cells were grown on a chamber slide™ . After the cells attained 70%–80% confluence, fresh RPMI 1640  medium containing 0.5% fetal bovine serum and antibiotics was added to each dish and subsequently treated with vehicle, Taxol®, and DHP23002 for 4 h. After a 4-h incubation period, cells were washed with PBS and fixed with 4% paraformaldehyde solution for 30 min at 4°C. After fixing cells, the chamber slide was washed again with PBS and incubated for 30 min with 3% BSA containing 0.2% triton X-100 (blocking solution). After washing with PBS, cells were further incubated for 1 h at 25°C with beta-tubulin antibody  in 3% BSA containing 0.2% triton X-100. Unbound antibodies were washed with PBS three times; the bound antibodies were stained for 0.5 h at 25°C with an anti-rabbit secondary antibody conjugated to fluorescein isothiocyanate (FITC)  by incubating them together in 3% BSA containing 0.2% triton X-100, which was prepared in PBS. After washing, a drop of 4ʹ,6-diamidino-2-phenylindole (300 nM in mounting solution) was added. Slides were viewed under K-1 imaging upright microscope  system using different filters.Immunohistochemical analysis was performed to examine the localization of β-tubulin proteins in untreated, Taxol6/mL) were seeded and incubated for 48 h. Taxol®-treated or DHP23002-treated BxPC-3 cells were collected after 24-h treatment. Apoptosis was detected using an FITC-Annexin V/propidium iodide (PI) kit , according to the manufacturer’s instructions. In brief, the cells were washed with cold PBS and subsequently stained with a combination of 1.25 μL FITC-Annexin V and 10 μL PI for each 100-μL cell suspension and incubated at room temperature for 15 min. Apoptotic cells were immediately analyzed using a flow cytometer . The apoptosis of samples was given as the sum of early and late apoptosis.Apoptosis was assessed by staining with annexin-V and propidium iodide (PI), which is a well-known method for apoptosis detection. BxPC-3 cells . Paired T-test was used to determine the statistical significance of the results. A P-value, 0.05 was considered statistically significant and at least 5~8 mice per group were used for each ®) resulted in a very low bioavailability of approximately 0%. However, the mean AUC values of DHP23002, which was developed for enhancing the oral absorption of paclitaxel, at doses of 62.5 mg/kg and 125 mg/kg was approximately 13.63±2.7 μg∙h/mL and 22.26±5.68 μg∙h/mL, respectively. Moreover, Cmax of DHP23002 at 62.5 mg/kg and 125 mg/kg was approximately 6.9±0.98 μg/mL and 9.2±1.23 μg/mL, respectively.Plasma concentration–time profiles for the oral paclitaxel, DHP 23002, were obtained using different paclitaxel doses to evaluate whether it is an orally active drug. DHP23002 formulation consisted of three safe lipid ingredients, one surfactant, and paclitaxel at a concentration of 25 mg/mL. Its solubility was comparable to any commercially available paclitaxel formulation. As shown in 3, p<0.01), 62.5 mg/kg , and 125 mg/kg  not only decelerated tumor growth but also reduced tumor size as opposed to the treatment of control mice with vehicle (213±38.6 mm3). Moreover, the average tumor volume in mice 7 weeks after i.v. administration of Taxol® was 113.1±50.4 mm3.To monitor the effect of the oral paclitaxel DHP23002 on tumor growth, we explored tumor retardation effect on tumor-bearing mice. As shown in ® and oral DHP23002 treatment; it is interesting to note that administration of oral DHP23002 for the first 3 weeks affected tumor growth for 7 weeks. As shown in ® group, the tumors size increased again at 7 weeks. Further, the minimum dose of DHP23002 used in this study to inhibit tumor growth was 25 mg/kg; this led to a significant inhibition in tumor growth. After 3 weeks of drug administration or 4 weeks after drug discontinuation, the tumor size of group treated with 25 mg/kg DHP23002 decreased by 52.3% and 81% of tumor size, respectively, when compared with the vehicle control.Tumor reduction was observed at 7 days in case of both Taxol® revealed that at 6 h after Taxol® intravenous injection, paclitaxel concentration in blood was low at 46.6±13.8 ng/mL, followed by a general elimination pattern until 48 h. Contrastingly, PK studies of oral DHP23002 revealed blood concentrations of 98.5±10.7, 170.8±9.1, and 577.0±182.8 ng/mL for doses of 25, 62.5, and 125 mg/kg, respectively, at 6 h after administration. However, at 24 and 48 h after administration, the concentration of DHP23002 in blood for each dose was reduced to nearly the same concentration of intravenously administered Taxol® (10 mg/kg) in blood  as opposed to treatment with vehicle in the control group Click here for additional data file.S2 Fig(DOCX)Click here for additional data file.


