Experimental verification of a conserved intronic microRNA located in the human <Emphasis Type="Italic">TrkC</Emphasis> gene with a cell type-dependent apoptotic function

Tropomyosin receptor kinase C (TrkC) is involved in cell survival, apoptosis induction and tumorigenesis. We hypothesized that, similar to p75^NTR receptor, some of the diverse functions of TrkC could be mediated by a microRNA (miRNA) embedded within the gene. Here, we experimentally verified the expression and processing of two bioinformatically predicted miRNAs named TrkC-miR1-5p and TrkC-miR1-3p. Transfecting a DNA fragment corresponding to the TrkC-premir1 sequence in HEK293t cells caused ~300-fold elevation in the level of mature TrkC-miR1 and also a significant downregulation of its predicted target genes. Furthermore, endogenous TrkC-miR1 was detected in several cell lines and brain tumors confirming its endogenous generation. Furthermore, its orthologous miRNA was detected in developing rat brain. Accordingly, TrkC-miR1 expression was increased during the course of neural differentiation of NT2 cell, whereas its suppression attenuated NT2 differentiation. Consistent with opposite functions of TrkC, TrkC-miR1 overexpression promoted survival and apoptosis in U87 and HEK293t cell lines, respectively. In conclusion, our data report the discovery of a new miRNA with overlapping function to TrkC.     

miR-124-3p is a chronic regulator of gene expression after brain injury

Traumatic brain injury (TBI) initiates molecular and cellular pathologies that underlie post-injury morbidities, including hippocampus-related memory decline and epileptogenesis. Non-coding small RNAs are master regulators of gene expression with the potential to affect multiple molecular pathways. To evaluate whether hippocampal gene expression networks are chronically regulated by microRNAs after TBI, we sampled the dentate gyrus of rats with severe TBI induced by lateral fluid-percussion injury 3 months earlier. Ingenuity pathway analysis revealed 30 upregulated miR-124-3p targets, suggesting that miR-124-3p is downregulated post-TBI (z-score?=?? 5.146, p?<?0.05). Droplet digital polymerase chain reaction (ddPCR) and in situ hybridization confirmed the chronic downregulation of miR-124-3p (p?<?0.05). Quantitative PCR analysis of two targets, Plp2 and Stat3, indicated that their upregulation correlated with the miR-124-3p downregulation (r?=?? 0.647, p?<?0.05; r?=?? 0.629, p?<?0.05, respectively). Immunohistochemical staining of STAT3 confirmed the increased protein expression. STRING analysis showed that 9 of the 30 miR-124-3p targets belonged to a STAT3 network. Reactome analysis and data mining connected the targets especially to inflammation and signal transduction. L1000CDS2 software revealed drugs (e.g., importazole, trichostatin A, and IKK-16) that could reverse the observed molecular changes. The translational value of our data was emphasized by in situ hybridization showing chronic post-traumatic downregulation of miR-124-3p in the dentate gyrus of TBI patients. Analysis of another brain injury model, status epilepticus, highlighted the fact that chronic downregulation of miR-124 is a common phenomenon after brain injury. Together, our findings indicate that miR-124-3p is a chronic modulator of molecular networks relevant to post-injury hippocampal pathologies in experimental models and in humans.     

Dicer generates a regulatory microRNA network in smooth muscle cells that limits neointima formation during vascular repair

