Integrated glycomic analysis of ovarian cancer side population cells

Additional file 2. The category of representative lectins for glycan profiling

Microglia Mediate Synaptic Material Clearance at the Early Stage of Rats With Retinitis Pigmentosa

Resident microglia are the main immune cells in the retina and play a key role in the pathogenesis of retinitis pigmentosa (RP). Many previous studies on the roles of microglia mainly focused on the neurotoxicity or neuroprotection of photoreceptors, while their contributions to synaptic remodeling of neuronal circuits in the retina of early RP remained unclarified. In the present study, we used Royal College of Surgeons (RCS) rats, a classic RP model characterized by progressive microglia activation and synapse loss, to investigate the constitutive effects of microglia on the synaptic lesions and ectopic neuritogenesis. Rod degeneration resulted in synapse disruption and loss in the outer plexiform layer (OPL) at the early stage of RP. Coincidentally, the resident microglia in the OPL increased phagocytosis and mainly engaged in phagocytic engulfment of postsynaptic mGluR6 of rod bipolar cells (RBCs). Complement pathway might be involved in clearance of postsynaptic elements of RBCs by microglia. We pharmacologically deleted microglia using a CSF1 receptor (CSF1R) inhibitor to confirm this finding, and found that it caused the accumulation of postsynaptic mGluR6 levels and increased the number and length of ectopic dendrites in the RBCs. Interestingly, the numbers of presynaptic sites expressing CtBP2 and colocalized puncta in the OPL of RCS rats were not affected by microglia elimination. However, sustained microglial depletion led to progressive functional deterioration in the retinal responses to light in RCS rats. Based on our results, microglia mediated the remodeling of RBCs by phagocytosing postsynaptic materials and inhibiting ectopic neuritogenesis, contributing to delay the deterioration of vision at the early stage of RP

Expression und Regulation des Neuronalen Kalziumsensorproteins VILIP-1 und die Auswirkung auf den α4β2 nAChR in hippocampalen Neuronen der Ratte und in Zelllinien: Implikationen für synaptische Plastizität