PADI4, whose G allele is associated with reduced PAD4 in neutrophils, correlates with NETs, anti-histone antibodies, and rheumatoid arthritis susceptibility in North Americans. Control and rheumatoid arthritis subjects, divided into anti-cyclic citrullinated peptide (CCP) antibody positive and negative groups, were genotyped at rs2240335. In homozygotes, in vitro NETosis was quantified in immunofluorescent images and circulating NET and anti-histone antibody levels by enzyme linked immunosorbent assay (ELISA). Results were compared by t-test and correlation of rheumatoid arthritis diagnosis with rs2240335 by Armitage trend test. NET levels did not significantly correlate with genotype. G allele homozygotes in the CCP− rheumatoid arthritis group had reduced anti-native and anti-citrullinated histone antibodies. However, the G allele conferred increased risk for rheumatoid arthritis diagnosis, suggesting a complex role for PAD4 in human rheumatoid arthritis.Autoantibodies against citrullinated proteins are a hallmark of rheumatoid arthritis, a destructive inflammatory arthritis. Peptidylarginine deiminase 4 (PAD4) has been hypothesized to contribute to rheumatoid arthritis by citrullinating histones to induce neutrophil extracellular traps (NETs), which display citrullinated proteins that are targeted by autoantibodies to drive inflammation and arthritis. Consistent with this theory, PAD4-deficient mice have reduced NETs, autoantibodies, and arthritis. However, PAD4′s role in human rheumatoid arthritis is less clear. Here, we determine if single nucleotide polymorphism rs2240335 in  Rheumatoid arthritis is an autoimmune destructive arthritis with a lifetime risk of 2.6% . Most rhCitrullination, the post-translational modification of arginines to citrullines, is catalyzed by the peptidylarginine deiminases (PADs). In activated neutrophils, PAD4 citrullinates histones enhancing chromatin unraveling and NET formation ,8, and PPADI4, the gene encoding PAD4, that are associated with rheumatoid arthritis [Understanding the role of a protein in human disease is challenging. One method for approaching this problem is to seek genetic variants associated with disease. Genome-wide association studies have identified several single nucleotide polymorphisms (SNPs) in rthritis ,20,21. Srthritis . Interesrthritis ,20, the rthritis ,22,23. TThe objective of this study is to determine if rs2240335 is associated with NETosis, anti-histone antibodies, and rheumatoid arthritis risk in a North American cohort to clarify the role of PAD4 in human rheumatoid arthritis. We found that the G allele of rs2240335 does not correlate with NETs, but does correlate with reduced anti-histone antibodies and increased risk of rheumatoid arthritis, suggesting that PAD4 has a complex role in human rheumatoid arthritis.Given the association of the G allele of rs2240335 with reduced PAD4 in human neutrophils , we eval− rheumatoid arthritis, and CCP+ rheumatoid arthritis subjects, in order to choose which group would be ideal for our experiments. We found that antibodies against histone H1 were not significantly different between control and rheumatoid arthritis subjects , where the G allele conferred increased risk for rheumatoid arthritis diagnosis compared to the T allele. Comparing the GG and TT homozygous genotypes, the odds ratio was calculated to be 2.14 with 95% CI .Finally, as noted above, the G allele of rs2240335 was associated with rheumatoid arthritis risk in East Asian populations ,20, yet PADI4 and increased rheumatoid arthritis risk in East Asians [Given the association between the G allele of rs2240335 in t Asians ,20, the t Asians ,17, and t Asians , we dete−/− mice [For example, we detected no significant difference in NET levels between GG and TT homozygotes at rs2240335. There are several possible explanations for this finding. First, unlike the