Thymosin beta-4 (Tβ4) is known to induce hair growth and hair follicle (HF) development; however, its mechanism of action is unknown. We generated mice that overexpressed Tβ4 in the epidermis, as well as Tβ4 global knockout mice, to study the role of Tβ4 in HF development and explore the mechanism of Tβ4 on hair growth. To study Tβ4 function, we depilated control and experimental mice and made tissue sections stained with hematoxylin and eosin (H&E). To explore the effect of Tβ4 on hair growth and HF development, the mRNA and protein levels of Tβ4 and VEGF were detected by real-time PCR and western blotting in control and experimental mice. Protein expression levels and the phosphorylation of P38, ERK and AKT were also examined by western blotting. The results of depilation indicated that hair re-growth was faster in Tβ4-overexpressing mice, but slower in knockout mice. Histological examination revealed that Tβ4-overexpressing mice had a higher number of hair shafts and HFs clustered together to form groups, while the HFs of control mice and knockout mice were separate. Hair shafts in knockout mice were significantly reduced in number compared with control mice. Increased Tβ4 expression at the mRNA and protein levels was confirmed in Tβ4-overexpressing mice, which also had increased VEGF expression. On the other hand, knockout mice had reduced levels of VEGF expression. Mechanistically, Tβ4-overexpressing mice showed increased protein expression levels and phosphorylation of P38, ERK and AKT, whereas knockout mice had decreased levels of both expression and phosphorylation of these proteins. Tβ4 appears to regulate P38/ERK/AKT signaling via its effect on VEGF expression, with a resultant effect on the speed of hair growth, the pattern of HFs and the number of hair shafts.

Although the various biological roles of thymosin β4 (Tβ4) have been studied widely, the effect of Tβ4 and Tβ4-expressing cells in the liver remains unclear. Therefore, we investigated the expression and function of Tβ4 in chronically damaged livers. CCl4 was injected into male mice to induce a model of chronic liver disease. Mice were sacrificed at 6 and 10 weeks after CCl4 treatment, and the livers were collected for biochemical analysis. The activated LX-2, human hepatic stellate cell (HSC) line, were transfected with Tβ4-specific siRNA and activation markers of HSCs were examined. Compared to HepG2, higher expression of Tβ4 in RNA and protein levels was detected in the activated LX-2. In addition, Tβ4 was up-regulated in human liver with advanced liver fibrosis. The expression of Tβ4 increased during mouse HSC activation. Tβ4 was also up-regulated and Tβ4-positive cells were co-localized with α-smooth muscle actin (α-SMA) in the livers of CCl4-treated mice, whereas such cells were rarely detected in the livers of corn-oil treated mice. The suppression of Tβ4 in LX-2 cells by siRNA induced the down-regulation of HSC activation-related genes, tgf-β, α-sma, collagen, and vimentin, and up-regulation of HSC inactivation markers, ppar-γ and gfap. Immunofluorescent staining detected rare co-expressing cells with Tβ4 and α-SMA in Tβ4 siRNA-transfected cells. In addition, cytoplasmic lipid droplets were observed in Tβ4 siRNA-treated cells. These results demonstrate that activated HSCs expressed Tβ4 in chronically damaged livers, and this endogenous expression of Tβ4 influenced HSC activation, indicating that Tβ4 might contribute to liver fibrosis by regulating HSC activation.