0.Title 1.Abstract 2.Introduction 3.Aim 4.Materials and Methods 5.Results 6.Discussion 7.Conclusion and Prospects 8.Zusammenfassung 9.Reference 10.AppendixVisinin-like protein-1 (VILIP-1) is a neuronal EF-hand Ca2+-binding protein. Here, I describe the distribution of the immunoreactivity detected by specific antibodies against VILIP-1 protein in rat peripheral tissue and in the brain at the light microscopic level. VILIP-1 antibodies stain neurons, but not glial cells, in the whole rat hippocampal formation, including principal cells in the CA1 and CA3 region of the Ammon's horn and in the dentate gyrus. Moreover, to analyze the precise distribution of VILIP-1 in hippocampal interneurons, immunofluorescence co-staining of VILIP-1 with the GABAergic neuronal marker, glutamic acid decarboxylase 67 (GAD67), and with different well defined neurochemical markers for perisomatic inhibitory cells (parvalbumin, PV), dendritic inhibitory cells (calbindin-D28k), and interneurons specialized to innervate other interneurons (calretinin, CR), were performed in rat hippocampal slices. Additionally, the co-localization of VILIP-1 with one of its interaction partners, the α4β2 nicotinic acetylcholine receptor (nAChR) has been studied. The α4β2 nAChR is the most abundant nAChR subtype with high-affinity for nicotine in the brain. The α4β2 nAChR is crucial for nociception, nicotine addiction and the beneficial effects of nicotine on cognition. VILIP-1, recently shown to affect clathrin- dependent receptor trafficking, interacts with the cytoplasmic loop of the α4 subunit. VILIP-1 enhances ACh responsiveness of hippocampal neurons, possibly comprising a novel form of physiological up-regulation of α4β2 nAChR. In the hippocampal formation VILIP-1 is co-localized with α4β2 nAChR in a distinct neuronal subpopulation consisting particularly of interneurons. Furthermore, enhancement of α4β2 nAChR induced by VILIP-1 leads to enhancement of inhibitory postsynaptic currents (IPSCs). Thus, VILIP-1 in conjunction with the α4β2 receptor might play an important role in modulating hippocampal network activity and synaptic plasticity. Interestingly in this context, induction of long term potentiation in the hippocampus correlates with increased expression levels of neuronal calcium sensor (NCS) proteins. We have investigated mGluR- and time-dependent changes in the expression of two different NCS proteins. Following DHPG application in vivo NCS-1 and VILIP-1 expression increased, with significant levels reached after 8 and 24h. Furthermore, the physiological effects of VILIP-1 upregulation were studied using patch-clamp recording from rat hippocampal neurons in vitro, indicating effects of VILIP-1 on neuronal excitability of hippocampal neurons.In der vorliegenden Arbeit beschreibe ich auf lichtmikroskopischer Ebene die Immunreaktivität spezifischer Antikörper gegen Visinin-like protein-1 (VILIP-1), ein neuronales Kalzium (Ca2+)-bindendes Protein, in peripherem Gewebe sowie in Hirngewebe der Ratte. VILIP-1 Antikörper markieren Neurone in der ganzen Hippokampusformation, beispielsweise die Prinzipalzellen der CA1- und CA3-Region des Ammonshorns und Nervenzellen des Gyrus Dentatus, jedoch keine Gliazellen. Desweiteren wurden in hippokampalen Schnitten der Ratte Immunfluoreszenz-Doppelfärbungen durchgeführt, um das Vorkommen von VILIP-1 in Interneuronen des Hippocampus genau zu analysieren. Dazu wurden der GABAerge neuronale Marker GAD67 (glutamic acid decarboxylase 67) und verschiedene gut charakterisierte neurochemische Marker für perisomatische inhibitorische Zellen (Parvalbumin, PV), für dendritische inhibitorische Zellen (Calbindin- D28k) und für Interneurone, die spezifische andere Interneurone innervieren (Calretinin, CR) eingesetzt. Weiterhin wurde die Kolokalisation von VILIP-1 mit einem seiner Interaktionspartner, dem α4β2 nikotinischen Azetylcholinrezeptor (nAChR), untersucht. Der α4β2 nAChR ist der häufigste Subtyp dieses Rezeptors im Gehirn mit einer hohen Affinität für Nikotin. Dieser Subtyp spielt eine wichtige Rolle bei der Nocizeption, bei der Nikotin- Abhängigkeit aber auch den positiven Effekten von Nikotin auf Kognition. VILIP-1, für das kürzlich gezeigt wurde, dass es den Clathrin-abhängige Rezeptortransport beeinflusst, interagiert mit der zytoplasmatischen Schleife der α4-Untereinheit des Rezeptors. Das Kalziumsensorprotein verstärkt das Reaktionsvermögen hippokampaler Neurone auf ACh wahrscheinlich über einen neuen Weg der funktionellen Hochregulierung des α4β2 nAChR. In der Hippokampusformation ist VILIP-1 mit dem α4β2 nAChR in einer neuronalen Subpopulation kolokalisiert, die insbesondere aus Interneuronen besteht. Die Verstärkung der Rezeptorantworten, die durch VILIP-1 ausgelöst wird, führt zu einer Verstärkung inhibitorischer postsynaptischer Ströme (IPSCs). Daher ist VILIP-1 in Verbindung mit dem α4β2 nAChR ein möglicher Modulator hippokampaler Netzwerkaktivität und synaptischer Plastizität. In diesem Zusammenhang ist interessant, dass die Induktion von Langzeitpotenzierung im Hippokampus mit einer erhöhten Expression von Kalciumsensorproteinen (NCS) korreliert. Wir haben mGluR- und zeitabhängige Veränderungen in der Expressionsstärke von zwei verschiedenen NCS-Proteinen untersucht. Als Resultat der Applikation von DHPG in vivo erhöhte sich die Expression von NCS-1 und VILIP-1 nach 8 und nach 24 Stunden signifikant. Zusätzlich wurden zur Analyse des physiologischen Effektes der erhöhten VILIP-1 Expression elektrophysiologische Messungen ( Patch-Clamp -Ganzzellableitungen) an hippokampalen Neuronen der Ratte in vitro durchgeführt, deren Ergebnisse auf einen Einfluss von VILIP-1 auf die neuronale Erregbarkeit hippokampaler Neurone hindeuten

Homeostatic Plasticity Mediated by Rod-Cone Gap Junction Coupling in Retinal Degenerative Dystrophic RCS Rats

Rod-cone gap junctions open at night to allow rod signals to pass to cones and activate the cone-bipolar pathway. This enhances the ability to detect large, dim objects at night. This electrical synaptic switch is governed by the circadian clock and represents a novel form of homeostatic plasticity that regulates retinal excitability according to network activity. We used tracer labeling and ERG recording in the retinae of control and retinal degenerative dystrophic RCS rats. We found that in the control animals, rod-cone gap junction coupling was regulated by the circadian clock via the modulation of the phosphorylation of the melatonin synthetic enzyme arylalkylamine N-acetyltransferase (AANAT). However, in dystrophic RCS rats, AANAT was constitutively phosphorylated, causing rod-cone gap junctions to remain open. A further b/a-wave ratio analysis revealed that dystrophic RCS rats had stronger synaptic strength between photoreceptors and bipolar cells, possibly because rod-cone gap junctions remained open. This was despite the fact that a decrease was observed in the amplitude of both a- and b-waves as a result of the progressive loss of rods during early degenerative stages. These results suggest that electric synaptic strength is increased during the day to allow cone signals to pass to the remaining rods and to be propagated to rod bipolar cells, thereby partially compensating for the weak visual input caused by the loss of rods

Next-Generation Sequencing Panel Analysis of Clinically Relevant Mutations in Circulating Cell-Free DNA from Patients with Gestational Trophoblastic Neoplasia: A Pilot Study

