extfieldsofstudy: Biology Medicine

s2fieldsofstudy: Biology Medicine

text:
IGFBP7 inhibits cell proliferation by suppressing AKT activity and cell cycle progression in thyroid carcinoma

Background Thyroid cancer is the most common malignant endocrine tumor and is classified into papillary thyroid cancer (PTC), follicular thyroid cancer (FTC) and anaplastic thyroid cancer (ATC), which have substantially different characteristics. Insulin-like growth factor binding protein 7 (IGFBP7) has recently been recognized as a tumor suppressor in many cancer types. However, the expression pattern of IGFBP7 and its biological function in various types of thyroid carcinoma remain poorly understood. Results We found that the protein levels of IGFBP7 in FTC and ATC tissues were significantly lower or even absent compared with those in normal thyroid, benign thyroid adenoma and classical PTC tissues. Moreover, overexpression of IGFBP7 in two undifferentiated ATC cell lines, ARO and FRO, and one differentiated FTC cell line, WRO, significantly inhibited cell proliferation in vitro. In vivo experiments revealed that ectopic IGFBP7 expression markedly suppressed growth of tumor xenografts derived from these thyroid cancer cell lines, while IGFBP7 silencing accelerated tumor growth. At the mechanistic level, overexpression of IGFBP7 dramatically suppressed phosphorylation-mediated activation and kinase activity of AKT, causing an upregulation of cyclin-dependent kinase (CDK) inhibitors p27Kip1 and p21Cip1 and induction of G1/S cell cycle arrest, while silencing IGFBP7 exerted the opposite effects. Conclusions IGFBP7 expression is decreased or even absent in FTC and ATC. Acting as a cell cycle repressor, IGFBP7 plays an important tumor-suppressive role in human thyroid cancer, especially in FTC and ATC subtypes and may represent a promising biomarker and therapeutic target for human thyroid cancer treatment. Electronic supplementary material The online version of this article (10.1186/s13578-019-0310-2) contains supplementary material, which is available to authorized users.

cases, respectively [3]. Differentiated thyroid carcinoma (DTC), including PTC and FTC, has a favorable prognosis due to slow growth, well differentiation and lowgrade malignancy. However, approximately 20-30% of PTC patients will develop recurrence, and patients with recurrent disease have poor prognoses. Notably, in contrast, ATC is characterized by poor differentiation and is notorious for its aggressive clinical behavior and tendency to rapidly metastasize and develop intrinsic resistance to chemotherapy, making it one of the most malignant tumors [4]. Even with aggressive therapy, the 5-year survival rate of ATC patients is less than 10%. Due to the lack of effective biomarkers for well-differentiated and poorly differentiated thyroid carcinoma, inappropriate excessive treatments have been used in clinical therapy. Therefore, there is an urgent need to investigate the molecular mechanisms underlying the development and progression of thyroid cancer for identifying potential molecular biomarkers to distinguish various subtypes of thyroid cancer and develop effective therapeutic strategies.
The insulin-like growth factor (IGF) signaling pathway is important for regulating cell proliferation, differentiation and apoptosis in mammals and for promoting tumor development and progression. Insulin-like growth factor binding proteins (IGFBPs) affect the interaction between IGF and its receptor and regulate the activity of the IGF signaling pathway. The IGFBP family consists of seven structurally homologous proteins (IGFBP1-IGFBP7), which bind insulin-like growth factor I (IGF-I) and insulin-like growth factor 2 (IGF-II) with high affinity, carry and transfer these IGFs, and extend their halflives, thus regulating the biological function of IGFs. Among the IGFBPs, IGFBP7, a soluble protein identified in normal meningeal cells and mammary epithelium cells in humans, has been found to bind IGFs with low affinity and to bind insulin with high affinity. In addition, IGFBP7 can participate in a variety of pathophysiological processes, such as cell growth, differentiation, development, senescence and carcinogenesis, independent of the IGF signaling pathway. IGFBP7 is highly expressed in senescent breast epithelial cells [5]. Accumulating evidence suggests that IGFBP7 serves as a tumor suppressor in various types of cancers. Downregulation and even deletion of IGFBP7 have been found in breast cancer, lung cancer, bladder cancer, colorectal cancer, prostate cancer and melanoma, and low levels of IGFBP7 in tumor tissues are correlated with poor prognosis [6][7][8][9][10]. Overexpression of IGFBP7 in lung cancer cells inhibits anchorage-independent growth and xenograft tumor growth [11]. Similar findings have been obtained in other cancer types, such as colon cancer, prostate cancer and melanoma [12][13][14]. However, the expression pattern of IGFBP7 and its biological function in various types of thyroid carcinoma have not been clearly elucidated.
In this study, we found that IGFBP7 expression is greatly decreased and even absent in follicular and anaplastic thyroid carcinoma compared to that in normal thyroid tissue, adenoma and classical PTC. Overexpression of IGFBP7 in two undifferentiated human anaplastic thyroid carcinoma cell lines, ARO and FRO, and one differentiated follicular human thyroid carcinoma cell line, WRO, significantly inhibits tumor proliferation both in vitro and in vivo, whereas knockdown of endogenous IGFBP7 in WRO cells substantially increases proliferation and tumor growth. Furthermore, IGFBP7 dramatically suppresses the phosphorylation-mediated activation and kinase activity of AKT, causing upregulation of cyclin-dependent kinase (CDK) inhibitors, p27 Kip1 and p21 Cip1 , and induction of G1/S cell cycle arrest. Taken together, our findings suggest that IGFBP7 plays an important tumor-suppressive role in human thyroid cancer, especially in FTC and ATC subtypes and may represent a promising biomarker and therapeutic target for the disease.

IGFBP7 expression is downregulated in the FTC and ATC subtypes of thyroid cancer
To determine the protein levels of IGFBP7 in different subtypes of thyroid tumors, we employed immunohistochemical staining in our collection of 112 paraffin-embedded, archived thyroid neoplasm specimens, including 18 cases of thyroid adenoma, 63 cases of papillary thyroid carcinoma (PTC), 15 cases of follicular thyroid carcinoma (FTC), and 16 cases of anaplastic thyroid carcinoma (ATC). As shown in Fig. 1a, b, positive expression of IGFBP7 was detected in 66.7% of thyroid adenoma tissues and 65.0% of PTC tissues, but IGFBP7 was detectable only in 13.4% of FTC and 12.5% of ATC tissues. Notably, classical papillary thyroid carcinoma (CPTC) tissues showed much strong IGFBP7 immunostaining, whereas follicular papillary thyroid carcinoma (FVPTC) tissues hardly showed IGFBP7 expression (Fig. 1c). These data showed that the expression of IGFBP7 was significantly lower or even absent in FTC and ATC as compared with that in normal thyroid tissues, benign thyroid tumors and classical PTC, suggesting that IGFBP7 may represent a valuable biomarker for well-differentiated and poorly differentiated thyroid carcinoma.

IGFBP7 inhibits the proliferation of thyroid cancer cells in vitro
To examine whether IGFBP7 is functionally involved in the development and progression of thyroid cancer, we used two undifferentiated human anaplastic thyroid carcinoma cell lines, ARO and FRO, both of which have undetectable levels of IGFBP7, to ectopically overexpress IGFBP7, and we employed one differentiated follicular human thyroid carcinoma cell line, WRO, which shows moderate IGFBP7 expression, to ectopically overexpress and knockdown IGFBP7 (Fig. 2a). After plating the same number of cells, we observed that vector control cells grew to reach 80-90% confluence within 3 or 5 days, whereas IGFBP7-overexpressing cells reached approximately 20% confluence. Moreover, knockdown of IGFBP7 with two specific shRNAs in WRO cells exhibited a more than twofold increase in cell number compared with that in the vector control cells (Fig. 2b, c). Consistent with these results, both cell counting and MTT assays revealed that while ectopic overexpression of IGFBP7 significantly suppressed proliferation of thyroid cancer cells, silencing of IGFBP7 accelerated thyroid cancer cell growth (Fig. 2d, e). Furthermore, we assessed the effect of IGFBP7 deregulation on anchorage independent growth using soft agar assay, and the results showed that thyroid cancer cells overexpressing IGFBP7 formed fewer and smaller cell colonies in the 3-D setting, and IGFBP7-silenced thyroid cancer cells produced more and larger colonies (Fig. 2f ). Similarly, colony formation assays showed that the ability of cells to form cellular colonies in the 2-D setting was  significantly suppressed upon IGFBP7 overexpression in comparison with that of their corresponding vector-control cells, whereas silencing of IGFBP7 apparently promoted formation of cellular colonies (Additional file 1: Figure S1). These results suggest that IGFBP7 strongly inhibits the proliferation of FTC and ATC cells in vitro.

IGFBP7 suppresses tumor growth of thyroid cancer cells in vivo
Next, we assessed whether IGFBP7 inhibits growth of thyroid cancer cell-derived tumors using subcutaneous xenografts. As shown in Fig. 3a, IGFBP7-overexpressing ARO, FRO and WRO cells grew much more slowly than their corresponding vector control cells following subcutaneous inoculation, while IGFBP7-silenced WRO cells showed a much higher tumor growth rate than vector control WRO cells. In parallel, at the experimental endpoint, excised tumors formed by IGFBP7-overexpressing ARO, FRO and WRO cells had smaller size and lower weight than tumors formed by control cells, and IGFBP7 silencing produced the opposite effect (Fig. 3b). Notably, while four out of five mice inoculated with vector control WRO cells presented detectable tumor xenografts, only two out of five mice inoculated with WRO-IGFBP7 cells presented tumor growth (Fig. 3b), suggesting that IGFBP7 overexpression apparently suppressed not only the tumor growth rate, but also the tumorigenicity of thyroid cancer cells. Furthermore, the histological types of respective subcutaneous tumors were confirmed by H&E staining (Fig. 3c). Moreover, the proportion of proliferative Ki67-positive cells was significantly decreased in tumor tissues of IGFBP7-overexpressing xenografts and notably increased in IGFBP7-silenced tumor tissues (Fig. 3c). Taken together, these data suggest that IGFBP7 strongly suppresses the growth of ATC and FTC cell-derived tumors in vivo and that IGFBP7 represents a potent tumor suppressor preventing the development and progression of thyroid cancer.