Thymosin beta-4 (Tβ4) is an ubiquitous multi-functional regenerative peptide, related to many critical biological processes, with a dynamic and flexible conformation which may influence its functions and its subcellular distribution. For these reasons, the intracellular localization and trafficking of Tβ4 is still not completely defined and is still under investigation in in vivo as well as in vitro studies. In the current study we used HepG2 cells, a human hepatoma cell line; cells growing in normal conditions with fetal bovine serum expressed high levels of Tβ4, restricted to the cytoplasm until 72 h. At 84 h, a diffuse Tβ4 cytoplasmic immunostaining shifted to a focal perinuclear and nuclear reactivity. In the absence of serum, nuclear reactivity was localized in small granules, evenly dispersed throughout the entire nuclear envelop, and was observed as earlier as at 48 h. Cytoplasmic immunostaining for Tβ4 in HepG2 cells under starvation appeared significantly lower at 48 h and decreased progressively at 72 and at 84 h. At these time points, the decrease in cytoplasmic staining was associated with a progressive increase in nuclear reactivity, suggesting a possible translocation of the peptide from the cytoplasm to the nuclear membrane. The normal immunocytochemical pattern was restored when culture cells submitted to starvation for 84 h received a new complete medium for 48 h. Mass spectrometry analysis, performed on the nuclear and cytosolic fractions of HepG2 growing with and without serum, showed that Tβ4 was detectable only in the cytosolic and not in the intranuclear fraction. These data suggest that Tβ4 is able to translocate from different cytoplasmic domains to the nuclear membrane and back, based on different stress conditions within the cell. The punctuate pattern of nuclear Tβ4 immunostaining associated with Tβ4 absence in the nucleoplasm suggest that this peptide might be localized in the nuclear pores, where it could regulate the pore permeability.

The objective of this study was to investigate the expression and localisation of thymosin β4 (Tβ4) in the developing human heart. Tβ4 is a cardioprotective protein which may have therapeutic potential. While Tβ4 is an endogenously produced protein with known importance during development, its role within the developing human heart is not fully understood. Elucidating the localisation of Tβ4 within the developing heart will help in understanding its role during cardiac development and is crucial for understanding its potential for cardioprotection and repair in the adult heart.

A serious limitation of numerous antitumor drugs is the incapacity to penetrate solid tumors. However, addition of an RGD fragment to peptide drugs might solve this problem. In this study, we explored whether the introduction of a permeability-enhancing sequence, such as iRGD (CRGDK/RGPD/EC) fragments, would enhance the activity of thymosin alpha 1 (Tα1). The modified Tα1 (Tα1-iRGD) was successfully expressed and purified, and the in vitro assay showed that Tα1-iRGD presented a similar activity as Tα1 in promoting proliferation of mouse splenocytes. Meanwhile, cell adhesion analysis revealed that Tα1-iRGD exhibited more specific and greater binding with tumor cells compared with Tα1. Furthermore, the iRGD fragment evidently enhanced the basal ability of Tα1 to inhibit proliferation of cancer cells in vitro, particularly of mouse melanoma cell line B16F10 and human lung cancer cell line H460. Our findings indicated that the addition of an iRGD fragment increased the anti-proliferative activity of Tα1 against cancer cells by improving the ability of Tα1 to penetrate the tumor cells. This study highlighted the important roles of an iRGD sequence in the therapeutic strategy of Tα1-iRGD. Thus, Tα1-iRGD could be a novel drug candidate for cancer treatment.

Pulmonary hypertension (PH) is a progressive vascular disease of pulmonary arteries that impedes ejection of blood by the right ventricle. As a result there is an increase in pulmonary vascular resistance and pulmonary arterial pressure causing right ventricular hypertrophy (RVH) and RV failure. The pathology of PAH involves vascular cell remodeling including pulmonary arterial endothelial cell (PAEC) dysfunction and pulmonary arterial smooth muscle cell (PASMC) proliferation. Current therapies are limited to reverse the vascular remodeling. Investigating a key molecule is required for development of new therapeutic intervention. Thymosin beta-4 (Tβ4) is a ubiquitous G-actin sequestering protein with diverse biological function and promotes wound healing and modulates inflammatory responses. However, it remains unknown whether Tβ4 has any protective role in PH. The purpose of this study is to evaluate the whether Tβ4 can be used as a vascular-protective agent. In monocrotaline (MCT)-induced PH mouse model, we showed that mice treated with Tβ4 significantly attenuated the systolic pressure and RVH, compared to the MCT treated mice. Our data revealed for the first time that Tβ4 selectively targets Notch3-Col 3A-CTGF gene axis in preventing MCT-induced PH and RVH. Our study may provide pre-clinical evidence for Tβ4 and may consider as vasculo-protective agent for the treatment of PH induced RVH.

