Identification of Abundant and Functional dodecaRNAs (doRNAs) Derived from Ribosomal RNA

Using a modified RNA-sequencing (RNA-seq) approach, we discovered a new family of unusually short RNAs mapping to ribosomal RNA 5.8S, which we named dodecaRNAs (doRNAs), according to the number of core nucleotides (12 nt) their members contain. Using a new quantitative detection method that we developed, we confirmed our RNA-seq data and determined that the minimal core doRNA sequence and its 13-nt variant C-doRNA (doRNA with a 5′ Cytosine) are the two most abundant doRNAs, which, together, may outnumber microRNAs. The C-doRNA/doRNA ratio is stable within species but differed between species. doRNA and C-doRNA are mainly cytoplasmic and interact with heterogeneous nuclear ribonucleoproteins (hnRNP) A0, A1 and A2B1, but not Argonaute 2. Reporter gene activity assays suggest that C-doRNA may function as a regulator of Annexin II receptor (AXIIR) expression. doRNAs are differentially expressed in prostate cancer cells/tissues and may control cell migration. These findings suggest that unusually short RNAs may be more abundant and important than previously thought.

Affinity interactions of Actin with HS1 and Annexin I, II, IV, V and VI in RAW264.7 macrophages in vitro and in the Fc-receptor complex

Actin binding proteins HS1 and Annexin I, II, III, IV, V and VI proteins were examined in RAW macrophages in vitro using affinity chromatography, LC-MS/MS, western blot, cell staining and expression of fluorescent proteins. Actins and many related actin isoforms were observed in the phagosome. However, actins were also observed on polystyrene beads incubated with crude extracts readily bound to NHS affinity chromatography beads in the absence of anti-actin antibodies. Annexin II and V and the haematopoietic specific proteins HS1 (also called Lyn substrate) were prominently observed in isolated phagosomes and not observed in control beads incubated with crude extracts of RAW macrophages. Annexin II and V as well as G3BP, FLT1, FARP2, STARD13, RAB25, and FRAP1 were observed to bind actin in affinity chromatography experiments using LC-MS/MS and western blot. Cell staining and/or expression of GFP constructs showed that actin, HS1 and Annexin V prominently accumulate at the nascent Fc-receptor complex of RAW macrophages.

Affinity interactions of Actin with HS1 and Annexin I, II, IV, V and VI in RAW264.7 macrophages in vitro and in the Fc-receptor complex

Actin binding proteins HS1 and Annexin I, II, III, IV, V and VI proteins were examined in RAW macrophages in vitro using affinity chromatography, LC-MS/MS, western blot, cell staining and expression of fluorescent proteins. Actins and many related actin isoforms were observed in the phagosome. However, actins were also observed on polystyrene beads incubated with crude extracts readily bound to NHS affinity chromatography beads in the absence of anti-actin antibodies. Annexin II and V and the haematopoietic specific proteins HS1 (also called Lyn substrate) were prominently observed in isolated phagosomes and not observed in control beads incubated with crude extracts of RAW macrophages. Annexin II and V as well as G3BP, FLT1, FARP2, STARD13, RAB25, and FRAP1 were observed to bind actin in affinity chromatography experiments using LC-MS/MS and western blot. Cell staining and/or expression of GFP constructs showed that actin, HS1 and Annexin V prominently accumulate at the nascent Fc-receptor complex of RAW macrophages.

Proteomic Analysis of Differentially Expressed Proteins in Endothelial Cells Under Low Shear Stress

Background: Shear stress (SS) affects the morphology, proliferation, differentiation and migration of endothelial cells, and regulates protein expression.Methods: This study aims to conduct a proteomic analysis of human umbilical vein endothelial cells (HUVECs) under low SS. HUVECs were cultured on glass slides (test group n=30; control group n=30) and transferred to parallel-plate flow chamber. The test group was subjected to low SS at 4.58 dyne/cm2 for 2 hours, and the control group was maintained under static conditions. Approximately 2×107 cells were collected from each group and analyzed by two-dimensional electrophoresis. The protein from each spot were identified by mass spectrometry. The biological functions and processes associated with the identified proteins were evaluated using the NCBInr, SwissProt, and DAVID databases. The protein expression of 14-3-3 proteins, annexin I, annexin II, zyxin, filamin A, filamin B, Ras-related nuclear protein (Ran), and talin were assessed by Western blotting.Results: A total of 234 proteins were analyzed; 14 proteins were upregulated and 78 were downregulated in the test group compared with the control group. The expression of 14-3-3 proteins, annexin I, annexin II, zyxin, filamin A, and filamin B was significantly decreased, whereas the expression of Ran and talin was significantly increased in the test group.Conclusion: The current results provide evidence that SS changes the protein profile of HUVECs.