IGFBP7 induces the expression of the cell-cycle inhibitors p21 Cip1 and p27 Kip1 , leading to cell cycle arrest
To reveal the mechanism underlying the anti-proliferative effect of IGFBP7 on thyroid cancer, we analyzed the expression levels of cell cycle regulators. As shown in Fig. 4a, ectopic expression of IGFBP7 dramatically upregulated the levels of two cell-cycle inhibitors, namely, p21 Cip1 and p27 Kip1 , accompanied by decreased levels of phosphorylated Rb, although the expression of CDK2 and Cyclin E was not altered. Additionally, the opposite results were observed in IGFBP7-silenced cells, indicating that IGFBP7 might inhibit cell-cycle progression in thyroid cancer cells. As it is well known that p21 Cip1 and p27 Kip1 play important inhibitory roles in the transition from G1 to S phase, we further assessed whether IGFBP7 influences G1/S progression using the 5-bromo-2-deoxyuridine (BrdU) incorporation assays and flow cytometry analyses. ARO and FRO cell lines were synchronized by serum starvation for 36 h and then induced to re-enter the cell cycle by the addition of 10% serum. As shown in Fig. 4b, the proportion of BrdU-positive ARO-IGFBP7 (10.6%), FRO-IGFBP7 (21.2%) and WRO-IGFBP7 (7.7%) cells were much lower than that of their corresponding vector control cells (44.4%, 34.0% and 16.0% for ARO, FRO and WRO cells, respectively), whereas BrdU-positive signals were prominently enhanced in IGFBP7-silenced cells (26.7% and 37.1% for WRO cells transduced with the two IGFBP7-targeting shRNAs) in contrast to vector control WRO cells (15.7%) (Fig. 4b). Consistent with these results, flow cytometry analysis demonstrated that ectopic expression of IGFBP7 in ATC and FTC cell lines increased the percentage of cells in the G0/ G1 phase of the cell cycle and decreased the percentage of cells in the S phase (Fig. 4c). In contrast, IGFBP7 silencing facilitated cell cycle progression through G1 to S phase (Fig. 4c). Taken together, these data suggest that IGFBP7 is able to induce the expression of the critical cell-cycle inhibitors p21 Cip1 and p27 Kip1 , causing G1-S phase arrest during the cell cycle and subsequent growth inhibition.

IGFBP7 suppresses the phosphorylation-mediated activation and kinase activity of AKT in thyroid cancer
Numerous studies have illustrated that AKT crucially regulates the expression of p21 Cip1 and p27 Kip1 and cell cycle progression. We thus further investigated the effect of IGFBP7 on both phosphorylation levels and kinase activity of AKT. As expected, overexpression of IGFBP7 markedly reduced the phosphorylation of AKT (Ser473 and Thr308) in various thyroid cancer cells, whereas silencing of IGFBP7 enhanced AKT phosphorylation (Ser473 and Thr308) (Fig. 5a), indicating that IGFBP7 suppresses phosphorylation-mediated activation of AKT. Meanwhile, an in vitro AKT kinase activity assay was employed to verify the inhibitory effect of IGFBP7 on AKT activity. As shown in Fig. 5b, the kinase activity assay demonstrated that silencing of IGFBP7 in various thyroid cancer cells markedly promoted, whereas overexpression of IGFBP7 suppressed, endogenous AKT activity, as demonstrated by the phosphorylation levels of a common AKT substrate, GSK3β. Collectively, our data suggest that IGFBP7 suppresses proliferation through the inhibition of both

Discussion
Thyroid cancer is the most commonly diagnosed endocrine malignancy. Thyroid cancer is an excellent example of how technological advancements in diagnostic imaging have led to the detection of indolent disease.
Although an increase in incidence is largely confined to the more indolent histological subtype and to early tumor stages, thyroid cancer is a disease with several diverse subtypes. The various subtypes, mainly PTC, FTC and ATC, are different in their unique biology, natural history, prognosis, and available therapeutic options [15][16][17][18][19][20].  [24]. Since IGFBP7 is a secretory protein that could be easily detected in clinical specimens such as blood, it is of great interest to detect levels of serum IGFBP7 in patients with benign thyroid adenoma Western blotting analysis of p-GSK-3β (Ser21/9) and total AKT in the indicated cells. All data were obtained from three independent experiments with similar results or malignant thyroid carcinoma of various subtypes, such that serum IGFBP7 levels might be used as valuable biomarkers for the early diagnosis and clinical therapy of thyroid neoplasms. Different types of thyroid cancer have unique gene mutations, suggesting that different molecular mechanisms may be related to the development and progression of thyroid cancer. Point mutations in the BRAF or RAS gene or RET/PTC rearrangements, all of which lead to constitutive activation of the mitogen-activated protein kinase (MAPK) signaling pathway, can be found in approximately 70% of thyroid cancer patients. Nevertheless, it is not common to have two or more mutations in the same patient at the same time [25,26]. Among these mutations, BRAFV600E is considered to be the marker for the diagnosis and prognosis of papillary thyroid cancer and is closely related to extrathyroidal infiltration, lymph node metastasis, clinical stage, recurrence and mortality of thyroid cancer [27]. It has been reported that IGFBP7 inhibits the BRAF-MEK-ERK pathway in an autocrine or paracrine manner and causes apoptosis in BRAF mutation-positive melanoma cells. However, we detected the phosphorylated levels of MEK and ERK in thyroid cancer cells with overexpression or knockdown of IGFBP7 and found that IGFBP7 did not impact on the BRAF-MEK-ERK pathway (data not shown). In parallel, the various thyroid cancer tissues collected in our study showed loss of IGFBP7 expression regardless of BRAF status. In fact, an increasing number of studies suggest that hyperactivation of the PI3K/AKT signaling pathway is as important as the BRAF-MEK-ERK pathway during tumor development and progression. For example, Maria et al. demonstrated that the PI3K/AKT signaling pathway is activated in more than 50% of thyroid cancer cases [28]. Several reports have shown that gene mutations in the PI3K/AKT pathway are mainly present in poorly differentiated thyroid cancer tissues [29][30][31], suggesting that the activation of the PI3K/AKT signaling pathway may play an important role in the development and progression of thyroid cancer. In support of this notion, our current study reveals that IGFBP7 potently suppresses the growth of ATC and FTC cell-derived tumors and inhibits both phosphorylation-mediated activation and kinase activity of AKT. Therefore, our data suggest that the loss of IGFBP7 causes overactivation of the AKT signaling pathway, an effect essentially equivalent to that of the well-documented genetic mutations causing overactivation of the BRAF-MEK-ERK pathway. Notably, as IGFBP7 is a secreted protein and has low affinity for IGF, how IGFBP7 blocks the activation of AKT signaling remains to be investigated.
From a biological point of view, tumors, characterized by hyperproliferation, disrupted cell cycle and dysregulated of signal transduction pathways, are indicative of progressive disorders in cell cycle regulation. In normal cells, each stage of the cell cycle is controlled by a series of regulatory mechanisms to ensure that the cell cycle is carried out in a strict and orderly fashion. Three key classes of regulatory molecules, namely, cyclins, cyclin-dependent kinases (CDKs) and cyclin-dependent kinase inhibitors (CKIs), determine the progress of a cell through the cell cycle. Therefore, it is important to determine which types of these regulatory molecules are involved under a certain condition. In this study, we found that ectopic expression of IGFBP7 dramatically upregulates the levels of p27 Kip1 and p21 Cip1 and that the silencing of IGFBP7 causes the opposite effects in ATC and FTC cells. Notably, the cell cycle regulator p27 Kip1 is a tumor-suppressive protein that broadly inhibits CDKs and plays an important role in resting cells in the G1 phase during the cell cycle [32]. P21 Cip1 which halts the cell cycle in the G1 phase by binding to and inactivating cyclin-CDK complexes is also a negative regulator of cell cycle progression and a typical tumor suppressor gene [33]. After the inhibition of protein kinase activity in cyclin-CDK complexes by p27 Kip1 and p21 Cip1 , Rb is in a state of hypophosphorylation. Hypophosphorylated Rb can bind the critical transcription factor E2F, making it lose its function and causing cell cycle arrest at G1 phase [34]. Indeed, our data demonstrate that IGFBP7 overexpression significantly increases the percentage of cells in the G0/G1 phase and reduces the percentage of cells in the S phase of the cell cycle, and IGFBP7 silencing causes the opposite effects in ATC and FTC cells. Thus, we propose that IGFBP7 induces G1-S phase arrest during the cell cycle by upregulating the expression of two critical CKIs, namely, p27 Kip1 and p21 Cip1 , to potently inhibit the proliferation of ATC and FTC cells. Notably, the AKT signaling pathway has been widely reported to decrease the expression of p27 Kip1 and p21 Cip1 through inactivating its downstream substrates, such as FOXO transcriptional factors, and to promote cell cycle progression [35][36][37][38][39]. Therefore, whether and how IGFBP7 inhibits the AKT signaling pathway to upregulate the expression of p27 Kip1 and p21 Cip1 in thyroid cancer requires further investigation.
Although IGFBP7 is present in almost all normal tissues, IGFBP7 is downregulated or even absent in a variety of tumor types, including breast cancer, lung cancer, bladder cancer, colorectal cancer, prostate cancer, melanoma and thyroid cancers [40][41][42]. Similar to that for many other tumor suppressors, the downregulated expression of IGFBP7 in tumor tissues is mainly ascribed to genetic and epigenetic regulation. It was reported that loss of heterozygosity (LOH) causes loss of expression of IGFBP7 in breast cancer [6]. Accumulating evidence has demonstrated that hypermethylation of the IGFBP7 gene promoter leads to its loss of expression. Moreover, tumor cells lacking IGFBP7 expression could be restored by treatment with the DNA methylation inhibitor 5-aza-2′-deoxycytidine [7,43]. Therefore, it is of great interest to further investigate whether the significant downregulation of IGFBP7 in ATC and FTC tissues, but not in classical PTC tissues, is caused by LOH or hypermethylation.

Conclusions
Taken together, results from the present study showed that the expression of IGFBP7 is significantly downregulated and even absent in thyroid follicular carcinoma and anaplastic carcinoma tissues. IGFBP7 might act as a tumor suppressor by inhibiting the proliferation of thyroid cancer cells via the downregulation of the activity of AKT, leading to an increase in the expression of the cell cycle inhibitors p27 Kip1 and p21 Cip1 .

Tissue specimens
This study was conducted with a cohort of paraffinembedded sections of 112 thyroid tumor patients, who had undergone surgical resection in the first affiliated hospital of Sun Yat-sen university during the period of 2005 to 2010. The histologic types of the above tumor samples were as the following: 18 cases of thyroid adenomas, 63 cases of papillary thyroid cancer, 15 cases of follicular thyroid cancer, 16 cases of anaplastic thyroid cancer.