Thymosin beta-4 (Tβ4) is a ubiquitous protein with diverse functions relating to cell proliferation and differentiation that promotes wound healing and modulates inflammatory responses. The effecter molecules targeted by Tβ4 for cardiac protection remains unknown. The purpose of this study is to determine the molecules targeted by Tβ4 that mediate cardio-protection under oxidative stress.

Thymosin β(10) (Tβ(10)) regulates actin dynamics as a cytoplasm G-actin sequestering protein. Previously, we have shown that Tβ(10) diminishes tumor growth, angiogenesis, and proliferation by disrupting actin and by inhibiting Ras. However, little is known about its mechanism of action and biological function. In the present study, we establish a new gene therapy model using a genetically modified adenovirus, referred to as Ad.TERT.Tβ(10), that can overexpress the Tβ(10) gene in cancer cells. This was accomplished by replacing the native Tβ(10) gene promoter with the human TERT promoter in Ad.TERT.Tβ(10). We investigated the cancer suppression activity of Tβ(10) and found that Ad.TERT.Tβ(10) strikingly induced cancer-specific expression of Tβ(10) as well as apoptosis in a co-culture model of human primary ovarian cancer cells and normal fibroblasts. Additionally, Ad.TERT.Tβ(10) decreased mitochondrial membrane potential and increased reactive oxygen species (ROS) production. These effects were amplified by co-treatment with anticancer drugs, such as paclitaxel and cisplatin. These findings indicate that the rise in ROS production due to actin disruption by Tβ(10) overexpression increases apoptosis of human ovarian cancer cells. Indeed, the cancer-specific overexpression of Tβ(10) by Ad.TERT.Tβ(10) could be a valuable anti-cancer therapeutic for the treatment of ovarian cancer without toxicity to normal cells.

We identified a novel gene encoding a Bombyx mori thymosin (BmTHY) protein from a cDNA library of silkworm pupae, which has an open reading frame (ORF) of 399 bp encoding 132 amino acids. It was found by bioinformatics that BmTHY gene consisted of three exons and two introns and BmTHY was highly homologous to thymosin betas (Tβ). BmTHY has a conserved motif LKHTET with only one amino acid difference from LKKTET, which is involved in Tβ binding to actin. A His-tagged BmTHY fusion protein (rBmTHY) with a molecular weight of approximately 18.4 kDa was expressed and purified to homogeneity. The purified fusion protein was used to produce anti-rBmTHY polyclonal antibodies in a New Zealand rabbit. Subcellular localization revealed that BmTHY can be found in both Bm5 cell (a silkworm ovary cell line) nucleus and cytoplasm but is primarily located in the nucleus. Western blotting and real-time RT-PCR showed that during silkworm developmental stages, BmTHY expression levels are highest in moth, followed by instar larvae, and are lowest in pupa and egg. BmTHY mRNA was universally distributed in most of fifth-instar larvae tissues (except testis). However, BmTHY was expressed in the head, ovary and epidermis during the larvae stage. BmTHY formed complexes with actin monomer, inhibited actin polymerization and cross-linked to actin. All the results indicated BmTHY might be an actin-sequestering protein and participate in silkworm development.

Thymosin beta-4 (Tβ4) is a ubiquitous protein with many properties relating to cell proliferation and differentiation that promotes wound healing and modulates inflammatory mediators. The mechanism by which Tβ4 modulates cardiac protection under oxidative stress is not known. The purpose of this study is to dissect the cardioprotective mechanism of Tβ4 on H(2)O(2) induced cardiac damage.