Prognostic significance of annexin II , human epididymis protein (HE-4) and claudin in endometrial carcinoma

Introduction: Endometrial cancer (EC) is one of the most prevalent malignant tumors of the female reproductive system worldwide. Annexins are membrane binding proteins with important role in tumor development and progression. Human Epididymis Protein (HE-4) is a novel marker for gynecolgical tumors. Claudins are proteins of tight junction category playing an important role in cell adhesion and tumor spread.Material and methods: Seventy blocks of paraffin-embedded tissues of endometrial carcinoma cases. Immunohistochemical evaluation of Annexin II , HE- 4 and Claudin-7 staining was performed. Clinical follow-up to all cases was done every three months.Results: Positive Annexin II,HE-4 expression were observed in 88.6% and 77.1% of EC respectively. Significant correlation was found between expression of both Annexin II and HE-4 and FIGO stage, decreased both overall and disease free survival rates. Positive Claudin-7 expression was observed in 40% of EC, with significant correlation with high grade only, however, no correlation with other clinical parameters or survival analysis was detected.Conclusion: Annexin II, HE-4 and Claudin-7 are prognostic factors for endometrial carcinoma and could be used in molecular targeted therapy.

Proteomic analysis of differentially expressed proteins in endothelial cells under low shear stress

Background: Shear stress (SS) affects the morphology, proliferation, differentiation and migration of endothelial cells, and regulates protein expression.Methods: This study aims to conduct a proteomic analysis of human umbilical vein endothelial cells (HUVECs) under low SS. HUVECs were cultured on glass slides (test group n=30; control group n=30) and transferred to parallel-plate flow chamber. The test group was subjected to low SS at 4.58 dyne/cm2 for 2 hours, and the control group was maintained under static conditions. Approximately 2´107 cells were collected from each group and analyzed by two-dimensional electrophoresis. The protein from each spot were identified by mass spectrometry. The biological functions and processes associated with the identified proteins were evaluated using the NCBInr, SwissProt, and DAVID databases. The protein expression of 14-3-3 proteins, annexin I, annexin II, zyxin, filamin A, filamin B, Ras-related nuclear protein (Ran), and talin were assessed by Western blotting.Results: A total of 234 proteins were analyzed; 14 proteins were upregulated and 78 were downregulated in the test group compared with the control group. The expression of 14-3-3 proteins, annexin I, annexin II, zyxin, filamin A, and filamin B was significantly decreased, whereas the expression of Ran and talin was significantly increased in the test group.Conclusion: The current results provide evidence that SS changes the protein profile of HUVECs.

Abnormal Lysosomal Positioning and Small Extracellular Vesicle Secretion in Arterial Stiffening and Calcification of Mice Lacking Mucolipin 1 Gene

Recent studies have shown that arterial medial calcification is mediated by abnormal release of exosomes/small extracellular vesicles from vascular smooth muscle cells (VSMCs) and that small extracellular vesicle (sEV) secretion from cells is associated with lysosome activity. The present study was designed to investigate whether lysosomal expression of mucolipin-1, a product of the mouse Mcoln1 gene, contributes to lysosomal positioning and sEV secretion, thereby leading to arterial medial calcification (AMC) and stiffening. In Mcoln1−/− mice, we found that a high dose of vitamin D (Vit D; 500,000 IU/kg/day) resulted in increased AMC compared to their wild-type littermates, which was accompanied by significant downregulation of SM22-α and upregulation of RUNX2 and osteopontin in the arterial media, indicating a phenotypic switch to osteogenic. It was also shown that significantly decreased co-localization of lysosome marker (Lamp-1) with lysosome coupling marker (Rab 7 and ALG-2) in the aortic wall of Mcoln1−/− mice as compared to their wild-type littermates. Besides, Mcoln1−/− mice showed significant increase in the expression of exosome/ sEV markers, CD63, and annexin-II (AnX2) in the arterial medial wall, accompanied by significantly reduced co-localization of lysosome marker (Lamp-1) with multivesicular body (MVB) marker (VPS16), suggesting a reduction of the lysosome-MVB interactions. In the plasma of Mcoln1−/− mice, the number of sEVs significantly increased as compared to the wild-type littermates. Functionally, pulse wave velocity (PWV), an arterial stiffening indicator, was found significantly increased in Mcoln1−/− mice, and Vit D treatment further enhanced such stiffening. All these data indicate that the Mcoln1 gene deletion in mice leads to abnormal lysosome positioning and increased sEV secretion, which may contribute to the arterial stiffness during the development of AMC.

