Identification of Novel Biomarkers in Platelets for Diagnosing Parkinson’s Disease

<b><i>Background:</i></b> Parkinson’s disease (PD) is a common neurodegenerative disease affecting the elderly, but there is no blood test for PD diagnosis in the clinic currently. This study aimed to explore promising biomarkers in platelets (PLTs) for PD diagnosis. <b><i>Methods:</i></b> PLTs were isolated from whole blood samples of PD patients and healthy controls (HCs), and RNA was extracted for sequencing. RNA-seq was performed on the Illumina HiSeq platform. <b><i>Results:</i></b> A total of 2,221 genes with differential transcript levels (GDTLs) were identified between PD patients and HCs, 1,041 of which are upregulated genes and 1,180 of which are downregulated genes. <i>WASH5P</i> was the most upregulated gene and <i>AC114491.1</i> was the most downregulated gene. Among the top 12 most relevant genes, metastasis-associated lung adenocarcinoma transcript 1 (<i>MALAT1</i>), eukaryotic elongation factor 1A (<i>EEF1A1</i>), and cathepsin S (<i>CTSS</i>) were reported to be associated with PD. Furthermore, gene ontology analysis showed that the most significant term in biological processes was neutrophil degranulation; the most enriched term in cellular components was cytoplasmic vesicle lumen; and tumor necrosis factor receptor superfamily binding was the most significant term in molecular functions. In the Kyoto Encyclopedia of Genes and Genomes enrichment analysis, inflammation-related pathway accounts for the majority. <b><i>Conclusion:</i></b> Our findings demonstrated <i>WASH5P</i>, <i>MALAT1</i>, <i>EEF1A1,</i> and <i>CTSS</i> may be promising biomarkers in PD, which may contribute to improving the effectiveness and accuracy of diagnosis for PD in the future.

Network Pharmacology and Molecular docking Analysis on Mechanisms and Molecular Targets of LongChaiJiangXue Formula for Treatment of Polycythemia Vera

Background: LongChaiJiangXue formula (LCJX) has the effect of not only clearing up excessive erythrocytes but also relieving clinical symptoms of Polycythemia vera (PV). Material/Methods: The chemical constitution of LCJX was identified from Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP). Seven hundred and fifty-nine targets were identified and a total of 248 targets were screened out after discarding duplication and genes without any ID in databases. GeneCards database, OMIM database, and GEO database were searched for differential expression genes. The network was built by Cytoscape (3.7.2) software and the protein-protein interaction (PPI) networks of PV and LCJX were merged by https://string-db.org . Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment was processed by the R platform. The molecular docking technology was used to further analyze the intense of the assosiation of the compounds and targets. Results: 73 compounds of LCJX were chosen as the candidate active compounds. The compound-targets network contained 105 nods and 216 edges that presented the interaction of agents and targets. The PPI network of LCJX targets involved 59 nodes and 168 edges. The network showed that the key nodes were concentrated in signal transducer and activator of transcription-3(STAT-3), interleukin-6(IL-6), Janus kinase 2(JAK2), and vascular endothelial growth factor-A(VEGFA). The most enriched terms in the GO analysis in the GO biological processes(BP) were reactive oxygen species metabolic process, response to lipopolysaccharide, and response to oxidative stress. According to GO molecular functions(MF), the central nodes were generally enriched in cytokine receptor binding, cytokine activity, and heme binding. Regarding the GO cell components(CC), the terms included vesicle lumen, cytoplasmic vesicle lumen, and secretory granule lumen. In light of the KEGG enrichment analysis, the Hepatitis B, AGE-RAGE signaling pathway in diabetic complications, Kaposi sarcoma-associated herpesvirus infection, measles, Human cytomegalovirus infection, and JAK-STAT signaling pathway were significantly enriched. The molecular docking technology found that puerarin and saikosaponin A had relative stronger affinity with VEGFA, HIF-1A, JAK2 and STAT3 than other compounds in terms of the binding free energy. Conclusions: The effect of LCJX on PV is achieved through a series of complex mechanisms. Network pharmacology and molecular docking are powerful tools to reveal the effect of compound Chinese medicine on the disease.