Myocardial remodeling is a major contributor in the development of heart failure (HF) after myocardial infarction (MI). Integrin-linked kinase (ILK), LIM-only adaptor PINCH-1, and α-parvin are essential components of focal adhesions (FAs), which are highly expressed in the heart. ILK binds tightly to PINCH-1 and α-parvin, which regulates FA assembly and promotes cell survival via the activation of the kinase Akt. Mice lacking ILK, PINCH or α-parvin have been shown to develop severe defects in the heart, suggesting that these proteins play a critical role in heart function. Utilizing failing human heart tissues (dilated cardiomyopathy, DCM), we found a 2.27-fold (p<0.001) enhanced expression of PINCH, 4 fold for α-parvin, and 10.5 fold (p<0.001) for ILK as compared to non-failing (NF) counterparts. No significant enhancements were found for the PINCH isoform PINCH-2 and parvin isoform β-parvin. Using a co-immunoprecipitation method, we also found that the PINCH-1-ILK-α-parvin (PIP) complex and Akt activation were significantly up-regulated. These observations were further corroborated with the mouse myocardial infarction (MI) and transaortic constriction (TAC) model. Thymosin beta4 (Tβ4), an effective cell penetrating peptide for treating MI, was found to further enhance the level of PIP components and Akt activation, while substantially suppressing NF-κB activation and collagen expression--the hallmarks of cardiac fibrosis. In the presence of an Akt inhibitor, wortmannin, we show that Tβ4 had a decreased effect in protecting the heart from MI. These data suggest that the PIP complex and activation of Akt play critical roles in HF development. Tβ4 treatment likely improves cardiac function by enhancing PIP mediated Akt activation and suppressing NF-κB activation and collagen-mediated fibrosis. These data provide significant insight into the role of the PIP-Akt pathway and its regulation by Tβ4 treatment in post-MI.

Alzheimer's disease (AD) is a common amnestic cognitive impairment characterised by β-amyloid (Aβ) plaques deposit in the brain of the elderly. AD is a yet incurable disease due to its unknown exact pathogenesis and unavailability of effective remedies in clinical application. Thymosin β4 (Tβ4) is a housekeeping protein that plays important role in cell proliferation, migration and differentiation. It has the ability to protect and repair neurons however it is still unclear involvement in AD. Therefore, the aim of this study is to elucidate the role and mechanism of Tβ4 in mediating the improvement of AD. AD-like cell model was constructed in neuroblastoma cell line SH-SY5Y treated with Aβ. Overexpression of Tβ4 were done using lentivirus infection and downregulation through siRNA transfection. We performed western blot and flow cytometry to study the apoptosis and standard kits to measure the oxidative stress-associated biomarkers. There is significant increased in viability and decreased apoptosis in Tβ4 overexpression group compared to control. Furthermore, overexpression of Tβ4 suppressed the expression of pro-apoptotic markers such as Caspase-3, Caspase-8, and Bax meanwhile upregulated the expression of anti-apoptotic gene Bcl-2. Tβ4 alleviated oxidative damage by reducing MDA, LDH and ROS and increasing SOD and GSH-PX in Aβ-treated SH-SY5Y cells. We found that Tβ4 inhibit ERK/p38 MAPK pathway and intensify the expression of 5-HTR1A. Additionally, we showed that upregulation of 5-HTR1A dampened the Tβ4 to activate ERK signalling. In conclusion, our study revealed the neuroprotective role of Tβ4 in AD which may open up new therapeutic applications in AD treatment.