loss of NETs in the absence of PAD4 in PAD4−/− mice , the red−/− mice ,29,30,31− subjects homozygous for the G allele at rs2240335 had reduced autoantibodies against histones  and those with an anti-CCP test result that was twice the upper limit of normal (CCP+). Anti-CCP titers were determined using the Immunoscan CCPlus test kit  according to the manufacturer’s instructions or generation II anti-CCP or anti-CCP3 ELISA  in the UW Health clinical lab. Controls were excluded if they had an autoimmune disease, inflammatory disease, or hematologic malignancy. Control subjects, CCP− rheumatoid arthritis subjects, and CCP+ rheumatoid arthritis subjects had similar demographic features for the GG versus TT genotype, except for smoking in the CCP+ rheumatoid arthritis group . All clinical data and biologic samples were obtained from the University of Wisconsin (UW) Rheumatology Biorepository first described in . All cli. Brieflyis group .Peripheral blood was collected into ethylenediaminetetraacetic acid (EDTA) vacutainers . DNA was isolated using the Gentra Puregene Blood Kit  according to the manufacturer’s instructions and stored at −80 °C until use.g for 10 min at room temperature without braking. Plasma was transferred to a new tube, centrifuged at 2000× g for 5 min at room temperature with braking, transferred to a new tube, and stored at −80 °C until use.Peripheral blood was collected into EDTA vacutainers, centrifuged at 2000× At the University of Wisconsin-Madison Biotechnology Center, DNA concentration was verified with Quant-iT PicoGreen dsDNA kit . For the KASPar polymerase chain reaction (PCR) reaction, DNA samples were standardized to 0.5 ng/µL using epMotion 5075 and 10 mM Ultrapure Tris-HCl pH 7.5 (Life Technologies). A volume of 2 µL of 0.5 ng/µL DNA and 2 µL of KASPar reaction mix  and 13.2 µL assay mix ) were dispensed into a 384 dark-well plate . After brief centrifugation, 10 µL of Bio-Rad Chill-Out Liquid Wax was added and the following amplification protocol was used with an Eppendorf Mastercycler pro384. First, samples were incubated at 94 °C for 15 min. Then, a three-step protocol was repeated 20 times: 94 °C for 10 s, 59 °C for 5 s, 72 °C for 10 s. Then, another three-step protocol was repeated 25 times: 94 °C for 10 s, 59 °C for 20 s, 72 °C for 40 s, and finally at 10 °C until analysis. To determine the products of the PCR reaction, the plate was stored in darkness at room temperature until the wax melted and became transparent. Then a Synergy 2  plate reader measured fluorescence using Gen5 software (Biotek).2SO4. Absorbance was read at 450 nm with 540 nm plate correction, using a Synergy 2 plate reader (BioTek) and normalized to a neutrophil NET standard. The neutrophil NET standard was generated by incubating a known number of purified neutrophils with phorbol 12-myristate 13-acetate and ionomycin overnight at 37 °C with 5% CO2. After 100% NETosis was visualized, NETs were scraped from the plate and stored at −80 °C.Similar to a previous NET ELISA assay , Costar Neutrophils were purified and then incubated for 4 h without any stimulant, followed by fixation, Sytox staining, imaging, and semi-automated quantification of NETosis using DNA Area and NETosis Analysis (DANA) as previously .2 at room temperature overnight with citrullination confirmed  and H4 (MilliporeSigma) were citrullinated with recombinant human PAD4  at a ratio of 2 µg of PAD4 per mg of histone in a buffer of 100 mM Tris-HCl pH7.5, 1 mM DTT, and 5 mM CaClonfirmed .Corning 96 well plates were left uncoated or were coated with 10µg/mL of native or citrullinated histone, or the same concentration of PAD4 present in the citrullinated histone solution in phosphate buffered saline (PBS) overnight at 4 °C. After washing 3 times (0.1% Tween 20 in PBS), wells were incubated with block solution (5% nonfat dried milk in 0.2% Tween 20 in PBS) at room