Gestational trophoblastic neoplasia (GTN) originates from placental tissue and exhibits the potential for invasion and metastasis. Gene alterations in GTN have not been extensively studied because of a lack of qualified tumor specimens after chemotherapy. GTN has a rapid growth rate and is highly metastatic, which makes circulating tumor DNA (ctDNA) sequencing a promising modality for gene profiling. Accordingly, in this study, we performed targeted next-generation sequencing (NGS) of 559 tumor-associated genes using circulating cell-free DNA (cfDNA) collected prior to chemotherapy from 11 patients with GTN. All sequenced genes were associated with oncogenesis, progression, and targeted therapy. The average cfDNA level was 0.43 ± 0.22 ng/μL. Significant correlations were found between cfDNA concentration and maximum lesion diameter (r = 0.625, p=0.040) and time for human chorionic gonadotropin beta subunit (β-HCG) recovering to normal level (r = 0.609, p=0.047). There were no significant correlations between cfDNA concentrations and β-HCG expression level or lung metastasis. ctDNA mutations were detected in all patients, and 73 mutant genes were detected in 11 patients. BMPR1A (27.3%), LRP1B (27.3%), ERCC4 (18.2%), FGF14 (18.2%), HSP90AA1 (18.2%), KAT6A (18.2%), KMT2D (18.2%), MAP3K1 (18.2%), RANBP2 (18.2%), and ZNF217 (18.2%) mutations were detected as overlapping mutations. The mRNA and protein levels of bone morphogenetic protein receptor type 1A were significantly downregulated in human JAR and JEG-3 choriocarcinoma cells (p<0.0001), whereas mRNA and protein levels of mitogen-activated protein kinase kinase kinase 1 were upregulated in these two cell lines (p=0.0128, p=0.0012, respectively). These genes may play important roles in GTN initiation and progression and may be candidate targets for GTN treatment. These findings suggested that cfDNA levels could provide potential assessment value in disease severity of GTN and that ctDNA sequencing was a promising approach for identifying gene mutations in GTN

Retro-inverso follicle-stimulating hormone peptide-mediated polyethylenimine complexes for targeted ovarian cancer gene therapy

Background: The development of nanoparticle drug delivery systems with targeted ligands has the potential to increase treatment efficiency in ovarian cancer. Methods: We developed a 21-amino acid peptide, YTRDLVYGDPARPGIQGTGTF (L-FP21) conjugated to polyethylenimine (PEI) and methoxy polyethylene glycol (mPEG) to prepare a nanoparticle drug vehicle to target follicle-stimulating hormone receptor (FSHR) in ovarian cancer. At the same time, we optimized the ligand of the nanoparticle vehicle using D-peptides, which consist of D-amino acids (D-FP21). Nanoparticle vehicles carrying the therapeutic gene plasmid growth-regulated oncogene alpha (pGRO-α) short hairpin RNA (shRNA) (FP21-PEG-PEI/pGRO-α) were prepared for further investigation. Results: Compared with L-FP21, D-FP21 exhibited improved biological stability and higher uptake rate for FSHR-expressing ovarian cancer cells. The cytotoxicity of the L, D-FP21-PEG-PEI/pGRO-α complexes were significantly lower than that of the PEI/pGRO-α complex. The nanoparticle drug with the targeted ligand showed higher transfection efficiencies and improved anti-proliferation effects for ovarian cancer cells than that without the targeted ligand (mPEG-PEI/pGRO-α). Furthermore, an in vivo evaluation of an antitumor assay indicated that D-FP21-PEG-PEI/pGRO-α inhibited the growth of tumor spheroids considerably more than L-FP21-PEG-PEI/pGRO-α; their tumor inhibition rates were 58.5% and 33.3%, respectively. Conclusions: D-FP21-PEG-PEI/plasmid DNA is a safe and efficient gene delivery vehicle for ovarian cancer targeted therapy

PD-L1 Expression Predicts a Distinct Prognosis in Krukenberg Tumor with Corresponding Origins

Krukenberg tumor (KT) is an uncommon ovarian metastatic signet-ring cell adenocarcinoma that mostly metastasizes from gastrointestinal carcinoma. Optimal treatment options for KTs are limited. Programmed death-1 (PD-1)/programmed death-ligand 1 (PD-L1) inhibitors have shown remarkable activity in clinical trials for metastatic tumors. Here, we evaluated PD-L1 expression and T cell infiltration in KTs and their corresponding primary tumors. Positive tumor PD-L1 expression was detected in 9 (25.7%) KTs from gastric carcinomas (GCs) and in 20 (66.7%) KTs from colorectal carcinomas (CRCs). Patient survival was assessed according to the PD-L1 status and CD8+ T cell density. Positive tumor PD-L1 expression in KTs from GCs was associated with poor prognosis. In contrast, positive tumor PD-L1 expression in KTs from CRCs was associated with an improved prognosis. We analyzed copy number variations of the PD-L1 gene in KTs. PD-L1 expression was higher in cases with copy number gains. The T cell densities within KTs and their corresponding primary tumors were compared. The densities of CD8+ T cells correlated significantly between the primary tumors and KTs from the same case. Taken together, the research further highlighted targets for immune-based therapy in KTs from GCs and CRCs