Cell cultures
The undifferentiated thyroid cancer cell lines FRO and ARO, follicular thyroid cancer cell lines WRO were gifted from the surgical laboratory of the Chinese University of Hong Kong, and maintained in DMEM medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (HyClone, Logan, UT) and 1% penicillin/ streptomycin (Invitrogen, Carlsbad, CA), according to the protocols of previous reports [44]. All cell lines were authenticated by short tandem repeat fingerprinting at Medicine Laboratory of Forensic Medicine Department of Sun Yat-Sen University (Guangzhou, China), and were free of mycoplasma contamination.

Plasmids and transfection
IGFBP7 expression plasmid was generated by PCR subcloning of human IGFBP7 coding sequence into retroviral transfer plasmid pMSCV to generate plasmid pMSCV-retro-puro-IGFBP7. For depletion of IGFBP7, two human shRNA sequences were cloned into the pSuper-retro-puro plasmid to generate pSuper-retro-IGFBP7-RNAi(s), respectively. Retroviral production and infection were performed as previously described [45], and stable cell lines were selected by treatment with 0.5 μg/ml puromycin for 10-14 days, beginning at 48 h after infection.

3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay
According to previous studies [46], cell viability was determined using an MTT assay. The cells were seeded at a density of 5 × 10 3 cells per well in 96-well plates. Subsequently at 1, 2, 3, 4, 5 and 6 days, 20 μl MTT (Sigma-Aldrich, St. Louis, MO) was added to each well and incubated for 4 h. The culture medium was removed, and 200 μl dimethyl sulfoxide (DMSO) (Amresco, Solon, Ohio) was added to each well. The plates were then shaken at room temperature for 10 min, and the absorbance of stained cells was measured at 570 nm, with 655 nm as the reference wavelength. Each experiment was performed in triplicates and repeated for three times.

Colony formation and soft agar assays
For the 2-D colony formation assay, cells were plated into 6-well plates at the density of 200-500 per well. The cells were allowed to grow for 7-12 days and stained with crystal violet. The plates were photographed and the numbers of colonies formed by indicated cells were quantified using the Quantity One software package (Bio-Rad, Hercules, CA). For the soft agar assays, one or three thousand cells were trypsinized and suspended in 2 ml of culture medium containing 0.3% agar (Sigma). The agar-cell mixture was grown on top of 1% agar contained in culture medium. After 10 days, viable cellular colonies larger than the indicated diameter were counted. Each experiment was repeated for three times.

Bromodeoxyuridine labeling and immunofluorescence
Cells were plated on coverslips (Fisher, Pittsburgh, PA). After 24 h, cells were incubated with bromodeoxyuridine (BrdU) for 1 h and stained with anti-BrdU antibody (Upstate, Temecula, CA) according to the manufacturer's instruction. Immunostaining images were acquired under a laser scanning microscope (Axioskop 2 plus, Carl Zeiss Co. Ltd., Jena, Germany). Each experiment was repeated for three times.

Flow cytometry
Cells were harvested, washed with cold PBS and processed for cell cycle analysis using flow cytometry. Briefly, the cells were fixed in 75% ethanol and stored at − 20 °C for later analysis. The fixed cells were centrifuged at 1000 rpm and washed with cold PBS twice. RNase A (20 μg/ml final concentration) and propidium iodide staining solution (50 μg/ml final concentration) was added to the cells and incubated for 30 min at 37 °C in the dark. Fifty thousand cells were analyzed using a FACSC alibur instrument (BD Biosciences, San Jose, CA) equipped with CellQuest 3.3 software. ModFit LT 3.1 trial cell cycle analysis software was used to determine the percentage of cells in the different phases of the cell cycle. Each experiment was repeated for three times.

Immunohistochemical analysis (IHC)
A total of 112 paraffin-embedded thyroid tumor samples described above were used in the current study. For the use of these clinical materials for research purposes, prior patients' consents and approval from the Institutional Research Ethics Committee were obtained. After deparaffinization, sections were immunostained using anti-IGFBP7 monoclonal antibody. The resultant immunostaining images were captured using the AxioVision Rel.4.6 computerized image analysis system (Carl Zeiss, Oberkochen, Germany). The procedure was carried out according to previously described methods [47]. The degree of immunostaining of formalin-fixed, paraffinembedded sections was reviewed and scored independently by two investigators, based on both the proportion of positively-stained tumor cells and the intensity of staining. The observers were blinded to the histopathological features and patient data of the samples.

AKT kinase activity detection
The AKT Kinase Assay kit was purchased from the Cell Signaling (Danvers, MA, # 9840). As instructed by the provided protocol, immobilized AKT antibody was used to immune-precipitate AKT from the indicated cell extracts [48]. Immune-precipitated pellets were then incubated in the Kinase Buffer containing GSK-3 fusion protein and cold ATP. Levels of GSK-3 phosphorylation using the anti-phospho-GSK-3α/β (Ser21/9) antibody were measured by western blotting and chemiluminescent detection. All the experimental steps were carried out strictly according to the instructions of the kit. Each experiment was repeated for three times.

In vivo tumor growth assay
For the in vivo tumor growth assay, ARO-Vector and ARO-IGFBP7cells (2x10 6 cells each), FRO-Vector and FRO-IGFBP7 cells (5 × 10 6 cells each), WRO-Vector and WRO-IGFBP7 (8 × 10 7 cells each), WRO-RNAi-Vector and WRO-IGFBP7-RNAi-2 (7 × 10 7 cells each) were subcutaneously inoculated into the dorsal thighs of individual female BALB/C nude mice (6-7 weeks of age, 20-24 g). Tumor lengths (L) and widths (W) were measured every 2 days using a digital caliper, and tumor volumes were calculated using the equation volume (mm 3 ) = L*W 2 /2. After 3-4 weeks, mice were anesthetized and sacrificed, and tumors were removed completely, photographed and weighed. The tumor growth curves were plotted and statistically analyzed. The measurement data were expressed as mean value plus standard deviation (X ± S). The comparison between the two groups was conducted by t test. Moreover, the tumors were sectioned for HE staining and immunostaining of Ki-67. Animal experiments were approved by the Ethical Committee of Sun Yat-sen University.

Statistical analysis
All statistical analyses were performed with the SPSS 20.0 software package (IBM, Chicago, IL, USA). Comparative analysis between groups was performed with two-tailed paired Student's t test for statistical significance. The Chi square test was used to analyze the relationship between IGFBP7 expression and pathological characteristics. All bars represent the mean ± SD derived from three independent experiments. P values < 0.05 were considered significant.

Additional file
Additional file 1: Figure S1. IGFBP7 inhibits proliferation of thyroid cancer cells in vitro. (a) Representative micrographs (upper) and relative quantification (lower) of the indicated cells as evaluated by the colony formation assay. Data represent the mean ± S.D. of three independent experiments. A two-tailed Student's t-test was used for statistical analysis (*P < 0.05, **P < 0.01).


extfieldsofstudy: Chemistry Medicine

text:
Relative stabilities of wild-type and mutant glial fibrillary acidic protein in patients with Alexander disease

Alexander disease (AxD) is an often fatal astrogliopathy caused by dominant gain-of-function missense mutations in the glial fibrillary acidic protein (GFAP) gene. The mechanism by which the mutations produce the AxD phenotype is not known. However, the observation that features of AxD are displayed by mice that express elevated levels of GFAP from a human WT GFAP transgene has contributed to the notion that the mutations produce AxD by increasing accumulation of total GFAP above some toxic threshold rather than the mutant GFAP being inherently toxic. A possible mechanism for accumulation of GFAP in AxD patients is that the mutated GFAP variants are more stable than the WT, an attribution abetted by observations that GFAP complexes containing GFAP variants are more resistant to solvent extraction. Here we tested this hypothesis by determining the relative levels of WT and mutant GFAP in three individuals with AxD, each of whom carried a common but different GFAP mutation (R79C, R239H, or R416W). Mass spectrometry analysis identified a peptide specific to the mutant or WT GFAP in each patient, and we quantified this peptide by comparing its signal to that of an added [15N]GFAP standard. In all three individuals, the level of mutant GFAP was less than that of the WT. This finding suggests that AxD onset is due to an intrinsic toxicity of the mutant GFAP instead of it acting indirectly by being more stable than WT GFAP and thereby increasing the total GFAP level.

Astrocytes from human patients and animal models display multiple defects (reviewed in Ref. 7), including decreased glutamate and potassium transport (8), decreased proteasome activity (9), increased production of nitric oxide and proinflammatory chemokines (10 -13), defective calcium signaling (14), and aberrant mechanosensitive signaling (15). A widely held view of how the presence of mutant GFAP leads to these defects is that, rather than being toxic per se, it is more stable than the WT and thereby causes accumulation of GFAP above a critical toxic threshold (4,8,16,17). The origin of this view was the observation that mice expressing elevated levels of WT GFAP from a human GFAP transgene display features of AxD (18). In addition, the presence of this WT transgene dramatically enhances the pathology displayed by AxD model mice engineered to express an endogenous mutated GFAP (8,11,16). Also contributing to the toxic threshold hypothesis is the analysis of transgenic mouse lines expressing different levels of the AxD-causing human R239H mutant GFAP (19). This analysis led to the conclusion that expression of the mutant GFAP does not in itself cause Rosenthal fiber formation but that these aggregates form when the level of total GFAP increases by at least some amount between 2.7% to 30%. Other contributors to this hypothesis are multiple observations that multimeric forms of GFAP are more resistant to solubilization when they contain mutant GFAP (e.g. Ref. 6,[20][21][22][23].
As a test of this toxic threshold hypothesis, we determined whether mutant GFAP is indeed more stable than the WT. Using MS, the relative levels of mutant and WT GFAP were analyzed in three AxD patients, each of whom carried one of the more prevalently occurring disease-causing GFAP mutations: R79C, R239H, or R416W. The level of mutant GFAP in all three patients was found to be less than that of the WT. This suggests that mutant GFAP is not inherently more stable than the WT and, consequently, that onset of AxD is due to the intrinsic toxicity of mutant GFAP rather than an increase in the total GFAP pool.

General approach
We investigated whether mutant GFAP is more stable than WT GFAP by using MS to determine the relative amounts of mutant and WT GFAP in AxD patients harboring the R79C, R239H, or R416W mutation. A peptide unique to either the mutant or WT protein was quantified, and its molar concentration was compared to that of total GFAP obtained by quantify-This work was supported by NINDS, National Institutes of Health Grant P01NS42803 (to M. B.). Funds for the Sciex 5600 TripleTOF mass spectrometer came from National Institutes of Health Shared Instrumentation Grant S10 RR027822 (to S. B.). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. This article contains cro ARTICLE ing several peptides common to both the mutant and WT forms.

