Shumin Zhou

tRNA-Derived Small Non-Coding RNAs in Response to Ischemia Inhibit Angiogenesis

Ischemic injuries will lead to necrotic tissue damage, and post-ischemia angiogenesis plays critical roles in blood flow restoration and tissue recovery. Recently, several types of small RNAs have been reported to be involved in this process. In this study, we first generated a rat brain ischemic model to investigate the involvement of new types of small RNAs in ischemia. We utilized deep sequencing and bioinformatics analyses to demonstrate that the level of small RNA fragments derived from tRNAs strikingly increased in the ischemic rat brain. Among these sequences, tRNAVal- and tRNAGly-derived small RNAs account for the most abundant segments. The up-regulation of tRNAVal- and tRNAGly-derived fragments was verified through northern blot and quantitative PCR analyses. The levels of these two fragments also increased in a mouse hindlimb ischemia model and cellular hypoxia model. Importantly, up-regulation of the tRNAVal- and tRNAGly-derived fragments in endothelial cells inhibited cell proliferation, migration and tube formation. Furthermore, we showed that these small RNAs are generated by angiogenin cleavage. Our results indicate that tRNA-derived fragments are involved in tissue ischemia, and we demonstrate for the first time that tRNAVal- and tRNAGly-derived fragments inhibit angiogenesis by modulating the function of endothelial cells.

Comprehensive profiling of accessible surface glycans of mammalian sperm using a lectin microarray

It is well known that cell surface glycans or glycocalyx play important roles in sperm motility, maturation and fertilization. A comprehensive profile of the sperm surface glycans will greatly facilitate both basic research (sperm glycobiology) and clinical studies, such as diagnostics of infertility. As a group of natural glycan binders, lectin is an ideal tool for cell surface glycan profiling. However, because of the lack of effective technology, only a few lectins have been tested for lectin-sperm binding profiles. To address this challenge, we have developed a procedure for high-throughput probing of mammalian sperm with 91 lectins on lectin microarrays. Normal sperm from human, boar, bull, goat and rabbit were collected and analyzed on the lectin microarrays. Positive bindings of a set of ~50 lectins were observed for all the sperm of 5 species, which indicated a wide range of glycans are on the surface of mammalian sperm. Species specific lectin bindings were also observed. Clustering analysis revealed that the distances of the five species according to the lectin binding profiles are consistent with that of the genome sequence based phylogenetic tree except for rabbit. The procedure that we established in this study could be generally applicable for sperm from other species or defect sperm from the same species. We believe the lectin binding profiles of the mammalian sperm that we established in this study are valuable for both basic research and clinical studies.

Purification and Characterization of a 4-Hydroxybenzoate Decarboxylase from Chlamydophila pneumoniae AR39

Chlamydophila pneumoniae AR39 is an obligate intracellular pathogen that causes human acute and chronic respiratory tract diseases. One protein from C. pneumoniae AR39 was assigned as 4-hydroxybenzoate decarboxylase (HBDC). Assays done with the purified oxygen-sensitive protein showed that the optimum pH and temperature were 7.5 and 30°C, respectively. The Km and Vmax obtained for 4-hydroxybenzoate were approximately 0.21 mM and 11.9 nM min−1 mg−1, respectively. During the period of 4-hydroxybenzoate decarboxylation, overall activity of the thermal-sensitive protein was 5.06 nM min−1 mg−1 protein. The 4-hydroxybenzoate decarboxylation was promoted by Mg2+, Fe2+, Mn2+, and Ca2+ but not by Cu2+ or Zn2+. The enzyme also slowly catalyzed the reverse reaction, which was phenol carboxylation.

Downregulation of the Long Non-Coding RNA Meg3 Promotes Angiogenesis After Ischemic Brain Injury by Activating Notch Signaling.