temperature for 2–4 h, then with serum (prepared as in ) dilutedp < 0.05 considered statistically significant. The Armitage trend test with permutation routine was calculated using the XLISP-STAT programming language; 1 M iterations of the permutation were performed to obtain a permuted p-value.T-tests and analysis of variance (ANOVA) were performed using Prism  with PADI4 correlates with reduced anti-histone antibodies and increased rheumatoid arthritis risk in North Americans. Taken together with the work of others, our data suggest that PAD4 may play a complex role in human rheumatoid arthritis. Additionally, we have provided a detailed evaluation of anti-histone antibodies in CCP− and CCP+ rheumatoid arthritis.We demonstrate that rs2240335 in


AbstractCosberella is described and illustrated from a temperate forest of Citlaltépetl formation, Veracruz State. It is characterized by the following combination of characters: Th I with 2 + 2 dorsal setae; 2 + 2 axial setae on Th II–III; two capitate tenent hairs on each leg; unguiculus half the length of unguis; unguis with tooth; six dental setae and Abd VI without anal spines. A key for the species of the genus is included.A new species of  Cosberella, was erected by Wray in 1963, and includes several species originally described in the subgenus Mucrella Fjellberg, 1985, and in Hypogastrura Bourlet, 1839 or Achorutes Templeton, 1835. All of them were transferred to the genus Cosberella by The genus The abbreviations used in this paper are:Ant antennal segment;a anterior row of setae;Abd abdominal segment;m median row of setae;p posterior row of seta.PAO postantennal organ;sgd dorsal guard sensillum;sgv ventral guard sensillum;Th thoracic segment.Maxillary lamellae are numbered according to Taxon classificationAnimaliaCollembolaHypogastruridaeWray, 1963Ant III and IV. Six to eight sensilla on Ant IV. Ventral sensory file on Ant IV, when present, with 30 or fewer modified setae. Mandible with four or five apical teeth and well-developed molar plate. Maxilla with six lamellae, the fourth one reduced, not exceeding apex of the fifth one. Guard setae of labial palpus pointed; lateral process present or absent. Postantennal organ small, with four small distinct lobes, or simply oval with lobes indistinct; accessory tubercle absent. Unguis with or without tooth, tenent hairs acuminate or clavate. Unguiculus with or without lamella. Pronotum usually with 2 + 2, rarely with 3 + 3 setae. Ventral tube with 4 + 4 or 5 + 5 setae. Tenaculum with 4 + 4 teeth. Dens with six or seven setae, one proximal seta longer than others; mucro with a latero-external lamella of variable shape, its apex tapering, curved. Anal spines minute or absent. Antenna with or without eversible sac between Cosberellaconatoa Wray, 1963.Cosberella. It recalls also the genus Choreutinula by the absence of anal spines and a small PAO with four flattened lobes. However, this genus lacks of unguiculus, or has only a short thin bristle in its place, while the unguiculus of the new species is well developed, straight and half as long as claw, and the fourth maxillary lamella is reduced, not exceeding apex of the fifth one, which are characteristics of genus Cosberella.The Mexican species described here is assigned to the genus Taxon classificationAnimaliaCollembolaHypogastruridaehttp://zoobank.org/21837BAF-AF8F-4D66-B0C8-C3191DC0A69B19°08'30N, 97°12'26W, 2,225 m a.s.l. ex litter from pitfall traps, F. Álvarez leg.México: Veracruz State (Atotonilco) 7 km from Parque Nacional del Pico de Orizaba. Pine-oak temperate forest, Holotype male mounted on slide. Original label: “21/2/12 México, Veracruz State, Pico de Orizaba, 2,225 m snm. F. Álvarez col. Pitfall 8” [printed label]. Collection number 22304.9 paratypes males and 1 juvenile mounted on slides, with the same data. Type material is kept at Colección de Microartrópodos, Facultad