Identification of WT-and mutant-specific peptides
The mutant and WT diagnostic peptides were selected based on their efficient detection by LC-MS/MS and sequence uniqueness to GFAP in the predicted human proteome using the PROWL algorithm ProteinInfo tool to search the NCBI database (http://prowl.rockefeller.edu). 4 For the R79C case, we observed a GFAP tryptic peptide specific for WT GFAP (FASYIEK, residues 80 -86). For the R416W patient, we observed a tryptic peptide specific for the mutant protein (TVEMWDGEVIK, residues 412-422). For the R239H patient, we were unable to detect either a WT-specific or mutant-specific peptide using trypsin digestion, but by using chymotrypsin, we detected the mutant-specific peptide KEIHTQY (residues 236 -242). Spectra for these three diagnostic peptides are shown in Fig. 1.

Identification of common peptides
The total amount of GFAP in the patient samples was measured by identifying proteotypic GFAP peptides not affected by the mutations. For the R79C and R416W patients, these were the tryptic peptides FADLTDAAAR (residues 261-270), LADVYQAELR (residues 112-121), ALAAELNQLR (residues 96 -105), and LLEGEENR (residues 369 -376). For the R239H patient sample, these were the chymotryptic peptides RQEADEATL (residues 173-181) and ARQQVHVEL (residues 216 -224). Because of difficulty in detecting ARQQVHVEL (Table 1), for R239H we also used IEKVRF (residues 84 -89), which is also present in desmin and peripherin. Both of these nontarget proteins are at extremely low concentrations in the brain compared to GFAP (The Human Protein Atlas, https://www.proteinatlas. org (44)). 4 The fragment ions used for quantitation of each common peptide are listed in Table S1.

Examination of degraded GFAP
GFAP Immunoblots of the patient brain extracts displayed the typically observed pattern of a major band at the expected position for intact human GFAP of 50 kDa and a series of bands in the 35-to 45-kDa range that are presumed to be partially degraded GFAP (Fig. 2) (24 -27). Because the WT and mutant GFAP composition of the degraded protein might be a sensitive indicator of their relative stabilities, this material was analyzed in addition to that in the intact region for the R79C and R416W patients (attempts to perform this analysis for the R239H patient were unsuccessful, probably because of the reduced amount of GFAP in the degraded size range coupled with decreased ionization efficiency or digestion of the chymotryptic peptides relative to the tryptic peptides used for the R79C and R416W patients).

Use of an internal standard for quantitation
The signals obtained for the various peptides were quantified by comparison to signals obtained from a known amount of full-length 15 N-labeled human GFAP added to each sample. The 15 N-labeled GFAP was either the mutant or WT form, depending on whether the type-specific peptide used for identification was from the mutant or the WT. Thus, WT 15 Nlabeled GFAP was added to the R79C patient sample, and 239H and 416W mutant 15 N-labeled GFAP was added to the R239H and R416W patient samples, respectively (see "Experimental procedures" for details). These 15 N-labeled GFAP standards were added to the samples prior to gel electrophoresis. However, because these were full-length protein standards lacking significant degradation products, and so would not be present in the degraded bands following electrophoresis (Fig. 2), the same amount of 15 N-labeled GFAP standard was run in a separate lane in the gel and then excised and combined with the excised patient degradation bands prior to extraction for analysis (see "Experimental procedures" for details).

Calculation method
The fraction of total GFAP that was mutant (or WT) was obtained by (A) determining the amount of the unique mutant (or WT) peptide in the patient sample relative to the same peptide in the 15 N-labeled GFAP internal standard, (B) determining the amount of total GFAP relative to the 15 N-labeled standard using GFAP peptides common to both mutant and WT forms, and (C) dividing A by B. For uniformity, results are presented in Table 1 as the percentage of total GFAP attributable to the mutant form (percent mutant); thus, for R79C, the experimentally determined WT percentage was subtracted from 100. The center and right sections of Table 1 show the data used to calculate these percentages (the signals from the diagnostic and control peptides in the patient samples normalized to those from the 15 N-labeled standard). An illustration of the quantitation procedure is provided in Fig. 3 for the peptide specific for the R416W mutant GFAP (TVEMWDGEVIK) and a peptide common to both the mutant and WT GFAP (FADLTDAAAR).

Control experiments
The calculation method used should yield the same result for any amount of 15 N-labeled standard added to the samples, provided that the amount was sufficient for its peptides to be detected. The data presented in Table 1 confirm this expectation. Varying the added 15 N-labeled standard up to 10-fold had no apparent effect on the percent mutant quantitation. Table 1 also shows results from testing whether the duration of proteolytic digestion was appropriate. As shown in Table 1, reducing the time of trypsin digestion from our standard of 40 h to 18 h or the time of chymotrypsin digestion from our standard of 72 h to 48 h had a marginal, if any, effect on quantitation.

Relative level of mutant GFAP in AxD patients
None of the three AxD patients had a higher percentage of mutant GFAP than WT in the intact band (Table 1), counter to expectation if mutant GFAP contributed to disease by having greater stability. The percentages of total GFAP attributed to the mutant form for the R79C patient (21%) and R239H patient (29%) were actually much lower than that of the WT, suggesting that these mutant GFAPs are less stable rather than more stable. Similarly, less mutant GFAP than WT was observed in the

Instability of mutant GFAP in Alexander disease patients
degraded bands. In the case of the R79C patient, the percentage of mutant GFAP in the degraded band was about the same as that in the intact band, whereas for the R416W patient, it was less than that in the intact band.

Discussion
The objective of this study was to test a proposed mechanism for AxD that posits that mutant GFAP is not directly toxic but has greater stability than WT GFAP and thus results in accumulation of GFAP to toxic levels. We used MS to determine the relative levels of mutant and WT GFAP by comparing the amount of a patient-specific peptide with that of common peptides, using recombinant 15 N-labeled GFAP as an internal stan-dard. A similar approach was used to determine the ratio of mutant to WT K-Ras proteins in clinical samples (28,29), except that in these other studies, internal standards were 13 C/ 15 N-labeled diagnostic peptides added after trypsin digestion. Our internal standards were full-length 15 N-labeled GFAP added to the initial homogenates, which provides correction for losses during sample processing (30).
Contrary to expectation for a mechanism based on greater stability of mutant GFAP, for each of the three AxD patients analyzed, the mutant GFAP was present at a similar or lower level than the WT form. This lower level of mutant GFAP could result from reduced transcription, mRNA stability, or translation rather than reduced protein stability, but this would require that each of the missense mutations acts at the nucleotide level. Evidence excluding decreased transcription or mRNA stability was obtained by Jones et al. (14), who found no difference in transcript levels of the mutant and WT GFAP mRNAs in astrocytes differentiated from induced pluripotent stem cells (iPSCs) isolated from AxD patients carrying either an R88C or R416W mutation. An effect on translation is also unlikely for these samples. In some organisms, codon use does affect translation, either by codon recognition efficiency or by dictating the secondary structure of the mRNA in the initiation region (reviewed in Ref. 31), but humans show no evidence of codon selection (32). Furthermore, the R79C and R239H mutations are to more frequently used codons rather than to ones that are rarer; the codons and their percentage of use in humans are CGC(0.79)3 UGC(0.92) for R79C, and CGC(0.79)3 CAC(1.15) for R239H (45). In addition, the nucleotide changes have no effect on the predicted mRNA structure in the region of the translation start site (data not shown; structures were analyzed using RNAfold via http://rna.tbi. univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi). 4 Thus, the most plausible explanation for the lower level of mutant GFAP is instability of the protein itself. Reduced stability provides an Table 1 Mutant GFAP as a percent of total GFAP in R79C, R416W, and R239H Alexander disease patients Total GFAP is the sum of the amount of patient GFAP and 15 N standard GFAP analyzed. The control peptides for trypsin digestion were FADLTDAAAR (1), LADVYQAELR (2), ALAAELNQLR (3), and LLEGEENR (4). The control peptides for chymotrypsin digestion were ARQQVHVEL (1), RQEADEATL (2), and IEKVRF (3). IEKVRF is also present in desmin and peripherin, but these proteins are not expected to be present in the brain at significant levels compared to GFAP. Were significant levels indeed present, their inclusion would lead to overestimation of the amount of WT GFAP present and, thus, underestimation of the percent mutant GFAP. With inclusion of IEKVRF, the calculated percent mutant GFAP was actually slightly higher than that obtained if it were excluded (29% versus 27%). ND, not detected in the patient sample, the 15

Instability of mutant GFAP in Alexander disease patients
explanation for the otherwise puzzling observation that total GFAP levels are actually lower in the spinal cord of R236H mice than in the WT (33). A mechanism for reduced stability of mutant GFAP arises from an analysis of the crystal structure of the human GFAP 1B rod domain (34). The structure obtained led the authors to conclude that many of the mutations in the central rod domain producing AxD would destabilize filament formation, particularly at the initial step of dimerization (a caveat, however, is that this prediction was also made for V115I, which is not believed to be disease-causing (35)). The suggested defect in polymerization of mutant GFAP would result in a greater fraction of the mutant form than WT being present as monomers and thus more available for degradation. This mechanism could explain not only the instability of R79C and R239H GFAP but also why the stability of R416W GFAP is similar to that of the WT; R79C and R239H are in the central rod domain and thus might affect dimerization, whereas R416W is in the tail region and is believed to only affect subsequent steps in polymerization (21). Several prior observations are consistent with compromised polymerization of mutant GFAP. Using a yeast two-hybrid system, Nielsen et al. (36) observed decreased dimerization for the several common AxD GFAP mutations they tested. Tian et al. (37) found that, in U251 cells expressing either GFP-tagged WT or tagged R239C GFAP, the fraction of GFAP present in the soluble fraction (monomers and short oligomers) was significantly greater for the mutant.
The possibility that analysis of the partially degraded forms of GFAP might provide a more sensitive measure of relative stabilities was not realized. Instead of containing more of the presumptively less stable R79C mutant GFAP, the percentage of R79C mutant GFAP in the partially degraded GFAP was similar to that in the intact GFAP (23% versus 21%). In the case of the R416W patient, the calculated percentage of mutant GFAP in the degraded band (21%) was even less than that in the intact band (46%) ( Table 1). A likely explanation for the lower calculated percentage of mutant GFAP in the degraded R416W GFAP is that some of the cleaved GFAPs had the R416W proteotypic peptide removed. The sequences of the degradation bands present in our AxD patient samples are not known, but the series of bands observed is quite similar to those seen in a variety of other control and diseased post-mortem samples (24 -27) and has been attributed to digestion by calpain (27,38,39). N-terminal sequencing of several of these large degradation products obtained from normal brain white matter, multiple sclerosis plaques, leucotomy scars (24) and spinal cords of patients with ALS (38), and by complete in vitro calpain digestion of GFAP (39), yielded cleavage sites after Ala-40, Asn-59, and Thr-383. Cleavage at Ala-40 or Asn-59 and/or Thr-383 could generate the series of degradation bands in our samples  (Table 1). A and B, elution chromatograms for detection of the product ions from the R416W mutant-specific peptide TVEMWDGEVIK that were used for quantitation. A, unlabeled patient GFAP. B, [ 15 N]GFAP internal standard. C and D, data for the common peptide FADLTDAAAR. Note that, as expected, the unlabeled and 15 N-labeled peptides coelute. The table insets in B and D list the product ions, the peak areas of their signals, and the ratios of the peak area of each unlabeled ion to that of the labeled ion. These ratios provide measures of the amount of mutant GFAP and total GFAP, respectively, in the patient sample relative to the 15 N standard. The ratio of these two values is the fraction of total patient GFAP that is the mutant form. The actual calculations summarized in Table 1 include additional values from other common peptides. Data for each individual peak area are provided in Table S1. (Fig. 2), with Thr-383 cleavage removing the R416W diagnostic peptide. Because this peptide identifies the mutant protein, its loss would result in underestimation of the contribution of mutant protein to the total. In contrast, the R79C site is expected to be retained in the degradation products examined. Two possible explanations for the percentage of R79C GFAP being similar in the intact and degraded bands are that the clipped mutant GFAPs may also be less stable than their WT counterparts, or that calpain degradation may occur predominantly post-mortem (39) so that the composition of the degradation products reflects that of the intact GFAP at the time of death.