Thymosin beta-4 (Tbeta4) is a ubiquitous protein with many properties relating to cell proliferation and differentiation that promotes wound healing and modulates inflammatory mediators. We studied the effects of chronic administration of Tbeta4 on the skeletal and cardiac muscle of dystrophin deficient mdx mice, the mouse model of Duchenne muscular dystrophy. Female wild type (C57BL10/ScSnJ) and mdx mice, 8-10 weeks old, were treated with 150 microg of Tbeta4 twice a week for 6 months. To promote muscle pathology, mice were exercised for 30 minutes twice a week. Skeletal and cardiac muscle function were assessed via grip strength and high frequency echocardiography. Localization of Tbeta4 and amount of fibrosis were quantified using immunohistochemistry and Gomori's tri-chrome staining, respectively. Mdx mice treated with Tbeta4 showed a significant increase in skeletal muscle regenerating fibers compared to untreated mdx mice. Tbeta4 stained exclusively in the regenerating fibers of mdx mice. Although untreated mdx mice had significantly decreased skeletal muscle strength compared to untreated wild type, there were no significant improvements in mdx mice after treatment. Systolic cardiac function, measured as percent shortening fraction, was decreased in untreated mdx mice compared to untreated wild type and there was no significant difference after treatment in mdx mice. Skeletal and cardiac muscle fibrosis were also significantly increased in untreated mdx mice compared to wild type, but there was no significant improvement in treated mdx mice. In exercised dystrophin deficient mice, chronic administration of Tbeta4 increased the number of regenerating fibers in skeletal muscle and could have a potential role in treatment of skeletal muscle disease in Duchenne muscular dystrophy.

The actin-sequestering protein thymosin beta-4 (Tβ4) is involved in various cellular and physiological processes such as proliferation, motility, growth and metastasis. Nitric oxide (NO) promotes tumor invasiveness and metastasis by activating various enzymes. Herein, we investigated whether hypoxia-inducible NO regulates Tβ4 expression and cancer cell migration using HeLa cervical cancer cells. NO production and Tβ4 expression were increased in a hypoxic condition. The treatment with N-(β-D-Glucopyranosyl)-N2-acetyl-S-nitroso-D, L-penicillaminamide (SNAP-1), to generate NO, enhanced the transcription of Tβ4 and cancer cell migration. SNAP-1-induced cell migration was decreased by the inhibition of Tβ4 with small interference (si) RNA. In a hypoxic condition, treatment with N(G)-monomethyl-L-arginine (L-NMMA), nitric oxide synthase (NOS) inhibitor, reduced Tβ4 transcriptional activity, and hypoxia-inducible factor (HIF)-1α. Hypoxia-induced cancer cell migration was also decreased by L-NMMA treatment. In a normoxic condition, Tβ4 transcriptional activity was decreased in the cells incubated in the presence of L-NMMA after co-transfection with Tβ4 promoter and GST-conjugated HIF-1α. Collectively, these results suggest that NO could regulate the expression of Tβ4 by direct or indirect effect of HIF-1α on Tβ4 promoter.

Due to its actin-sequestering properties, thymosin beta-4 (Tβ4) is considered to play a significant role in the cellular metabolism. Several physiological properties of Tβ4 have been reported;, however, many questions concerning its cellular function remain to be ascertained. To better understand the role of this small peptide we have analyzed by means of transmission immunoelectron microscopy techniques the ultrastructural localization of Tβ4 in HepG2 cells. Samples of HepG2 cells were fixed in a mixture of 3% formaldehyde and 0.1% glutaraldehyde in 0.1 M cacodylate buffer and processed for standard electron microscopic techniques. The samples were dehydrated in a cold graded methanol series and embedded in LR gold resin. Ultrathin sections were labeled with rabbit antibodies to Tβ4, followed by gold-labeled goat anti-rabbit, stained with uranyl acetate and bismuth subnitrate, observed and photographed in a JEOL 100S transmission electron microscope. High-resolution electron microscopy showed that Tβ4 was mainly restricted to the cytoplasm of HepG2 growing in complete medium. A strong Tβ4 reactivity was detected in the perinuclear region of the cytoplasmic compartment where gold particles appeared strictly associated to the nuclear membrane. In the nucleus specific Tβ4 labeling was observed in the nucleolus. The above electron microscopic results confirm and extend previous observations at light microscopic level, highlighting the subcellular distribution of Tβ4 in both cytoplasmic and nuclear compartments of HepG2 cells. The meaning of Tβ4 presence in the nucleolus is not on the best of our knowledge clarified yet. It could account for the interaction of Tβ4 with nucleolar actin and according with this hypothesis, Tβ4 could contribute together with the other nucleolar acting binding proteins to modulate the transcription activity of the RNA polymerases.