Traumatic-Induced Coagulopathy as a Systems Failure: A New Window into Hemostasis

Traumatic-induced coagulopathy (TIC) is often associated with significant bleeding, transfusion requirements, inflammation, morbidity, and mortality. This review considers TIC as a systems failure, not as a single-event manifestation of trauma. After briefly reviewing the meaning of TIC and the bewildering array of fibrinolysis phenotypes, we will discuss the role of platelets and fibrinogen in coagulopathy. Next, we will review the different TIC hypotheses and drill down to a single mechanistic domain comprising (1) thrombin's differential binding to thrombomodulin, (2) the expression of annexin II-S100A10 complex, and (3) the functional integrity of the endothelial glycocalyx. This triad forms the basis of the “switch” hypothesis of TIC. We will next address the potential limitations of current practice in treating a coagulation or fibrinolytic defect, and the next defect, and so on down the line, which often leads to what U.S. surgeon William C. Shoemaker considered “an uncoordinated and sometimes contradictory therapeutic outcome.” The treat-as-you-go approach using sequential, single-target treatments appears to be a by-product of decades of highly reductionist thinking and research. Lastly, we will present a unified systems hypothesis of TIC involving three pillars of physiology: the central nervous system (CNS)–cardiovascular system, the endothelial glycocalyx, and mitochondrial integrity. If CNS control of ventriculoarterial coupling is maintained close to unity following trauma, we hypothesize that the endothelium will be protected, mitochondrial energetics will be maintained, and TIC (and inflammation) will be minimized. The Systems Hypothesis of Trauma (SHOT) also helps to answer why certain groups of severely bleeding trauma patients are still dying despite receiving the best care. Currently, no drug therapy exists that targets the whole system.

Syncope as Initial Presentation in an Undifferentiated Type Acute Myeloid Leukemia Patient with Acute Intracranial Hemorrhage

Intracranial hemorrhage (ICH) is a catastrophic complication in patients with acute myeloid leukemia (AML). AML cells, especially in the acute promyelocytic leukemia subtype, may release microparticles (MPs), tissue factor (TF), and cancer procoagulant (CP) to promote coagulopathy. Hyperfibrinolysis is also triggered via release of annexin II, t-PA, u-PA, and u-PAR. Various inflammatory cytokines from cancer cells, such as IL-1β and TNF-α, activate endothelial cells and promote leukostasis. This condition may increase the ICH risk and lead to poor clinical outcomes. Here, we present a case under a unique situation with acute ICH detected prior to the diagnosis of AML. The patient initially presented with two episodes of syncope. Rapidly progressive ICH was noted in follow-up computed tomography (CT) scans. Therefore, we highlight that AML should be among the differential diagnoses of the etiologies of ICH. Early diagnosis and timely intervention are very important for AML patients.

Proteomic analysis of differentially expressed proteins in endothelial cells under low shear stress

Shear stress (SS) affects the morphology, migration, differentiation, and proliferation of endothelial cells, and regulates protein expression. The objective of this study was to perform a proteomic analysis of human umbilical vein endothelial cells (HUVECs) under low SS. HUVEC lines were cultured on glass slides (test group n=30; control group n=30) and transferred to a parallel-plate flow chamber. The test group was subjected to low SS at 4.58 dyne/cm2 for 2 hours, and the control group was maintained under static conditions. Approximately 2×107 cells were collected from each group and analyzed by two-dimensional electrophoresis. The proteins from each spot were identified by mass spectrometry. The biological functions and processes associated with the identified proteins were evaluated using the NCBInr, SwissProt, and DAVID databases. The protein expression of 14-3-3 proteins, annexin I, annexin II, zyxin, filamin A, filamin B, Ras-related nuclear protein (Ran), and talin were assessed by Western blotting. A total of 234 proteins were analyzed; 14 proteins were upregulated and 78 were downregulated in the test group compared with the control group. The expression of 14-3-3 proteins, annexin I, annexin II, zyxin, filamin A, and filamin B was significantly decreased, whereas the expression of Ran and talin was significantly increased in the test group. It is known that SS induces the remodeling of endothelial cells by modulating protein expression. However, the mechanisms underlying this modulation are unknown. The present results provide evidence that SS changes the protein profile of HUVECs.