Instability of mutant GFAP in Alexander disease patients
A remaining question is whether our finding of less mutant than WT GFAP applies to all astrocytes or only to those affected (or unaffected) by the disorder. Using Rosenthal fiber content as an indication of affected astrocytes, our data, in conjunction with that of Heaven et al. (6), suggest that the status of the astrocytes is not a critical parameter. Using urea extraction to distinguish between GFAP in normal filaments and that in Rosenthal fibers, that study estimated that about two-thirds of the GFAP in the R79C sample was associated with Rosenthal fibers, but little to none was in the R239H sample, whereas the observed percentage of mutant GFAP was similar in both samples (21% and 29%, respectively). However, a more rigorous examination of these variables with a larger sample size is required to establish these relationships.
Our finding that the levels of mutant GFAP are similar to or lower than WT levels suggests that the mutant GFAP does not initiate AxD by causing accumulation of normally functioning GFAP, but that the mutations per se produce a toxic response. This same conclusion was reached by Jones et al. (14) in their study of astrocytes derived from AxD patient iPSCs. They observed that, compared with their gene-corrected controls, these cells displayed marked differences in both their RNA transcripts and their ability to propagate intercellular calcium waves but no difference in their GFAP protein levels, prompting the comment that "mutations in GFAP are sufficient." An example of an effect due to a GFAP mutation rather than GFAP quantity is the finding of Tang et al. (9) that proteasome activity is inhibited more strongly by soluble R239C GFAP than by WT GFAP. Another is the finding of Jany et al. (33) that, although GFAP levels in the spinal cord of R236H mice were decreased compared with the WT, the level of Gfap mRNA was increased. Thus, similar to the findings of Jones et al. (14), mutant GFAP initiates changes in gene transcription in the absence of an increase in total GFAP levels. There are also multiple reports that, at comparable levels, mutant GFAP causes significantly more physiological changes, including aggregate formation, than WT GFAP (e.g. 10,19,23,40).
If mutation of GFAP per se causes disease, then how does one account for the AxD-like characteristics of mice overexpressing WT GFAP? A model proposed by Li et al. (41) provides a resolution of this seeming paradox. They hypothesized that a ratelimiting step in GFAP polymer formation is sensitive to the presence of the GFAP mutations, a suggestion now supported by the crystal structure analysis (34). Thus, either elevated production of WT GFAP or the presence of mutant GFAP would cause abnormal accumulation of precursors. Some of these pre-cursors might then be diverted into an alternative, aberrant pathway to produce toxic complexes. Consistent with this explanation, Tang et al. (9) suggested that formation of abnormal GFAP oligomers is responsible for proteasome inhibition. In light of this kinetic bottleneck model, we suggest an alternative to the conclusion of Tanaka et al. (19) that an increase in total GFAP of between 2.7% and 30%, caused by expression of a mutant transgene, is sufficient to produce Rosenthal fiber formation. We suggest that, rather than such a modest increase in total GFAP being toxic, their observations instead define the minimum percent of total GFAP that must be the mutant form to significantly interfere with polymerization. Our data suggest that having about 20% of the total GFAP as the mutant form is sufficient to produce disease (Table 1), and a prior report of an AxD mutation that caused occasional exon skipping suggests that about 10% mutant form is sufficient (42).
The greater resistance of mutant GFAP to solubilization by various solvents was another observation that contributed to the concept that the total amount of GFAP is the critical factor for toxicity in AxD. Although our data indicate that the stability of mutant GFAP is similar to or less than that of the WT, it remains possible that GFAP complexes containing even a minority of the mutant form have increased stability. However, several observations suggest that the GFAP extraction data, which were obtained in vitro, may not reflect differences in GFAP stability in vivo. Moody et al. (43) found that GFAP in the R236H mouse actually turns over more rapidly than in the WT, and in a Drosophila AxD model, Wang et al. (23) observed no change in GFAP levels when the solvent extraction of mutant GFAP was restored to that of the WT by overexpression of ␣B-crystallin.
In conclusion, we found that in three AxD patients the level of mutant GFAP is lower than that of the WT, indicating that mutant GFAP is less stable rather than more stable than the WT. This observation, together with those cited above of other laboratories, suggests a reordering of events in the development of AxD. Instead of mutant GFAP having increased stability and serving simply to increase the total level of GFAP above some toxic threshold, we suggest that the initial event is toxicity of mutant GFAP per se. Although this view places toxic effects of mutant GFAP as an event occurring prior to a rise in total GFAP levels, the importance of increasing levels of total GFAP is not discounted. A number of positive feedback loops have been proposed whereby pathological changes caused by mutant GFAP, such as induction of a stress response and inhibition of proteasome activity, result in increasing GFAP production (reviewed in Ref. 2). An irreversible point in disease progression may be reached when activation of the positive feedback loops by mutant GFAP more than compensates for its relative loss because of its instability.

Tissue samples
Use of post-mortem human tissue for this study was approved by the University of Alabama at Birmingham Institutional Review Board and abided by the Declaration of Helsinki principles. The sources and characteristics of the tissues are Instability of mutant GFAP in Alexander disease patients described in Table 2. They were stored at Ϫ80°C until use. For sample preparation, ϳ20 mg of tissue was homogenized by pipetting up and down 20 times in 1 ml of a total homogenate protein buffer consisting of 2% SDS, 6.25 mM Tris-HCl (pH 7.5), 5 mM EDTA, and Roche Complete Protease Inhibitor Mixture, as described previously (9). The resulting homogenates were boiled for 30 min, followed by centrifugation at 10,000 ϫ g for 5 min, and the supernatant was used in subsequent experiments. Protein concentrations were determined by BCA assay (Pierce).

Production of [ 15 N]GFAP standards
pET23b plasmids containing the WT and mutant human GFAP complementary DNA sequences were provided by Roy Quinlan (Durham University) (see Ref. 21 for the method of construction). The plasmids were transfected into Bl21pLysS Escherichia coli and grown in medium having more than 98% 15 N (Cambridge Isotope Laboratories, Andover, MA), and the recombinant GFAP was purified as described by Der Perng et al. (21).

Immunoblotting
Immunoblotting was used to estimate the GFAP concentrations in patient samples and to determine the gel position of the intact and degraded GFAP bands. Purified recombinant human GFAP, made as described above, was used as a quantitation standard. Protein samples were boiled in Laemmli sample buffer for 5 min and electrophoresed for 60 min in a 4%-20% SDS-PAGE gel (Pierce) at 120 V and subsequently transferred to nitrocellulose membranes for 1 h at room temperature at 100 V, followed by blocking with 5% BSA in PBS for 1 h at room temperature. The membranes were then incubated overnight with agitation in a 1:5,000 dilution of Z0334 anti-GFAP rabbit polyclonal antibody (Dako, Carpinteria, CA), washed three times in 5% nonfat dry milk/0.1% (v/v) Tween 20/PBS (diluent buffer), incubated for 1 h at room temperature in a 1:15,000 dilution of an anti-rabbit 800 CW secondary antibody (Li-Cor Biosciences, Lincoln, NE), washed three times with diluent buffer and once with PBS, and then imaged on a Li-Cor Odyssey imager.

Sample processing for LC-MS/MS
Samples were processed by gel electrophoresis, excision of bands of interest, proteolytic digestion, and analysis by LC-MS/ MS. Four analyses were performed for each patient sample. The same amount of patient GFAP was used for each patient sample set, but the amount of [ 15 N]GFAP standard or the time of proteolysis varied (Table 1). Because the [ 15 N]GFAP standard comigrates with the intact patient GFAP, to provide an internal standard for the degraded GFAP bands, the same amount of [ 15 N]GFAP standard for each sample was electrophoresed in separate lanes and then combined with the corresponding excised band of degraded GFAP. Electrophoresis was performed by placing the samples in Laemmli sample buffer and running on a 4%-20% SDS-PAGE gel (Pierce) for 45 min at 150 V, followed by Coomassie staining for 30 min and destaining in 15% (v/v) methanol/10% (v/v) glacial acetic for 2 h at room temperature, as described previously (6). The intact GFAP bands, corresponding to ϳ45-55 kDa, and degraded GFAP bands, corresponding to ϳ35-45 kDa (Fig. 2), were excised separately from the patient samples, and each gel slice containing degraded GFAP was combined with the corresponding intact GFAP band of the [ 15 N]GFAP standard.
For trypsin digestion of the R79C and R416W patient samples, the gel slices were further destained overnight in 100 mM ammonium bicarbonate/50% acetonitrile. The following day, they were washed three times in 100 mM ammonium bicarbonate, followed by dehydration in 100% acetonitrile. The fully dried gel slices were then digested at 37°C for 18 or 40 h (Table 1) with sequencinggrade trypsin (Promega, Madison, WI) with a 1:20 trypsin:protein ratio, determined by comparing the stained gel region with that for 1.5 g of BSA run on the same gel.
For chymotrypsin digestion of the R239H patient samples, the gel slices were further destained overnight in 50 mM ammonium bicarbonate/50% acetonitrile, washed three times in 100 mM Tris-HCl and 10 mM CaCl 2 (pH 8.0), followed by dehydration in 100% acetonitrile. The fully dried gel slices were then digested at 25°C for 48 or 72 h with sequencing-grade chymotrypsin (Promega), starting with a 1:10 chymotrypsin:protein ratio. Fresh aliquots of chymotrypsin at the same 1:10 ratio were added every 24 h.
Following proteolysis, the digestion solutions were transferred to separate tubes, and the gel slices were extracted further in buffer consisting of 5% formic acid/50% acetonitrile for 1 h at room temperature. The extraction buffer was then combined with the corresponding peptide digest solution, vacuumcentrifuged to dryness, and analyzed after being resuspended in 0.1% formic acid as the ion-pairing agent. The tissues analyzed were from typical cases of the infantile form of AxD that have been reported previously as indicated. The R79C and R239H tissues were obtained from the University of Maryland Brain and Tissue Bank (UMB BTB). The R79C sample was a 1-cm-thick coronal section of parietal cerebrum from section 12R, produced by sectioning protocol 1. The R239H sample was frontal neocortical gray matter from section 3L, produced by sectioning protocol 2. The R416W tissue was a small fragment of neocortex and underlying white matter provided by James Goldman (Columbia Medical School); its post-mortem interval (PMI) is unknown. Although the exact locations of the tissues excised for analysis are not known, information about their Rosenthal fiber content is provided in Ref. 6, which used adjacent samples in a proteomics study of Rosenthal fibers. A fraction enriched for Rosenthal fibers contained 87% of the total GFAP in the R416W sample, 73% in the R79C sample, and 3% in the R239H sample, whereas 1% to 8% was observed in controls. Thus, most of the GFAP analyzed here for the R416W and R79C samples was presumably in Rosenthal fibers, but little if any of these aggregates were present in the R239H sample.