MicroRNAs (miRNAs) coordinate vascular repair by regulating injury-induced gene expression in vascular smooth muscle cells (SMCs) and promote the transition of SMCs from a contractile to a proliferating phenotype. However, the effect of miRNA expression in SMCs on neointima formation is unclear. Therefore, we studied the role of miRNA biogenesis by Dicer in SMCs in vascular repair. Following wire-induced injury to carotid arteries of Apolipoprotein E knockout (Apoe ^?/?) mice, miRNA microarray analysis revealed that the most significantly regulated miRNAs, such as miR-222 and miR-21-3p, were upregulated. Conditional deletion of Dicer in SMCs increased neointima formation by reducing SMC proliferation in Apoe ^?/? mice, and decreased mainly the expression of miRNAs, such as miR-147 and miR-100, which were not upregulated following vascular injury. SMC-specific deletion of Dicer promoted growth factor and inflammatory signaling and regulated a miRNA–target interaction network in injured arteries that was enriched in anti-proliferative miRNAs. The most connected miRNA in this network was miR-27a-3p [e.g., with Rho guanine nucleotide exchange factor 26 (ARHGEF26)], which was expressed in medial and neointimal SMCs in a Dicer-dependent manner. In vitro, miR-27a-3p suppresses ARHGEF26 expression and inhibits SMC proliferation by interacting with a conserved binding site in the 3′ untranslated region of ARHGEF26 mRNA. We propose that Dicer expression in SMCs plays an essential role in vascular repair by generating anti-proliferative miRNAs, such as miR-27a-3p, to prevent vessel stenosis due to exaggerated neointima formation.     

Protein levels of clusterin and glutathione synthetase in platelets allow for early detection of colorectal cancer

Colorectal cancer (CRC) is one of the most frequent malignancies in the Western world. Early tumor detection and intervention are important determinants on CRC patient survival. During early tumor proliferation, dissemination and angiogenesis, platelets store and segregate proteins actively and selectively. Hence, the platelet proteome is a potential source of biomarkers denoting early malignancy. By comparing protein profiles of platelets between healthy volunteers (n = 12) and patients with early- (n = 7) and late-stage (n = 5) CRCs using multiplex fluorescence two-dimensional gel electrophoresis (2D-DIGE), we aimed at identifying differentially regulated proteins within platelets. By inter-group comparisons, 94 differentially expressed protein spots were detected (p < 0.05) between healthy controls and patients with early- and late-stage CRCs and revealed distinct separations between all three groups in principal component analyses. 54 proteins of interest were identified by mass spectrometry and resulted in high-ranked Ingenuity Pathway Analysis networks associated with Cellular function and maintenance, Cellular assembly and organization, Developmental disorder and Organismal injury and abnormalities (p < 0.0001 to p = 0.0495). Target proteins were validated by multiplex fluorescence-based Western blot analyses using an additional, independent cohort of platelet protein samples [healthy controls (n = 15), early-stage CRCs (n = 15), late-stage CRCs (n = 15)]. Two proteins—clusterin and glutathione synthetase (GSH-S)—featured high impact and were subsequently validated in this independent clinical cohort distinguishing healthy controls from patients with early- and late-stage CRCs. Thus, the potential of clusterin and GSH-S as platelet biomarkers for early detection of CRC could improve existing screening modalities in clinical application and should be confirmed in a prospective multicenter trial.     

Extracellular vesicles regulate the human osteoclastogenesis: divergent roles in discrete inflammatory arthropathies

Objective

Extracellular vesicles (EVs) are subcellular signalosomes. Although characteristic EV production is associated with numerous physiological and pathological conditions, the effect of blood-derived EVs on bone homeostasis is unknown. Herein we evaluated the role of circulating EVs on human osteoclastogenesis.

Methods

Blood samples from healthy volunteers, rheumatoid arthritis (RA) and psoriatic arthritis (PsA) patients were collected. Size-based EV sub-fractions were isolated by gravity-driven filtration and differential centrifugation. To investigate the properties of EV samples, resistive pulse sensing technique, transmission electron microscopy, flow cytometry and western blot were performed. CD14⁺ monocytes were separated from PBMCs, and stimulated with recombinant human M-CSF, RANKL and blood-derived EV sub-fractions. After 7 days, the cells were fixed and stained for tartrate-resistant acid phosphatase and counted.

Results

EVs isolated by size-based sub-fractions were characterized as either microvesicles or exosomes (EXO). Healthy (n = 11) and RA-derived (n = 12) EXOs profoundly inhibited osteoclast differentiation (70%, p < 0.01; 65%, p < 0.01, respectively). In contrast, PsA-derived (n = 10) EXOs had a stimulatory effect (75%, p < 0.05). In cross-treatment experiments where EXOs and CD14⁺ cells were interchanged between the three groups, only healthy (n = 5) and RA (n = 5)-derived EXOs inhibited (p < 0.01, respectively) the generation of osteoclasts in all groups, whereas PsA (n = 7)-derived EXOs were unable to mediate this effect.