In vivo real-time imaging of the liver with confocal endomicroscopy permits visualization of the temporospatial patterns of hepatocyte apoptosis

Apoptosis is a dynamic process of programmed cell death and is involved in multiple diseases. However, its mechanisms and sequence of events are still incompletely understood, partly because of the inability to visualize single cells continuously in vivo. The aim of the present study was to monitor hepatocyte apoptosis with confocal endomicroscopy in living rodents. In 73 anaesthetized mice, apoptotic liver injury was induced by injection of the CD95-agonistic antibody Jo2. Individual hepatocytes were followed for up to 240 min with a handheld confocal probe (FIVE1; Optiscan) providing 0.7 μm resolution (1,000-fold magnification). Different fluorescence staining protocols were used for cellular staining, vascular and cellular barrier function imaging, and caspase activation visualization. The time course of apoptosis could be visualized in vivo while liver perfusion and tissue integrity were maintained. In contrast to most ex vivo studies, initial cell swelling was observed that coincided with early defects in barrier function of sinusoids and hepatocytes. Cytoplasmic vesicle formation, nuclear condensation, cellular disintegration, and macrophage infiltration were captured sequentially. Labeling of caspases allowed molecular imaging. Our study allowed for the first time to continuously follow distinct morphological, functional, and molecular features of apoptosis in a solid organ in vivo and at high resolution. Intravital confocal microscopy may be a valuable tool to study the effects of therapeutic intervention on apoptosis in animal models and humans.

Cytoplasmic control of nuclear assembly

The reconstitution of a replication-competent, transcriptionally active nucleus following mitosis, fertilization or nuclear transplantation involves a stepwise series of reactions, most (if not all) of which are controlled by the cytoplasmic environment. This review discusses the nature of cytoplasmic contributions to the development of the male pronucleus at fertilization, and the effect of altering the cytoplasmic environment on nuclear assembly. The system used to investigate these regulations consists of permeabilized sea urchin sperm nuclei incubated under controlled conditions in a cell-free extract of fertilized sea urchin eggs. (1) In egg cytoplasmic extract, male pronuclear formation is initiated by the disassembly of the sperm nuclear lamina as a result of lamin phosphorylation by a cytosolic protein kinase C. (2) Sperm histones are phosphorylated by an as yet unidentified soluble kinase. (3) The conical sperm nucleus decondenses into a spherical pronucleus in an ATP-and cytosolic pH-dependent manner. (4) Chromatin decondensation is associated with the replacement of sperm histones by maternal histones. (5) Nuclear membranes form by ATP-dependent binding of vesicles to chromatin and GTP-dependent fusion of these vesicles to one another. (6) Three cytoplasmic vesicle populations with distinct biochemical, chromatin-binding and fusion properties are required for nuclear envelope assembly. (7) Targeting of the bulk of nuclear membrane vesicles to chromatin is mediated by an integral membrane protein similar to human lamin B receptor. (8) The last step of male pronuclear formation, nuclear swelling, is promoted by the assembly of nuclear pores, nuclear import of soluble lamins and growth of the nuclear membranes. (9) Once inside the nucleus, lamin B associates with lamin B receptors, presumably to tether the inner nuclear membrane with the lamina. Overall, these processes are similar to those characterizing nuclear reconstitution after mitosis in somatic cells or nuclear remodeling following transplantation into oocytes or eggs. The influence of the egg cytoplasmic environment on some aspects of nuclear remodeling after nuclear transplantation is also discussed.

Differential localization of Acanthamoeba myosin I isoforms.

Acanthamoeba myosins IA and IB were localized by immunofluorescence and immunoelectron microscopy in vegetative and phagocytosing cells and the total cell contents of myosins IA, IB, and IC were quantified by immunoprecipitation. The quantitative distributions of the three myosin I isoforms were then calculated from these data and the previously determined localization of myosin IC. Myosin IA occurs almost exclusively in the cytoplasm, where it accounts for approximately 50% of the total myosin I, in the cortex beneath phagocytic cups and in association with small cytoplasmic vesicles. Myosin IB is the predominant isoform associated with the plasma membrane, large vacuole membranes and phagocytic membranes and accounts for almost half of the total myosin I in the cytoplasm. Myosin IC accounts for a significant fraction of the total myosin I associated with the plasma membrane and large vacuole membranes and is the only myosin I isoform associated with the contractile vacuole membrane. These data suggest that myosin IA may function in cytoplasmic vesicle transport and myosin I-mediated cortical contraction, myosin IB in pseudopod extension and phagocytosis, and myosin IC in contractile vacuole function. In addition, endogenous and exogenously added myosins IA and IB appeared to be associated with the cytoplasmic surface of different subpopulations of purified plasma membranes implying that the different myosin I isoforms are targeted to specific membrane domains through a mechanism that involves more than the affinity of the myosins for anionic phospholipids.