Instability of mutant GFAP in Alexander disease patients Targeted LC-MS/MS acquisition
Peptides were loaded onto a 15 cm ϫ 75 m ChromXP C18-CL 3-m, 300 Å cHiP Nanoflex System (Eksigent, Foster City, CA) with a 250 nl/min flow rate and eluted using a 0%-50% acetonitrile/0.1% formic acid gradient over 30 min. Data were acquired using a TripleTOF 5600 system (Sciex) with an ion spray voltage of 2.3 kV, a declustering potential of 60 V, curtain gas of 20 p.s.i., nebulizer gas of 10 p.s.i., and an interface heating temperature of 120°C. MS survey scans were acquired for 250 ms from 300 -1250 m/z, and product ion scans targeting unlabeled and 15 N-labeled GFAP peptides were collected with an accumulation time of 100 ms from 100 -2,000 m/z.


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RNA-seq analysis of the salivary glands and midgut of the Argasid tick Ornithodoros rostratus

Ornithodoros rostratus is a South American argasid tick which importance relies on its itchy bite and potential as disease vector. They feed on a wide variety of hosts and secrete different molecules in their saliva and intestinal content that counteract host defences and help to accommodate and metabolize the relatively large quantity of blood upon feeding. The present work describes the transcriptome profile of salivary gland (SG) and midgut (MG) of O. rostratus using Illumina sequencing. A total of 8,031 contigs were assembled and assigned to different functional classes. Secreted proteins were the most abundant in the SG and accounted for ~67% of all expressed transcripts with contigs with identity to lipocalins and acid tail proteins being the most representative. On the other hand, immunity genes were upregulated in MG with a predominance of defensins and lysozymes. Only 10 transcripts in SG and 8 in MG represented ~30% of all RNA expressed in each tissue and one single contig (the acid tail protein ORN-9707) represented ~7% of all expressed contigs in SG. Results highlight the functional difference of each organ and identified the most expressed classes and contigs of O. rostratus SG and MG.

Argasid (soft) and ixodid (hard) ticks are hematophagous arthropods that feed on a wide variety of hosts. They are of medical and veterinary importance due to the blood spoliation and transmission of pathogens that cost significant financial losses worldwide in the animal industry 1,2 .
Unlike long term feeding hard ticks, in which feeding process can take up to two weeks, the soft ticks normally feed in minutes. In addition, while adults of hard ticks feed only once, adults of soft ticks are intermittent feeders, as they can take bloodmeal more than 10 times during their lifetime. As in all blood-sucking arthropods, to have a successful bloodmeal, ticks should bypass haemostatic, inflammatory and immune responses in the host's skin 3 . This task is carried mainly by the salivary glands that produce and secrete several bioactive compounds into the host via saliva. The midgut also plays a key role in hematophagy. In addition to acting on the management of blood meal (storage and digestion) and protection against host immunity and pathogen infections, it has also been shown that the behaviour of blood in its lumen can affect the ingestion rate of the arthropod 4,5 . As some blood-sucking arthropods ingest with host blood part of the released saliva, it is likely that some of the biological activities present in the digestive system are due to salivary molecules. Digestion in ticks occurs intracellularly, inside lysosomal vesicles of midgut enterocytes, at acidic pH 6 . It goes through several modifications to accommodate and metabolize the relatively large amount of blood that it receives upon feeding. Although blood bolus volume is significantly reduced because of water excretion by the tick salivary (hard ticks) or coxal (soft ticks) glands, a tick will increase in total volume and mass by many times its original size and weight during a meal.
In hard ticks, which can remain attached for several days to the host to complete a blood meal, several thousands of unique contigs have being described in extensive sequencing transcriptomes 7 , which is believed to be the highest variety of proteins produced at the salivary glands among hematophagous arthropods. To study the Results and Discussion transcriptome overview. Sequencing of SG and MG libraries produced 22,395,831 Illumina reads. De novo assembly generated 40058 putative contigs that were further selected down to 8031 contigs based on the presence of an ORF and any similarities to other proteins found in the Refseq invertebrate and Acari databases deposited at NCBI's Genbank or at SwissProt database. The presence of a signal peptide was also considered as an additional positive selection criteria. Contig mean size in the final assembly is 1449.43 nucleotides long (largest contig is 29079 bases and the smallest is 155 bases). BUSCO analysis to access the transcriptome quality show complete BUSCOs of 85.6% (75.5% single-copy orthologs and 10.1% duplicated orthologs), 2.1% fragmented genes and 12.3% missing orthologs. Contigs were classified into six major categories namely housekeeping, secreted, immunity, transposable elements, viral and unknown (Supplementary Table 1). To visualize the differential expression of contigs, sequences were analysed using normalized transcripts per million (TPM). Such normalization reduced the biases caused by reads with few sequences and enables a more trustworthy comparison of contigs expression. The contigs classified as secreted were the most abundant with 886 contigs and 46.51% of the TPM, followed by housekeeping genes which had more contigs (5,325 contigs) but lower abundance (35.54%) of the TPM. Immunity related reads were assembled into 127 contigs and represented 8.41% of the TPM, while reads of the unknown category were assembled in 1,528 contigs but had lower levels of expression and their total TPM represented 9.29% of the library. Viral and transposable elements represented together 202 contigs (TPM of 2.51%) (Supplementary Table 1).
Housekeeping related transcripts accounted for almost 2/3 of the contigs, but the TPM corresponded to only 22.80% in the SG and 40.41% in the MG. Secreted transcripts were considerably more representative in the SG (717 contigs) but representing 66.72% of the TPM, while in the MG, the 596 contigs identified represented only 18.38%. On the other hand, immunity related transcripts had similar levels of contigs in both tissues but were ~2-fold more abundant in the MG in comparison to the SG (TPM of 3.00% and 1.06%, respectively). Other groups (viral, transposable element and unknown) were more abundant in MG than in SG, with an emphasis on transcripts with unknown function that had a high number of contigs (>1400) and were ~3-fold more representative in MG than in SG (Fig. 1). the topmost abundant transcripts in sG and MG. To find the main transcripts in each tissue, we ordered contigs by abundance (TPM) and evaluated those that, together, contributed to ~30% of all mRNA present in each tissue (Tables 1 and 2). In both cases, it is intriguing that so few transcripts (10 in SG and 8 in MG) could represent ~30% of all mRNA in each tissue. Nine out of the ten most expressed transcripts in SG belongs to the Secreted proteins group, distributed mostly in two functional classes -Acid Tail Proteins and Lipocalins (three transcripts each). The two most expressed SG transcripts were acid tail proteins (ORN-9707 and ORN-8689) representing more than 10% of all transcripts in SG (Table 1). Our data suggests that these transcripts are SG specific (we found no reads in the MG library -Supplementary dataset). The Lipocalins were also abundant with three contigs representing ~7% of the total SG TPM. The transcript ORN-13158 is the only secreted transcript with unknown function among the top ten expressed transcripts. However, BLAST and evolutionary analysis (Supplementary Fig. 1) strongly suggests that this transcript belongs to the 7DB-family of proteins and might be misannotated in the final transcriptome assembly.
On the other hand, the MG had two immunity and two housekeeping transcripts among the most expressed, while the SG had none of those categories. Remarkably, a single defensin (ORN-7204) represents almost 12% of all transcripts in MG (total read count also suggests that this is the most abundant transcript in the whole dataset -see Supplementary dataset). Our data suggest that this defensin is not expressed in SG at all, playing a specific role in MG. It is also worth to mention the level of expression of a Ferritin (ORN-7176) in the MG, which is ~19x more abundant in MG than in SG.
Functional coherence and abundance of transcripts by functional class in salivary gland and midgut. To globally visualize the families of contigs expressed in each tissue, we classified the transcripts of each major category into putative protein functional classes and evaluated the relative abundance of transcripts for each class in each tissue (Supplementary Tables 2-4). The Housekeeping contigs were attributed to 21 classes, while Secreted and Immunity contigs were attributed to 40 and 14 functional classes, respectively. Here, we briefly comment interesting findings in each major class.
Housekeeping. The TPM abundance for the housekeeping classes was relatively similar between tissues ( Fig. 2 and Supplementary Table 2). The classes signal transduction and transcription Machinery were the ones with more contigs (1076 and 644, respectively) and the most abundant (~20% of all housekeeping TPM). The classes energy metabolism, protein synthesis machinery and protein modification were also among the six most abundant in both tissues. Storage transcripts were also abundant in the MG while protein export transcripts were abundant in the SG.  Table 3). SG presented TPM almost 3-fold higher than the MG (692,248.44 × 258,101.68). It is remarkable that the number of hypothetical secreted proteins contigs is similar in both tissues (385 and 359 in SG and MG, respectively), but the overall abundance of such transcripts is considerably lower in the SG (~13%) in comparison to the MG (~46%). Acid tail proteins and lipocalins seems to have an important role in SG, since together these proteins represented more than 50% of the total SG TPM. On the other hand, the most abundant secreted protein class in MG was the cytotoxin-like family, which corresponded to ~25% of all the secreted TPM. A notable difference was also seen among the abundance of enzymes. SG had high levels of metalloproteases  www.nature.com/scientificreports www.nature.com/scientificreports/ while cathepsins, cysteine proteases and serine proteases were more abundant in MG. Concerning the protease inhibitors, cystatins and proteins with Kunitz and Kazal domains were abundant in both tissues, while serine protease inhibitors and thyropin were also more represented in the MG (both with TPM above 1.5%).
Immunity. The number of immunity related contigs in each tissue was relatively similar, but the total TPM was ~15-fold higher in MG than in SG. Again, remarkable abundance differences were seen among the functional classes between tissues ( Fig. 2 Table 4). ML-domain proteins in the SG and defensins in the MG are by far the most abundant immunity related transcripts in each tissue (53% and 78%, respectively). A single defensin transcript (ORN-7204) was responsible for ~45% of all immunity related transcripts in the MG. In addition to proteins with ML-domains, other four classes were well represented in the SG, as the ficolin/ixoderin, microplusins, gamma-interferon inducible lysosomal thiol reductase, cytokine associated and microplusins (which, together, accounts for >90% of immunity transcript abundance in SG). On the other hand, ~95% of the abundance of all immunity related transcripts in MG belonged to defensins, lysozymes and cytokine-associated proteins.