Thymosin β4 (Tβ4), a small G-actin sequestering peptide, mediates cell proliferation, migration, and angiogenesis. Whether embryonic stem (ES) cells, overexpressing Tβ4, readily differentiate into cardiac myocytes in vitro and in vivo and enhance cardioprotection following transplantation post myocardial infarction (MI) remains unknown. Accordingly, we established stable mouse ES cell lines, RFP-ESCs and Tβ4-ESCs, expressing RFP and an RFP-Tβ4 fusion protein, respectively. In vitro, the number of spontaneously beating embryoid bodies (EBs) was significantly increased in Tβ4-ESCs at day 9, 12 and 15, compared with RFP-ESCs. Enhanced expression of cardiac transcriptional factors GATA-4, Mef2c and Txb6 in Tβ4-EBs, as confirmed with real time-PCR analysis, was accompanied by the increased number of EB areas stained positive for sarcomeric α-actin in Tβ4-EBs, compared with the RFP control, suggesting a significant increase in functional cardiac myocytes. Furthermore, we transplanted Tβ4-ESCs into the infarcted mouse heart and performed morphological and functional analysis 2 weeks after MI. There was a significant increase in newly formed cardiac myocytes associated with the Notch pathway, a decrease in apoptotic nuclei mediated by an increase in Akt and a decrease in levels of PTEN. Cardiac fibrosis was significantly reduced, and left ventricular function was significantly augmented in the Tβ4-ESC transplanted group, compared with controls. It is concluded that genetically modified Tβ4-ESCs, potentiates their ability to turn into cardiac myocytes in vitro as well as in vivo. Moreover, we also demonstrate that there was a significant decrease in both cardiac apoptosis and fibrosis, thus improving cardiac function in the infarcted heart.

Thymosin proteins are well known for their actin-binding activity. Thymosin beta 4 (Tβ4) has been associated with biological activities in tissue repair and cell migration via interaction with ATP-synthase in vertebrates, while the information of similar thymosin functions in invertebrates is limited. We have shown previously that ATP-synthase is present on the surface of crayfish hematopoietic tissue (HPT) cells, and that astakine 1 (Ast1, an invertebrate cytokine) was found to interact with this β-subunit of ATP synthase. Here, we identified five different β-thymosins from Pacifastacus leniusculus, designated Pl-β-thymosin1-5. The two dominant isoforms in brain, HPT and hemocytes, Pl-β-thymosin1 and 2, were chosen for functional studies. Both isoforms could bind to the β-subunit of ATP-synthase, and Pl-β-thymosin1, but not Pl-β-thymosin2, significantly increased extracellular ATP formation. Moreover, Pl-β-thymosin1 stimulated HPT cell migration in vitro and Ast1 blocked this effect. Pl-β-thymosin2 increased the circulating hemocyte number at an early stage after injection. Additionally, in vivo injection of Pl-β-thymosin1 resulted in significant reduction of reactive oxygen species (ROS) production in crayfish HPT whereas Pl-β-thymosin2 had a similar but transient effect. Both Pl-β-thymosins induced the expression of Ast1 and superoxide dismutase (SOD) transcripts, while silencing of endogenous Pl-β-thymosin 1 and 2 by RNAi resulted in significant reduction of the Ast1 and SOD transcripts. The diverse effects exhibited by Pl-β-thymosin1 and Pl-β-thymosin2 indicates that these proteins are involved in a complex interaction that regulates the hematopoietic stem cell proliferation and differentiation.