Conclusions

Our data suggest that blood-derived EXOs are novel regulators of the human osteoclastogenesis and may offer discrete effector function in distinct inflammatory arthropathies.

     

Membrane-shed vesicles from the parasite <Emphasis Type="Italic">Trichomonas vaginalis</Emphasis>: characterization and their association with cell interaction

Trichomonas vaginalis is a common sexually transmitted parasite that colonizes the human urogenital tract, where it remains extracellular and adheres to epithelial cells. Infections range from asymptomatic to highly inflammatory, depending on the host and the parasite strain. Despite the serious consequences associated with trichomoniasis disease, little is known about parasite or host factors involved in attachment of the parasite-to-host epithelial cells. Here, we report the identification of microvesicle-like structures (MVs) released by T. vaginalis. MVs are considered universal transport vehicles for intercellular communication as they can incorporate peptides, proteins, lipids, miRNA, and mRNA, all of which can be transferred to target cells through receptor–ligand interactions, fusion with the cell membrane, and delivery of a functional cargo to the cytoplasm of the target cell. In the present study, we demonstrated that T. vaginalis release MVs from the plasma and the flagellar membranes of the parasite. We performed proteomic profiling of these structures demonstrating that they possess physical characteristics similar to mammalian extracellular vesicles and might be selectively charged with specific protein content. In addition, we demonstrated that viable T. vaginalis parasites release large vesicles (LVs), membrane structures larger than 1 µm that are able to interact with other parasites and with the host cell. Finally, we show that both populations of vesicles present on the surface of T vaginalis are induced in the presence of host cells, consistent with a role in modulating cell interactions.     

Germline-specific dgcr8 knockout in zebrafish using a BACK approach

Zebrafish is an important model to study developmental biology and human diseases. However, an effective approach to achieve spatial and temporal gene knockout in zebrafish has not been well established. In this study, we have developed a new approach, namely bacterial artificial chromosome-rescue-based knockout (BACK), to achieve conditional gene knockout in zebrafish using the Cre/loxP system. We have successfully deleted the DiGeorge syndrome critical region gene 8 (dgcr8) in zebrafish germ line and demonstrated that the maternal-zygotic dgcr8 (MZdgcr8) embryos exhibit MZdicer-like phenotypes with morphological defects which could be rescued by miR-430, indicating that canonical microRNAs play critical role in early development. Our findings establish that Cre/loxP-mediated tissue-specific gene knockout could be achieved using this BACK strategy and that canonical microRNAs play important roles in early embryonic development in zebrafish.     

Role of microRNAs in malignant mesothelioma

Malignant mesothelioma (MM) is an aggressive tumor, mainly derived from the pleura, which is predominantly associated with exposure to asbestos fibers. The prognosis of MM patients is particularly severe, with a median survival of approximately 9–12 months and latency between exposure and diagnosis ranging from 20–50 years (median 30 years). Emerging evidence has demonstrated that tumor aggressiveness is associated with genome and gene expression abnormalities; therefore, several studies have recently focused on the role of microRNAs (miRNAs) in MM tumorigenesis. miRNAs are small non-protein coding single-stranded RNAs (17–22 nucleotides) involved in numerous cellular processes that negatively regulate gene expression by modulating the expression of downstream target genes. miRNAs are often deregulated in cancer; in particular, the differential miRNA expression profiles of MM cells compared to unaffected mesothelial cells have suggested potential roles of miRNAs as either oncogenes or tumor suppressor genes in MM oncogenesis. In this review, the mechanism of MM carcinogenesis was evaluated through the analysis of the published miRNA expression data. The roles of miRNAs as diagnostic biomarkers and prognostic factors for potential therapeutic strategies will be presented and discussed.     