Differential expression in salivary gland and midgut.
To have an insight in differential expression, the mean expression level (mean z-score) for each functional class in each tissue was evaluated. Heatmap analysis showed clear differences in the overall expression profile between SG and MG for secreted and immunity protein families, while there were no differences in the expression profile for housekeeping functional classes (Fig. 3). We found suggestive evidence of expression compartmentalization for some enzyme and protease inhibitor classes between organs. Metalloproteases and TIL-domain proteins were practically exclusive of the SG, while the cysteine proteases and thyropin were virtually only expressed in the MG.
Expression analysis of secreted proteins suggests that genes from eight other functional classes were upregulated (z-score > 2) in SG and downregulated in MG (z-score < −2), such as lipocalins, acid tail proteins, 7DB-family proteins, 7-cys domain proteins, calcitonin/adrenomedulin proteins, 17 kDa, 8.9 kDa and 5.3 kDa secreted proteins. On the other hand, 13 kDa secreted proteins, cytotoxin-like and evasin have expression clearly upregulated in MG and downregulated in SG.
For immunity related transcripts we observed expression differences in defensins and ML-domain proteins which were considerably more expressed in MG than in SG, while G-interferon inducible thiol reductase seemed to be differentially more expressed in SG. It is important to note the case of ML-domain proteins. Heatmap analysis suggests that transcripts of this family are, on average, more expressed in MG than in SG (z-scores of 0.74 and −1.10, respectively). On the other hand, transcript abundance based on TPM shows that ML-domain transcripts represent ~53% of all immunity related transcripts in SG, while it accounts for less than 1% of the immunity transcripts in MG. We found a total of six ML-domain transcripts in the O. rostratus transcriptome. All six are expressed in the MG (TPM values from 4.62 to 528.37 with three of them with TPM above 200). On the other hand, only three ML-domain transcripts are expressed in SG (two of them with TPM bellow 40) and the ORN-33642 has a TPM of 5768.55. This single ML-domain transcript account for ~53% of all immunity related genes transcribed in the SG.