de Ciencias, UNAM. Collection numbers 22305–22314.Th I with 2 + 2 dorsal setae. Tenent hairs 2, 2, 2 in one whorl. Unguiculus without lamella and half the length of unguis. Unguiculus with a tooth in the apical third. Six dental setae, one proximal longer than others. Anal spines absent.Body length (average of 10 specimens) = 1.06 mm. Setae not differentiated in macro and microsetae, all of same size Figs , smooth Ant I with seven dorsal setae, Ant II with 12 setae. Ant III with 16 setae in two whorls, sense organ with two free club-shaped microsensilla, not covered by tegumentary fold; two long guard sensilla (sgd and sgv) of same shape and size, and one ventral microsensillum. No eversible sac between Ant III-IV. Ant IV with seven sensilla, subapical organite, lateral microsensillum and simple subapical bulb . Nevertheless, the structure of maxillary head with a partial reduction of the fourth lamella (main diagnostic trait of the genus Cosberella) unequivocally points to this genus as a better choice.The new species has an isolated position in the genus due to the combination of a number of characters, some of which are observed in other genera of C.arborea , the only other known species of the genus lacking anal spines. Cosberellamendozarum sp. n. can be easily distinguished by its specific chaetotaxy, first of all by the absence of cephalic seta c1, the presence of only 2+2 axial setae on Th II–III (vs 3+3 setae in C.arborea), the number of tibiotarsal tenent hairs on mid-leg (2 vs. 3 in two different whorls in C.arborea), the number of dental setae , and the setae as microsetae, contrary to C.arborea which has one macroseta on each side of head and laterally (p6) on each abdominal segment from I to IV. Cosberellaconatoa  differs from the new species by its minute anal spines, the absence of ungual tooth, different number of tenent hairs and the unguiculus with a strong lamella.The new species is most similar to PAO sometimes had 5 vesicles with a central part in circle, looking as a small flower. In four specimens retinaculum had 3 + 3 teeth instead of 4 + 4. Setae m1 on Abd IV vary asymmetrically, with a single seta on right or left side . One specimen subadult had the posterior row of setae on Abd V with left side lacking sensorial setae, p1 and p2. Ventral tube had 5 + 5 setae (4 specimens), 4 + 4 (3 specimens), 5 + 6 (1 specimen), 5 + 3 (1 specimen) and 5 + 0 (1 specimen).Ten specimens were studied. They present several setal asymmetries dorsally on abdomen. Few also have duplicated setae in one abdominal alveolus, and in a couple of cases one or two bifid setae on each alveolus of abdominal segments. Two specimens had 2 + 1 postlabial setae (instead of 2 + 2);


Such data proposes that blood is a rich source of potential biomarkers reflecting central nervous system pathophysiology and should be fully explored for biomarkers that show promise in CSF. Recently, soluble fragments of the triggering receptor expressed on myeloid cells 2 (sTREM2) protein in CSF have been reported to be increased in prodromal AD and also in individuals with TREM2 rare genetic variants that increase the likelihood of developing dementia.Results from recent clinical studies suggest that cerebrospinal fluid (CSF) biomarkers that are indicative of Alzheimer’s disease (AD) can be replicated in blood, e.g. amyloid-beta peptides  or AD.Our results indicate that there are no changes in plasma sTREM2 and NFL concentrations between Concentrations of plasma sTREM2 do not mimic the recent changes found in CSF sTREM2. Reduced40 ratio –7 are be40 ratio . NFL is 40 ratio . Increas40 ratio –12, owin40 ratio  but it s40 ratio . Likewis40 ratio , alpha-s40 ratio  and the 40 ratio , all havTREM2 strongly increase the likelihood of developing AD, frontotemporal dementia (FTD), Parkinson’s disease (PD) and amyotrophic lateral sclerosis (ALS) [TREM2 genetic