Angiogenesis after ischemic brain injury contributes to the restoration of blood supply in the ischemic zone. Strategies to improve angiogenesis may facilitate the function recovery after stroke. Recent researches have demonstrated that dysfunction of long non-coding RNAs are associated with angiogenesis. We have previously reported that long non-coding RNAs (lncRNAs) are aberrantly expressed in ischemic stroke. However, little is known about long non-coding RNAs and theirs role in angiogenesis after stroke. In this study, we identified a rat lncRNAs, Meg3, and found that Meg3 was significantly decreased after ischemic stroke. Overexpression of Meg3 suppressed functional recovery and decreased capillary density after ischemic stroke. Downregulation of Meg3 ameliorated brain lesion and increased angiogenesis after ischemic stroke. Silencing of Meg3 resulted in a proangiogenic effect evidenced by increased endothelial cell migration, proliferation, sprouting, and tube formation. Mechanistically, we showed that Meg3 negatively regulated notch pathway both in vivo and in vitro. Inhibition of notch signaling in endothelial cells reversed the proangiogenic effect induced by Meg3 downregulation. This study revealed the function of Meg3 in ischemic stroke and elucidated its mechanism in angiogenesis after ischemic stroke.

Discovering cancer biomarkers from clinical samples by protein microarrays

Cancer biomarkers are of potential use in early cancer diagnosis, anticancer therapy development, and monitoring the responses to treatments. Protein‐based cancer biomarkers are major forms in use, as they are much easier to be monitored in body fluids or tissues. For cancer biomarker discovery, high‐throughput techniques such as protein microarrays hold great promises, because they are capable of global unbiased monitoring but with a miniaturized format. In doing so, novel and cancer type specific biomarkers can be systematically discovered at an affordable cost. In this review, we give a relatively complete picture on protein microarrays applied to clinical samples for cancer biomarker discovery, and conclude this review with the future perspectives.

Protein microarrays for studies of drug mechanisms and biomarker discovery in the era of systems biology.

Protein microarray technology is one of the most powerful tools presently available for proteomic studies. Numerous types of protein microarrays have been widely and successfully applied for both basic biological studies and clinical researches, including those designed to characterize protein-protein, protein-nucleic acid, protein-drug/small molecule and antibody-antigen interactions. In the past decade, a variety of protein microarrays have been developed, including those spotted with whole proteomes, smaller peptides, antibodies, and lectins. Featured as high-throughput, miniaturized, and capable of parallel analysis, the power of protein microarrays has already been demonstrated many times in both basic research and clinical applications. In this review, we have summarized the latest developments in the production and application of protein microarrays. We discuss several of the most important applications of protein microarray, ranging from proteome microarrays for large scale identification of protein-protein interactions to lectin microarrays for live cell surface glycan profiling, with special emphasis on their use in studies of drug mechanisms and biomarker discovery. Already with tremendous success, we envision protein microarrays will become an indispensible tool for any systems-wide studies, fostering the integration of basic research observations to clinically useful applications.

Lectin RCA-I specifically binds to metastasis-associated cell surface glycans in triple-negative breast cancer.