Discussion on the main classes of secreted protein.
Lipocalins. Proteins of the lipocalin family are known mainly for their role as carriers and ligands of soluble molecules, especially hydrophobic molecules 20 . They are expressed in the SG of insects and ticks and can perform a variety of functions. Lipocalins are particularly abundant in triatomine transcriptomes where they usually represent more than 50% of the secreted proteins, reaching up to 90% in some species 21 . In ticks, lipocalins are produced in the SG of all studied species, with described functions that varies from interfering with host hemostasis 22,23 , inhibition of complement factor C5 activation 24 , platelet and neutrophil aggregation inhibition 25,26 and inhibition of histamine-mediated inflammation 27 . The family diversification in ticks has been observed, with the number of lipocalins genes ranging between 34 to 52 25,28 .
Almost 31% of all SG secreted proteins corresponded to lipocalins, which demonstrates the importance of these molecules. Of the 48 lipocalins found in O. rostratus, 25 of them seem to be differentially expressed in the SG (z-scores > 1.8; Supplementary dataset), while 32 of them were not expressed in MG at all (TPM = 0.0). Phylogenetic analysis suggests at least four expansions of this family in O. rostratus (Fig. 4, clades LI to LIII and MIII). Our phylogenetic analysis suggests at least six O. rostratus lipocalins belongs to the moubatin family (Fig. 4), and these proteins could be classified in three distinct groups (MI to MIII). One sequence in specific  www.nature.com/scientificreports www.nature.com/scientificreports/ (ORN-18500) is more related to moubatins from the MI clade. Functional studies showed that moubatins from this clade have roles in the inhibition of the activation of C5 of vertebrate complement system 24 ; platelet aggregation inhibition by scavenging thromboxane A 2 and neutrophil aggregation inhibition by scavenging leukotriene B 4 25,29 . Other two contigs (ORN-4729 and ORN-21881) seems to be related to TSGP-4 family (tick salivary gland peptide 4) which are implicated in the scavenging of cysteinyl rich leukotrienes 26   www.nature.com/scientificreports www.nature.com/scientificreports/ and histamine indicates they suppress inflammation during blood feeding 27,[30][31][32] . Interestingly, the lipocalin ORN-18741 (related to SHBP-I) is among the top most expressed genes in O. rostratus salivary glands (Table 1). On the other hand, the other two highly expressed lipocalins (ORN-7555 and ORN-4748) does not seems to belong to any lipocalin family with known function.
Acid tail transcripts. Acid tail proteins are molecules with unknown function that shares a common characteristic of having a predominance of glutamate. Acid tail proteins were overexpressed only in SG. Overexpression of acid tail proteins was also reported in I. ricinus, with expression levels > 10-fold higher in SG compared to the MG 33 . From the seven acid tail transcripts of O. rostratus, four showed differential expression in SG with z-scores > 2.0 (Supplementary dataset). All the seven acid tail proteins presented a conserved PFAM domain named TSGP1 (tick salivary peptide group 1). One acid-tail protein, ORN-9707, caught our attention as the most abundant transcript in SG. This transcript showed a z-score of 3.09, corresponding for > 7% of all SG transcripts, and for ~10% of all secreted transcripts. Phylogenetic analysis suggests that ORN-9707 is a duplication of another acid tail protein (ORN-32876) and a possible ortholog of O. brasiliensis acid tail proteins JAT78798 and JAT78738 (Fig. 5). The acid tail protein ORN-32876 showed a z-score = 1.80. Such relationship could indicate a recent duplication with possible selection for the ORN-9707. However, our data do not allow us to speculate further.
Basic Tail Proteins. Basic tail proteins represented 6.11% and 1.38% of the secreted transcripts in the SG and MG, respectively (Supplementary Table 2). The name "basic tail" is related to a cluster of basic amino acids at the carboxy terminal end of the protein. This family was already reported for both soft and hard ticks 34 . In argasids, this class was also abundant in the saliva of O. coriaceus and O. parkeri 11,12 . From the nine O. rostratus basic tail contigs, ORN-24121 and ORNSIGP-21115 seems to be differentially expressed in MG (z-scores > 1.8) and  www.nature.com/scientificreports www.nature.com/scientificreports/ presented similarities with RNA recognition motif-containing protein and secreted PAPA repeat protein, respectively. In SG, three basic tail transcripts showed z-scores > 2.0 (ORN-21032; ORN-4112 and ORN-10496).
Mucins. Similarly, to basic tail proteins, mucins were overexpressed in both organs. We found 28 contigs and two of them presented high level of expression in SG (ORNSIGP-6290) or MG (ORNSIGP-25718). Mucins are proteins with a C-terminal serine-threonine rich domain capable of binding to N-acetyl-galactosamine 35 . After glycosylation, mucins are incorporated to a mucus produced by the saliva of ticks that has a possible function as a coating layer and, consequently, protection of buccal parts during blood feeding, besides the possible interaction with proteins of the host extracellular matrix 34 . The presence of salivary mucins has been observed in sialomes of other hematophagous arthropods, among them species of ticks of the genus Ornithodoros as O. parkeri and O. coriaceus 11,12 .
Enzymes. High number of enzyme transcripts is a common finding in the SG and MG transcriptomes of all studied ticks 11,12,33,[36][37][38] . In O. rostratus, enzymes were highly represented in both tissues and a notorious distinction of classes were seen between SG and MG. Metalloproteases was the most expressed protein subclass in the SG (Fig. 3) while cysteine proteases, cathepsin and serine proteases were more expressed in the MG. Metalloproteases are proteolytic enzymes that require a metal ion (Zn 2+ ) for catalysis reactions and are very abundant in the venom of animals 39 . The main purpose of metalloproteases in the tick SG would be the degradation of the host tissues at the bite site to form the feeding pool. This class of enzyme is also active in the degradation of fibrinogen and fibrin thus avoiding blood coagulation and maintaining the fluidity of blood 40 . They have a key role in tick feeding and have been described as a potential antigen to be used as a tick vaccine 41 .
On the other hand, the main purpose of the enzymes produced in the MG is the digestion of the diet. Digestion in ticks occurs intracellularly in lysosomes 6 by a multi enzymatic cascade 42 . Several midgut proteases of ixodids 43 and argasids of the genus Ornithodoros 44,45 have already being identified. Three enzyme classes were highly expressed in the MG: cathepsin, serine proteases and cysteine proteases. Those protease classes were also upregulated in the midgut of O. erraticus 24 hrs after blood ingestion 16 . Among the O. rostratus cathepsins, types B, D, I and F were described, and the most abundant ones were two transcripts with 71% identity to I. scapularis Cathepsin B endopeptidase and with 57% identity to I. ricinus cathepsin D.
Protease inhibitors. Secreted protease inhibitors were also well represented in the O. rostratus SG and MG. Their high expression levels reflect the importance of this class involved in blocking (or reducing) host reparatory responses, signal pathways or activation cascades triggered by the feeding process. We found 24 protease inhibitors transcripts containing kunitz domains (five of them with two kunitz domains). Protease inhibitors with a kunitz domain are usually responsible to block serine proteases involved in host hemostasis 46 . Kunitz-type domain is characterized by short length (usually 50-60 amino acids), low molecular weight (6 kDa) and six invariantly spaced cysteine residues 47 . Sequences for proteins containing kunitz domains are frequently and abundantly found in tick sialotranscriptomes 11,34,36 . Saliva of O. moubata and O. kalahariensis contain, respectively. ornithodorin and savignin proteins presenting two kunitz domains that are inhibitors of thrombin activity 48,49 . In the hard tick I. scapularis there is two proteins, ixolaris and penthalaris, with kunitz domains that inhibit blood clotting by binding to factor Xa 50,51 . Other three protease inhibitor proteins were characterized as papilins. www.nature.com/scientificreports www.nature.com/scientificreports/ Papilins have been implicated in interacting with metalloproteases in Drosophila melanogaster and Caenorhabditis elegans as essential for embryonic development 52 . The presence of papilins in SG suggests a possible new role for these proteins.
Most of the other protease inhibitor contigs belonged to the classes of the serine protease inhibitors (Serpins), cystatins and thyropin and were all highly expressed in the MG. Those were also the main classes of protease inhibitors found in O. mimon MG 15 . Serpin domains were also found in MG of O. moubata 9 . Serpins have important roles in the modulation of tick-host interactions, such as suppression of host's immune system and blood coagulation 53,54 . In the MG, those activities should be suppressed in the ingested blood in order to accommodate the blood ingested upon feeding 4 and avoid immune responses against the gut wall 55 . Cystatins are involved in different biological processes, such as modulation of endogenous proteolysis, immune response and blood feeding 56 . In O. moubata, the cystatins have been described in the salivary glands and midgut, presenting important role in evasion of the host's immune system and midgut physiology. A salivary cystatin from O. moubata (OmC2) is responsible for suppressing host's immune system, inhibiting cathepsins and suppressing antigen presentation by dendritic cells, reducing the production of proinflammatory cytokines and T CD4 + cell proliferation 57 . Midgut cystatins of O moubata are inhibitors of cathepsins B and H, involved in the regulation of proteolytic targets in the tick digestive system and modulation of host immune response during blood feeding 58 . One contig coding a protein from the thryropin class was highly expressed in O. rostratus MG (ORN-37269; z-score of 2.18) (Supplementary table Excel). Thyropin is presented as a repeat of the amino terminal region of human thyroglobulin. These domains are normally described as cysteine-protease inhibitors and binding partners of heparin 59 . They have been found in transcriptomes of ticks' SG and MG, including the Ornithodoros genus 15,60 . Their function for ticks, however, remains unknown.
Discussion on the main classes of immunity transcripts. Six classes from the immunity related contigs were considered more representative in the SG or MG, which contained transcripts mostly involved in pathogen/antigen recognition, signalling of immune pathways or direct antimicrobial activity. Microplusins and cytokine-Associated were the classes with abundant contigs in both tissues, while ML-domain proteins and ficolins/ixodegrins were more abundant in the SG and defensins and lysozymes were more abundant in the MG (Fig. 2 and Supplementary Table 4). Interestingly, we observed that only three and four contigs accounted for ~75% (SG) and ~95% (MG) of the overall abundance of immunity transcripts in each tissue.
In the SG, the most abundant immunity related transcript was a ML-domain protein with homology (>70% amino acid identity) to a salivary lipid interacting protein from O. parkeri and O coriaceus. ML-domain proteins interact with specific lipids and have roles in the recognition of pathogens 61 . The other two abundant contigs in O. rostratus SG were a lectin of the ficolin class with 67% identity to O. moubata dorin M precursor and a microplusin highly expressed in both tissues (z-scores of 1.65 and 2.01 in SG and MG, respectively). The microplusin (ORN-44579) was homologous (>70% amino acid identity) to a hebrain-like protein from O. coriaceus and O. parkeri. Hebrain, from the ixodid tick A. hebraeum 62 , and microplusin, from R. microplus 63 , are antimicrobial peptides. Microplusin was shown to be a copper chelating peptide active against fungi and bacteria 64 .
Among the four contigs highly expressed in the MG, one (ORN-7204) showed ~80% amino acid identity with O. moubata defensin A. This transcript represented more than 75% of the total abundance of immunity related transcripts in MG and is the most abundant transcript in the MG transcriptome (12% of MG TPM). Defensins are one of the most important and widespread antibacterial peptides from invertebrates 65 . The mature defensins from O. moubata contain six conserved cysteine residues and have high identity to other scorpion defensins, but at a lesser degree to defensins of dragonflies and other ancient arthropods. In O. moubata, the defensin mRNA expression was up-regulated by blood-feeding 66 .
The second most abundant immunity related transcript in MG is a lysozyme precursor (ORNSIGP-6324). Together with the defensin, those two contigs accounted for almost 90% of the MG Immunity-related TPM. Lysozymes are bacteriolytic peptides which are part of the innate immunity of several groups of animals and plants 67 . The structure of this lysozyme indicates it is a typical Tick Gut Lysozyme (TGL), which are peptides also involved in digestion and with structure characteristic of c-type lysozymes that contain eight cysteine residues 68 . TGLs are also characterized by the presence of a histidine at position 52 which replaces the highly conserved tyrosine found in most c-type lysozymes. As defensins, the expression of TGL in the MG is also stimulated by the ingestion of blood in O. moubata 69 .
The other two of the most expressed immunity transcripts were classified as putative tumour necrosis factor receptor attributed to the Cytokine-associated class. TNF and their receptors are involved in immunity processes once they participate in several cellular signalling pathways that induce cell proliferation, survival, and differentiation processes 70 .
Final insights in the O. rostratus transcriptome. The analysis of the salivary gland and midgut transcriptome of O. rostratus can give us some insights on: (i) the role of each organ in the tick physiology; (ii) similarities and differences of both organs on gene expression; (iii) key-role genes for a successful bloodmeal; (iv) new targets for further functional studies and; (v) possible targets for tick vaccines. The first and most clear observation is the functional coherence of each organ. Our data shows that SG excels in the production of secreted proteins, especially those correlated to maintain homeostasis and recognition of antigens during blood-feeding (e.g. lipocalins, metalloproteases, kunitz-domain proteins, apyrases). On the other hand, besides blood digestion, the MG intensively produces immunity related genes (e.g. Defensins, lysozymes) suggesting the importance to control bacteria growth during blood digestion. A few genes caught our attention due to their high abundance in each tissue. Lipocalins and acid-tail proteins are the most predominant transcripts in the SG. While the role of lipocalins to maintain homeostasis during blood ingestion is well known, the role of acid-tail proteins in this process (and one acid-tail protein is the most abundant transcript in SG) need further studies. In MG, a single www.nature.com/scientificreports www.nature.com/scientificreports/ defensin-A is the most abundant gene in all transcriptome and, although the role of defensins is well known in other arthropods, new studies on the role of this gene in O. rostratus are needed. Such observations indicate those genes are extremely important for tick feeding and may constitute important targets to be focused on control programs such as vaccine antigens or drug targets.
The current study brings new knowledge on salivary and intestinal molecules of argasid ticks, specially from O. rostratus, which gene information in databases are scarce. Although this study has its limitations (e.g. expression is based in a pool of individuals in different physiological stages and lack of samples for statistical analysis on differential expression), the intention was to show an overview of molecules produced in the SG and MG of O. rostratus. The information provided by this study will help in the design of future experiments to better understand the role of the highly expressed genes and to improve our knowledge on the biology O. rostratus and soft ticks. salivary gland and midgut isolation. SG and MG were dissected from unfed and one and three-day fed fourth instar nymphs (two of each). Tick lateral cuticule was cut with micro scissors, the superior cuticule was folded up front and the interior organs of ticks were exposed. Tissues were collected with forceps, rinsed by immersion in saline solution (NaCl 0.9%), transferred to tubes containing 50 µL of RNA latter (Sigma Aldrich) and stored at 4 °C until use.
sequencing. Samples  Bioinformatic tools and procedures. Custom bioinformatic analysis were describe elsewhere 72 with modifications. Concisely, low quality reads were trimmed from Fastq files (<20) and contaminating adapter primer sequences removed. De novo assembly from reads was a result of Abyss (using k parameters from 21 to 91 in 5-fold increments) 73 and SOAP de novo-trans 74 assemblers. The combined fasta files were further assembled using an iterative blast and CAP3 pipeline as previously described 75 . The final transcriptome quality was quantitatively assessed using Benchmarking Universal Single-Copy Orthologues (BUSCO) v 3.0 76 against the arthropod database v9.0 with default parameters. Coding sequences (CDs) were extracted based on the existence of a signal peptide in the longer open reading frame (ORF) and by similarities to other proteins found in the Refseq invertebrate database from the National Center for Biotechnology Information (NCBI), proteins from Acari deposited at NCBI's Genbank and from SwissProt. Only contigs containing open reading frame or any similarity to sequences in the chosen databases were selected for further analysis (please see references 72,75 for details). Reads for each library were mapped on the deducted CDs using blastn with a word size of 25, 1 gap allowed and 95% identity or better required. Up to five matches were allowed if and only if the scores were the same as the largest score. Mapping of the reads was also included in the Excel spreadsheet. Values of the reads per kilobase of transcript per million mapped reads (TPM) 77 for each coding sequence were also mapped to the spreadsheet. Automated annotation of proteins was based on a vocabulary of nearly 350 words found in matches to various databases, including Swissprot, Gene Ontology, KOG, Pfam, and SMART, Refseq-invertebrates and the Acari subset of the GenBank sequences obtained by querying acari [organism] and retrieving all protein sequences. Raw reads were deposited on the Sequence Read Archive (SRA) of the National Center for Biotechnology Information (NCBI) under bioproject ID PRJNA270484. This Transcriptome Shotgun Assembly project has been deposited at DDBJ/ EMBL/GenBank under the accession GCJJ00000000.1. Differential expression analysis. For differential expression analysis, TPM values were transformed into z-scores, using data from both tissues to calculate the mean Log. For heatmap analysis, we calculated the mean z-score for each protein class based on their putative role (e.g. protein synthesis machinery, amino-acid metabolism, detoxification, etc). Heatmap of protein classes was constructed using Heatmapper 78 . Since we were interested only in having an insight in contig abundancy, we did not perform in depth differential expression analysis using common approaches (e.g. DEGseq or edgeR). Thus, we do not present and discuss any DE contigs in terms of statistical significance. evolutionary analysis. Protein sequences from other organisms were obtained at NCBI and aligned with Muscle 79 . Evolutionary analyses were conducted in MEGA7 80 . The evolutionary history was inferred using the Neighbour-Joining method (10.000 replicates; pairwise deletion) 81 . The evolutionary distances were computed using the Poisson correction method and are in the units of the number of amino acid substitutions per site. All accession numbers are shown in the respective figures.