Neuroprotective effects of the gliopeptide ODN in an in vivo model of Parkinson’s disease

Parkinson’s disease (PD) is a neurodegenerative disorder characterized by a progressive loss of dopamine (DA) neurons through apoptotic, inflammatory and oxidative stress mechanisms. The octadecaneuropeptide (ODN) is a diazepam-binding inhibitor (DBI)-derived peptide, expressed by astrocytes, which protects neurons against oxidative cell damages and apoptosis in an in vitro model of PD. The present study reveals that a single intracerebroventricular injection of 10 ng ODN 1 h after the last administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) prevented the degeneration of DA neurons induced by the toxin in the substantia nigra pars compacta of mice, 7 days after treatment. ODN-mediated neuroprotection was associated with a reduction of the number of glial fibrillary acidic protein-positive reactive astrocytes and a strong inhibition of the expression of pro-inflammatory genes such as interleukins 1β and 6, and tumor necrosis factor-α. Moreover, ODN blocked the inhibition of the anti-apoptotic gene Bcl-2, and the stimulation of the pro-apoptotic genes Bax and caspase-3, induced by MPTP in the substantia nigra pars compacta. ODN also decreased or even in some cases abolished MPTP-induced oxidative damages, overproduction of reactive oxygen species and accumulation of lipid oxidation products in DA neurons. Furthermore, DBI knockout mice appeared to be more vulnerable than wild-type animals to MPTP neurotoxicity. Taken together, these results show that the gliopeptide ODN exerts a potent neuroprotective effect against MPTP-induced degeneration of nigrostriatal DA neurons in mice, through mechanisms involving downregulation of neuroinflammatory, oxidative and apoptotic processes. ODN may, thus, reduce neuronal damages in PD and other cerebral injuries involving oxidative neurodegeneration.     

<Emphasis Type="Italic">Clostridium difficile</Emphasis>-related postinfectious IBS: a case of enteroglial microbiological stalking and/or the solution of a conundrum?

Post-infectious irritable bowel syndrome is a well-defined pathological entity that develops in about one-third of subjects after an acute infection (bacterial, viral) or parasitic infestation. Only recently it has been documented that an high incidence of post-infectious irritable bowel syndrome occurs after Clostridium difficile infection. However, until now it is not known why in some patients recovered from this infection the gastrointestinal disturbances persist for months or years. Based on our in vitro studies on enteric glial cells exposed to the effects of C. difficile toxin B, we hypothesize that persistence of symptoms up to the development of irritable bowel syndrome might be due to a disturbance/impairment of the correct functions of the enteroglial intestinal network.     

Tumor surveillance by circulating microRNAs: a hypothesis

A growing body of experimental evidence supports the diagnostic relevance of circulating microRNAs in various diseases including cancer. The biological relevance of circulating microRNAs is, however, largely unknown, particularly in healthy individuals. Here, we propose a hypothesis based on the relative abundance of microRNAs with predominant tumor suppressor activity in the blood of healthy individuals. According to our hypothesis, certain sets of circulating microRNAs might function as a tumor surveillance mechanism exerting continuous inhibition on tumor formation. The microRNA-mediated tumor surveillance might complement cancer immune surveillance.     

Regulation of microRNA biogenesis and turnover by animals and their viruses

MicroRNAs (miRNAs) are a ubiquitous component of gene regulatory networks that modulate the precise amounts of proteins expressed in a cell. Despite their small size, miRNA genes contain various recognition elements that enable specificity in when, where and to what extent they are expressed. The importance of precise control of miRNA expression is underscored by functional studies in model organisms and by the association between miRNA mis-expression and disease. In the last decade, identification of the pathways by which miRNAs are produced, matured and turned-over has revealed many aspects of their biogenesis that are subject to regulation. Studies in viral systems have revealed a range of mechanisms by which viruses target these pathways through viral proteins or non-coding RNAs in order to regulate cellular gene expression. In parallel, a field of study has evolved around the activation and suppression of antiviral RNA interference (RNAi) by viruses. Virus encoded suppressors of RNAi can impact miRNA biogenesis in cases where miRNA and small interfering RNA pathways converge. Here we review the literature on the mechanisms by which miRNA biogenesis and turnover are regulated in animals and the diverse strategies that viruses use to subvert or inhibit these processes.     