IntroductionTriple-negative breast cancer (TNBC) patients often face a high risk of early relapse characterized by extensive metastasis. Previous works have shown that aberrant cell surface glycosylation is associated with cancer metastasis, suggesting that altered glycosylations might serve as diagnostic signatures of metastatic potential. To address this question, we took TNBC as an example and analyzed six TNBC cell lines, derived from a common progenitor, that differ in metastatic potential.MethodsWe used a microarray with 91 lectins to screen for altered lectin bindings to the six TNBC cell lines. Candidate lectins were then verified by lectin-based flow cytometry and immunofluorescent staining assays using both TNBC/non-TNBC cancer cells. Patient-derived tissue microarrays were then employed to analyze whether the staining of Ricinus communis agglutinin I (RCA-I), correlated with TNBC severity. We also carried out real-time cell motility assays in the presence of RCA-I. Finally, liquid chromatography-mass spectrometry/tandem spectrometry (LC-MS/MS) was employed to identify the membrane glycoproteins recognized by RCA-I.ResultsUsing the lectin microarray, we found that the bindings of RCA-I to TNBC cells are proportional to their metastatic capacity. Tissue microarray experiments showed that the intensity of RCA-I staining is positively correlated with the TNM grades. The real-time cell motility assays clearly demonstrated RCA-I inhibition of adhesion, migration, and invasion of TNBC cells of high metastatic capacity. Additionally, a membrane glycoprotein, POTE ankyrin domain family member F (POTEF), with different galactosylation extents in high/low metastatic TNBC cells was identified by LC-MS/MS as a binder of RCA-I.ConclusionsWe discovered RCA-I, which bound to TNBC cells to a degree that is proportional to their metastatic capacities, and found that this binding inhibits the cell invasion, migration, and adhesion, and identified a membrane protein, POTEF, which may play a key role in mediating these effects. These results thus indicate that RCA-I-specific cell surface glycoproteins may play a critical role in TNBC metastasis and that the extent of RCA-I cell binding could be used in diagnosis to predict the likelihood of developing metastases in TNBC patients.

Baicalein attenuates hypertrophic scar formation via inhibition of the transforming growth factor-β/Smad2/3 signalling pathway

Hypertrophic scars (HPSs) are characterized by excessive fibrosis associated with aberrant function of fibroblasts. Currently no satisfactory treatment has been developed.

Role of Phosphorylated HDAC4 in Stroke-Induced Angiogenesis

Acetylation or deacetylation of chromatin proteins and transcription factors is part of a complex signaling system that is involved in the control of neurological disorders. Recent studies have demonstrated that histone deacetylases (HDACs) exert protective effects in attenuating neuronal injury after ischemic insults. Class IIa HDAC4 is highly expressed in the brain, and neuronal activity depends on the nucleocytoplasmic shuttling of HDAC4. However, little is known about HDAC4 and its roles in ischemic stroke. In this study, we report that phosphorylation of HDAC4 was remarkably upregulated after stroke and blockade of HDAC4 phosphorylation with GÖ6976 repressed stroke-induced angiogenesis. Phosphorylation of HDAC4 was also increased in endothelial cells hypoxia model and suppression of HDAC4 phosphorylation inhibited the tube formation and migration of endothelial cells in vitro. Furthermore, in addition to the inhibition of angiogenesis, blockade of HDAC4 phosphorylation suppressed the expression of genes downstream of HIF-VEGF signaling in vitro and in vivo. These data indicate that phosphorylated HDAC4 may serve as an important regulator in stroke-induced angiogenesis. The protective mechanism of phosphorylated HDAC4 is associated with HIF-VEGF signaling, implicating a novel therapeutic target in stroke.

A Human Lectin Microarray for Sperm Surface Glycosylation Analysis

Glycosylation is one of the most abundant and functionally important protein post-translational modifications. As such, technology for efficient glycosylation analysis is in high demand. Lectin microarrays are a powerful tool for such investigations and have been successfully applied for a variety of glycobiological studies. However, most of the current lectin microarrays are primarily constructed from plant lectins, which are not well suited for studies of human glycosylation because of the extreme complexity of human glycans. Herein, we constructed a human lectin microarray with 60 human lectin and lectin-like proteins. All of the lectins and lectin-like proteins were purified from yeast, and most showed binding to human glycans. To demonstrate the applicability of the human lectin microarray, human sperm were probed on the microarray and strong bindings were observed for several lectins, including galectin-1, 7, 8, GalNAc-T6, and ERGIC-53 (LMAN1). These bindings were validated by flow cytometry and fluorescence immunostaining. Further, mass spectrometry analysis showed that galectin-1 binds several membrane-associated proteins including heat shock protein 90. Finally, functional assays showed that binding of galectin-8 could significantly enhance the acrosome reaction within human sperms. To our knowledge, this is the first construction of a human lectin microarray, and we anticipate it will find wide use for a range of human or mammalian studies, alone or in combination with plant lectin microarrays.