Bimodal redistribution of surface transmembrane glycoproteins during Ca2+-dependent secretion (acrosome reaction) in boar spermatozoa

We used the acrosome reaction of boar sperm cells to study the dynamics of surface transmembrane glycoproteins (TMG) during a secretory process. The acrosome reaction is the Ca2+-dependent fusion of a large cytoplasmic vesicle (the acrosome) with the overlying segment of the plasma membrane (acrosomal cap) that leads to the release of the acrosomal enzymes. After triggering the acrosome reaction in vitro (2 mM-CaCl2 in the presence of 10 microM-A23187), we used freeze-fracture electron microscopy to follow the topographical rearrangement of a population of acrosomal-cap large intramembrane particles that correspond to transmembrane proteins that bind wheat germ agglutinin. We found that these TMG move in the direction of either one of two opposite poles, proximal and distal, of the acrosomal cap. This bimodal movement of the TMG reorganizes the acrosomal cap into three extensive domains. The first two, on the apical rim and on the equator, are membrane domains to which the TMG are directed and where they accumulate. The third, a large in-between area of protein clearing, corresponds to the region from which TMG were preferentially located before displacement induced by the Ca2+ effect. The topography of these new membrane domains of the acrosomal cap becomes coincident with that of the structural domains of the subjacent acrosomal membrane. Mirroring of the acrosomal membrane by the plasma membrane is followed by fusion between the two membranes, formation of an exquisite labyrinth of hybrid-membrane tubules, followed by fission and release of the acrosomal contents through intertubular fenestrae.

Ultrastructural localization of nonspecific esterase activity in guinea pig and human monocytes, macrophages, and lymphocytes

Using either hexazotized pararosaniline or new fuchsin as coupling agents, we investigated the ultrastructural localization of alpha- naphthyl acetate esterase (ANAE) activity in guinea pig bone marrow and peritoneal exudates, and in human peripheral blood cells. DFP- inhibitable ANAE activity was present on the cell surface of lymphocytes, monocytes, macrophages, neutrophils, eosinophils, basophils, megakaryocytes, platelets, and blasts. Demarcation lines in megakaryocytes and the perinuclear cisternae in normoblasts were also positive. In addition, lymphocytes, monocytes, and macrophages displayed ANAE activity associated with cytoplasmic-vesicle clusters (CVC). Reaction product was always present on the cytoplasmic surfaces of these vesicles and in the adjacent cytoplasm; vesicle interiors were invariably ANAE-negative. Small lymphocytes generally had a single large paranuclear ANAE-positive CVC, whereas mononuclear phagocytes had multiple discrete foci of similar appearing ANAE-positive CVC that sometime became confluent. ANAE activity was also found in the Gall bodies of human lymphocytes and in coated vesicles of macrophages. Cytoplasmic ANAE activity was increased in oil-induced guinea pig peritoneal macrophages. Both surface and cytoplasmic esterase activities had a neutral pH optimum. An identical distribution of reaction product was observed when alpha-naphthyl butyrate was employed as substrate. The function of these esterases, and their relation to known surface and cytoplasmic neutral proteases, awaits further investigation.

Ultrastructural localization of nonspecific esterase activity in guinea pig and human monocytes, macrophages, and lymphocytes

Using either hexazotized pararosaniline or new fuchsin as coupling agents, we investigated the ultrastructural localization of alpha- naphthyl acetate esterase (ANAE) activity in guinea pig bone marrow and peritoneal exudates, and in human peripheral blood cells. DFP- inhibitable ANAE activity was present on the cell surface of lymphocytes, monocytes, macrophages, neutrophils, eosinophils, basophils, megakaryocytes, platelets, and blasts. Demarcation lines in megakaryocytes and the perinuclear cisternae in normoblasts were also positive. In addition, lymphocytes, monocytes, and macrophages displayed ANAE activity associated with cytoplasmic-vesicle clusters (CVC). Reaction product was always present on the cytoplasmic surfaces of these vesicles and in the adjacent cytoplasm; vesicle interiors were invariably ANAE-negative. Small lymphocytes generally had a single large paranuclear ANAE-positive CVC, whereas mononuclear phagocytes had multiple discrete foci of similar appearing ANAE-positive CVC that sometime became confluent. ANAE activity was also found in the Gall bodies of human lymphocytes and in coated vesicles of macrophages. Cytoplasmic ANAE activity was increased in oil-induced guinea pig peritoneal macrophages. Both surface and cytoplasmic esterase activities had a neutral pH optimum. An identical distribution of reaction product was observed when alpha-naphthyl butyrate was employed as substrate. The function of these esterases, and their relation to known surface and cytoplasmic neutral proteases, awaits further investigation.