The identification of new biomarkers for preneoplastic pancreatic lesions (PanINs, IPMNs) and early pancreatic ductal adenocarcinoma (PDAC) is crucial due to the diseases high mortality rate upon late detection. To address this task we used the novel technique of matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) on genetically engineered mouse models (GEM) of pancreatic cancer. Various GEM were analyzed with MALDI IMS to investigate the peptide/protein-expression pattern of precursor lesions in comparison to normal pancreas and PDAC with cellular resolution. Statistical analysis revealed several discriminative m/z-species between normal and diseased tissue. Intraepithelial neoplasia (PanIN) and intraductal papillary mucinous neoplasm (IPMN) could be distinguished from normal pancreatic tissue and PDAC by 26 significant m/z-species. Among these m/z-species, we identified Albumin and Thymosin-beta 4 by liquid chromatography and tandem mass spectrometry (LC-MS/MS), which were further validated by immunohistochemistry, western blot, quantitative RT-PCR and ELISA in both murine and human tissue. Thymosin-beta 4 was found significantly increased in sera of mice with PanIN lesions. Upregulated PanIN expression of Albumin was accompanied by increased expression of liver-restricted genes suggesting a hepatic transdifferentiation program of preneoplastic cells. In conclusion we show that GEM of endogenous PDAC are a suitable model system for MALDI-IMS and subsequent LC-MS/MS analysis, allowing in situ analysis of small precursor lesions and identification of differentially expressed peptides and proteins.

We sought to identify serological markers capable of diagnosing preeclampsia (PE). We performed serum peptide analysis (liquid chromatography mass spectrometry) of 62 unique samples from 31 PE patients and 31 healthy pregnant controls, with two-thirds used as a training set and the other third as a testing set. Differential serum peptide profiling identified 52 significant serum peptides, and a 19-peptide panel collectively discriminating PE in training sets (n = 21 PE, n = 21 control; specificity = 85.7% and sensitivity = 100%) and testing sets (n = 10 PE, n = 10 control; specificity = 80% and sensitivity = 100%). The panel peptides were derived from 6 different protein precursors: 13 from fibrinogen alpha (FGA), 1 from alpha-1-antitrypsin (A1AT), 1 from apolipoprotein L1 (APO-L1), 1 from inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4), 2 from kininogen-1 (KNG1), and 1 from thymosin beta-4 (TMSB4). We concluded that serum peptides can accurately discriminate active PE. Measurement of a 19-peptide panel could be performed quickly and in a quantitative mass spectrometric platform available in clinical laboratories. This serum peptide panel quantification could provide clinical utility in predicting PE or differential diagnosis of PE from confounding chronic hypertension.

Xenopus laevis eggs are used as a biological model system for studying fertilization and early embryonic development in vertebrates. Most methods used for their molecular analysis require elaborate sample preparation including separate protocols for the water soluble and lipid components. In this study, laser ablation electrospray ionization (LAESI), an ambient ionization technique, was used for direct mass spectrometric analysis of X. laevis eggs and early stage embryos up to five cleavage cycles. Single unfertilized and fertilized eggs, their animal and vegetal poles, and embryos through the 32-cell stage were analyzed. Fifty two small metabolite ions, including glutathione, GABA and amino acids, as well as numerous lipids including 14 fatty acids, 13 lysophosphatidylcholines, 36 phosphatidylcholines and 29 triacylglycerols were putatively identified. Additionally, some proteins, for example thymosin β4 (Xen), were also detected. On the subcellular level, the lipid profiles were found to differ between the animal and vegetal poles of the eggs. Radial profiling revealed profound compositional differences between the jelly coat vitelline/plasma membrane and egg cytoplasm. Changes in the metabolic profile of the egg following fertilization, e.g., the decline of polyamine content with the development of the embryo were observed using LAESI-MS. This approach enables the exploration of metabolic and lipid changes during the early stages of embryogenesis.