variant carriers [The triggering receptor expressed on myeloid cells 2 (TREM2) is an innate immune receptor that guides essential functions of microglia. Rare variants in is (ALS) –23. TREMis (ALS) –27. The is (ALS) , 29. Recis (ALS) –34 and ais (ALS) . Interesis (ALS) . Moreovecarriers , 33.TREM2 rare variant carriers.Unlike CSF sTREM2, levels of sTREM2 in blood have been poorly investigated. In this study, we investigate plasma concentrations of sTREM2 in patients with AD and mild cognitive impairment (MCI) compared with aged-matched healthy controls. Furthermore, in a novel approach, we also report on blood concentrations of sTREM2 and NFL in n = 82) were from the longitudinal AD cohorts managed at King’s College London , MCI (n = 8) and AD (n = 31) samples were also included.Samples from a total of 97 participants were used for these analyses Table . The majNeuroMed , Kings HNeuroMed  and the NeuroMed ). Furthensortium , 40) andresponse ). InformPlasma sTREM2 was measured using an in-house electrochemoluminescent assay on the MesoScale Discovery SECTOR imager 6000 , Maryland, USA) using a method adapted from Kleinberger et al. . The capd), with a type error I (α) of 0.05 assuming 97 participants. In a medium effect size (Cohen’s d = 0.5), a power of 0.68 is achieved, whereas a large effect size (Cohen’s d = 0.8) computes a power of 0.97. Thus, we can reasonably state that there is a low probability of error type II in our results if the effect size was large.In CSF, the effect size of sTREM2 between AD and control ranges between 1.077 and 1.539 . In applying a type error I (α) of 0.05, we reach a power (1-β) of 0.99 in our sample size of 97 participants (G*Power). However, the effect size of plasma sTREM2 is likely to be considerably lower than that of CSF sTREM2. Therefore, we examined the achieved power as a function of effect size  whereas t test (sTREM2) or a one-way analysis of covariance  for gender and APOE status. Only age was a significant predictor of plasma NFL; the subsequent analyses were therefore conducted including age as a confounder. A t test or ANCOVA were conducted to determine clinical group differences between blood biomarkers. ANCOVA analyses were followed by a Bonferroni-corrected post hoc pairwise comparison where appropriate. A partial correlation, adjusted by age, tested the association between plasma sTREM2 and plasma NFL. Statistical analysis was performed using IBM SPSS Statistics, version 25 .Data normality was determined by the D’Agostino-Pearson test, and statistical evaluation was performed on logTREM2 rare variant carriers and 49 non-carriers were included in the study. The TREM2 rare variant carrier group comprised of 10 different variants: p.Q33X, p.D39N, p.R47H, p.P59L, p.R62H, p.D87N, p.T96K, p.Q691H, p.H703Y and p.L868R. The demographical characteristics of the cohort are described in Table TREM2 rare variant carriers  and non-carriers . The inclusion of gender was very similar across groups . Finally, there was no statistically significant difference in MMSE  or clinical diagnosis between the two groups , gender (P = 0.083), APOE ε4 status (P = 0.237) or MMSE . There were no differences in the levels of plasma sTREM2 between TREM2 rare variant carriers  and non-carriers  = 1.696, P = 0.093; Fig. TREM2 rare variants  = 1.68, P = 0.177, Fig. TREM2 rare variants with > 2 individuals per group in this analysis. Adding clinical diagnosis as covariate did not change the result (P = 0.171). Next, we tested whether plasma sTREM2 levels differ between clinical diagnoses, regardless of the TREM2 rare variant status, between AD , MCI  and controls . Plasma sTREM2 levels were not different between these groups  = 1.84, P = 0.164, Fig. In the whole cohort, plasma sTREM2 was not associated with age . After accounting for the effect of age, plasma NFL levels were found to be associated with MMSE  and