The emerging link between O-GlcNAcylation and neurological disorders

O-linked β-N-acetylglucosaminylation (O-GlcNAcylation) is involved in the regulation of many cellular cascades and neurological diseases such as Alzheimer’s disease (AD), Parkinson’s disease (PD), and stroke. In the brain, the expression of O-GlcNAcylation is notably heightened, as is that of O-linked N-acetylglucosaminyltransferase (OGT) and β-N-acetylglucosaminidase (OGA), the presence of which is prominent in many regions of neurological importance. Most importantly, O-GlcNAcylation is believed to contribute to the normal functioning of neurons; conversely, its dysregulation participates in the pathogenesis of neurological disorders. In neurodegenerative diseases, O-GlcNAcylation of the brain’s key proteins, such as tau and amyloid-β, interacts with their phosphorylation, thereby triggering the formation of neurofibrillary tangles and amyloid plaques. An increase of O-GlcNAcylation by pharmacological intervention prevents neuronal loss. Additionally, O-GlcNAcylation is stress sensitive, and its elevation is cytoprotective. Increased O-GlcNAcylation ameliorated brain damage in victims of both trauma-hemorrhage and stroke. In this review, we summarize the current understanding of O-GlcNAcylation’s physiological and pathological roles in the nervous system and provide a foundation for development of a therapeutic strategy for neurological disorders.     

Structural variations in wheat HKT1;5 underpin differences in Na<Superscript>+</Superscript> transport capacity

An important trait associated with the salt tolerance of wheat is the exclusion of sodium ions (Na⁺) from the shoot. We have previously shown that the sodium transporters TmHKT1;5-A and TaHKT1;5-D, from Triticum monoccocum (Tm) and Triticum aestivum (Ta), are encoded by genes underlying the major shoot Na⁺-exclusion loci Nax1 and Kna1, respectively. Here, using heterologous expression, we show that the affinity (K _m) for the Na⁺ transport of TmHKT1;5-A, at 2.66 mM, is higher than that of TaHKT1;5-D at 7.50 mM. Through 3D structural modelling, we identify residues D⁴⁷¹/a gap and D⁴⁷⁴/G⁴⁷³ that contribute to this property. We identify four additional mutations in amino acid residues that inhibit the transport activity of TmHKT1;5-A, which are predicted to be the result of an occlusion of the pore. We propose that the underlying transport properties of TmHKT1;5-A and TaHKT1;5-D contribute to their unique ability to improve Na⁺ exclusion in wheat that leads to an improved salinity tolerance in the field.     

Fehlermöglichkeiten bei der Bestimmung von Metabolitgehalten in Hefezellen

Summary The exact assay of metabolites like NAD, ATP and other glycolytic substances in intact yeast cells greatly depends on the method of cell extraction. Reproducible and high levels can be obtained after prolonged incubation at high temperatures or by freezing and thawing of the denatureted cellular material in perchloric acid, thus eliminating possible errors due to different recoveries in the cell-free extracts.     

Multiple overwintering mechanisms inChymomyza amoena larvae (Diptera: Drosophilidae) and laboratory induction of freeze tolerance

Summary Chymomyza amoena larvae in apples in summer were found to be either potentially freeze tolerant or to supercool. By late September only potentially freeze tolerant larvae were recovered from apples. Larvae from walnut husks in winter supercooled to avoid freezing. However, freeze tolerance could be induced in laboratory grown larvae by placing them in apples around the seed area and maintaining them at chilling temperatures for several weeks. Overwintering mechanisms employed byC. amoena larvae in Michigan appear to depend upon larval feeding site.Prof. Davis at Michigan Agricultural College placed 9C. amoena emerging from apples in August and Sept. 1891 along with codling moth in the MAC (=MSU) Entomological Museum collection.