tended to be higher in females  compared to males . Levels were not affected by APOE ε4 status (P = 0.899). There were no differences in plasma NFL between TREM2 rare variant carriers  and non-carriers   = 0.505, P = 0.479, Fig. TREM2 rare variants  = 0.113, P = 0.952, Fig. P = 0.633). As expected, differences in plasma NFL were observed between the three clinical groups  = 4.89, P = 0.010). The Bonferroni-corrected post hoc pairwise comparison demonstrated that the AD group  had significantly higher levels of NFL compared to controls  but not to MCI . However, this correlation is driven by the symptomatic individuals (MCI and AD) as no significant association of plasma sTREM2 and plasma NFL was observed in the control group  but a tendency in the symptomatic group .In the whole cohort, when correcting for age, a significant positive correlation between plasma sTREM2 and plasma NFL was observed  origin from microglia. This is in agreement with the ubiquitous expression pattern of TREM2 at both mRNA and protein levels in Human Protein Atlas (https://www.proteinatlas.org/ENSG00000095970-TREM2/tissue). TREM2 is highly expressed in cells from a myeloid lineage, such as monocytes, macrophages, Kupffer cells or osteoclasts [While sTREM2 has been extensively studied in CSF, only a few studies have reported sTREM2 in blood. Piccio and co-workers  demonstreoclasts –46.TREM2 mutations  that lead to Nasu-Hakola disease or an FTD-like syndrome [TREM2 rare variant carriers have a less obvious and inconsistent pattern of CSF sTREM2. While the TREM2 p.R47H rare variant is associated with increased CSF sTREM2, other TREM2 variants  have been reported to be associated with decreased or unchanged levels of CSF sTREM2 (e.g. R62H) [in press). Our findings could be explained by how these rare variants differently affect the processing and shedding of TREM2. For example, individual variants are believed to impact TREM2 production (Q33X), expression or turnover at the cell surface (R47H) [TREM2 rare variant carriers and non-carriers, even when introducing clinical diagnosis as a co-variable, suggesting that the neuronal injury is no different in AD irrespective of whether someone has a TREM2 rare variant. These findings are agreement with clinical data that find AD cases with a TREM2 p.R47H rare variant are clinically indistinguishable from other AD, albeit those with a TREM2 rare variant generally have an earlier age of onset [Previously, it was shown that both plasma and CSF sTREM2 were useful to detect those with homozygous syndrome , 48. In syndrome , 49. In g. R62H) , 33 (Deme (R47H) , 50; α-se (R47H) , 51; ande (R47H) –54. Surpof onset . Finallyof onset .TREM2 rare variant carriers to date, some individual variants were represented by only very small numbers, which precluded a comparison between them. Additionally, these samples did not have the core AD CSF biomarkers to confirm diagnosis, disease stage or link analyses individually to tau or amyloid burden, and hence, we were limited to using clinical diagnosis. The main strengths are the fact that we used very reliable and well-established assays for both sTREM2 and NFL. The study was designed in such a way that an important total number of TREM2 rare variants were carefully matched to a control group in terms of age, gender and clinical diagnosis.This study has some limitations. Despite our study recruiting the highest number of TREM2 rare variant carriers and non-carriers. Furthermore, we confirm previous reports that sTREM2 is not changed in AD or MCI compared with aged-matched controls. Therefore, we conclude that although plasma sTREM2 may be useful to detect TREM2 homozygous mutations, plasma sTREM2 is not a reliable biomarker to detect TREM2 rare variant status nor suspected AD.This study, for the first time, demonstrates that the levels of sTREM2 and NFL in plasma do not differ between