bluuebunny/crossref_metadata_2025 huggingface dataset:
Cold Atmospheric Plasma Treatment of Chondrosarcoma Cells Affects Proliferation and Cell Membrane Permeability
Authors: Haralambiev Lyubomir , Nitsch Andreas , Jacoby Josephine M. , Strakeljahn Silas , Bekeschus Sander , Mustea Alexander , Ekkernkamp Axel , Stope Matthias B.
Published: April 2020

Chondrosarcoma is the second most common malign bone tumor in adults. Surgical resection of the tumor is recommended because of its resistance to clinical treatment such as chemotherapy and radiation therapy. Thus, the prognosis for patients mainly depends on sufficient surgical resection. Due to this, research on alternative therapies is needed. Cold atmospheric plasma (CAP) is an ionized gas that contains various reactive species. Previous studies have shown an anti-oncogenic potential of CAP on different cancer cell types. The current study examined the effects of treatment with CAP on two chondrosarcoma cell lines (CAL-78, SW1353). Through proliferation assay, the cell growth after CAP-treatment was determined. A strong antiproliferative effect for both cell lines was detected. By fluorescein diacetate (FDA) assay and ATP release assay, alterations in the cell membrane and associated translocation of low molecular weight particles through the cytoplasmic membrane were observed. In supernatant, the non-membrane-permeable FDA and endogenously synthesized ATP detected suggest an increased membrane permeability after CAP treatment. Similar results were shown by the dextran-uptake assay. Furthermore, fluorescence microscopic G-/F-actin assay was performed. G- and F-actin were selectively dyed, and the ratio was measured. The presented results indicate CAP-induced changes in cell membrane function and possible alterations in actin-cytoskeleton, which may contribute to the antiproliferative effects of CAP.
MYC-Associated Factor MAX is a Regulator of the Circadian Clock
Authors: Blaževitš Olga , Bolshette Nityanand , Vecchio Donatella , Guijarro Ana , Croci Ottavio , Campaner Stefano , Grimaldi Benedetto
Published: April 2020

The circadian transcriptional network is based on a competition between transcriptional activator and repressor complexes regulating the rhythmic expression of clock-controlled genes. We show here that the MYC-associated factor X, MAX, plays a repressive role in this network and operates through a MYC-independent binding to E-box-containing regulatory regions within the promoters of circadian BMAL1 targets. We further show that this “clock” function of MAX is required for maintaining a proper circadian rhythm and that MAX and BMAL1 contribute to two temporally alternating transcriptional complexes on clock-regulated promoters. We also identified MAX network transcriptional repressor, MNT, as a fundamental partner of MAX-mediated circadian regulation. Collectively, our data indicate that MAX regulates clock gene expression and contributes to keeping the balance between positive and negative elements of the molecular clock machinery.
Increased Purinergic Responses Dependent on P2Y2 Receptors in Hepatocytes from CCl4-Treated Fibrotic Mice
Authors: Velázquez-Miranda Erandi , Molina-Aguilar Christian , González-Gallardo Adriana , Vázquez-Martínez Olivia , Díaz-Muñoz Mauricio , Vázquez-Cuevas Francisco G
Published: April 2020

Inflammatory and wound healing responses take place during liver damage, primarily in the parenchymal tissue. It is known that cellular injury elicits an activation of the purinergic signaling, mainly by the P2X7 receptor; however, the role of P2Y receptors in the onset of liver pathology such as fibrosis has not been explored. Hence, we used mice treated with the hepatotoxin CCl4 to implement a reversible model of liver fibrosis to evaluate the expression and function of the P2Y2 receptor (P2Y2R). Fibrotic livers showed an enhanced expression of P2Y2R that eliminated its zonal distribution. Hepatocytes from CCl4-treated mice showed an exacerbated ERK-phosphorylated response to the P2Y2R-specific agonist, UTP. Cell proliferation was also enhanced in the fibrotic livers. Hepatic transcriptional analysis by microarrays, upon CCl4 administration, showed that P2Y2 activation regulated diverse pathways, revealing complex action mechanisms. In conclusion, our data indicate that P2Y2R activation is involved in the onset of the fibrotic damage associated with the reversible phase of the hepatic damage promoted by CCl4.
The Encystment-Related MicroRNAs and Its Regulation Molecular Mechanism in Pseudourostyla cristata Revealed by High Throughput Small RNA Sequencing
Authors: Pan Nan , Bhatti Muhammad Zeeshan , Zhang Haiyang , Ni Bing , Fan Xinpeng , Chen Jiwu
Published: April 2020

MicroRNAs (miRNAs) regulate the expression of target genes in diverse cellular processes and play important roles in different physiological processes. However, little is known about the microRNAome (miRNAome) during encystment of ciliated protozoa. In the current study, we first investigated the differentially expressed miRNAs and relative signaling pathways participating in the transformation of vegetative cells into dormant cysts of Pseudourostyla cristata (P. cristata). A total of 1608 known miRNAs were found in the two libraries. There were 165 miRNAs with 1217 target miRNAs. The total number of differential miRNAs screened between vegetative cells and dormant cysts databases were 449 with p < 0.05 and |log2 fold changes| > 1. Among them, the upregulated and downregulated miRNAs were 243 and 206, respectively. Furthermore, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that some of the differentially expressed miRNAs were mainly associated with oxidative phosphorylation, two-component system, and biosynthesis of amino acids. Combining with our bioinformatics analyzes, some differentially expressed miRNAs including miR-143, miR-23b-3p, miR-28, and miR-744-5p participates in the encystment of P. cristata. Based on these findings, we propose a hypothetical signaling network of miRNAs regulating or promoting P. cristata encystment. This study shed new lights on the regulatory mechanisms of miRNAs in encystment of ciliated protozoa.
Exosomal MicroRNAs Released by Activated Astrocytes as Potential Neuroinflammatory Biomarkers
Authors: Gayen Manoshi , Bhomia Manish , Balakathiresan Nagaraja , Knollmann-Ritschel Barbara
Published: April 2020

Neuroinflammation is a hallmark of several neurodegenerative diseases and disorders, including traumatic brain injury (TBI). Neuroinflammation results in the activation of glial cells which exacerbates the neuroinflammatory process by secretion of pro-inflammatory cytokines and results in disruption of glial transmission networks. The glial cells, including astrocytes, play a critical role in the maintenance of homeostasis in the brain. Activated astrocytes release several factors as part of the inflammatory process including cytokines, proteins, and microRNAs (miRNAs). MiRNAs are noncoding RNA molecules involved in normal physiological processes and disease pathogenesis. MiRNAs have been implicated as important cell signaling molecules, and they are potential diagnostic biomarkers and therapeutic targets for various diseases, including neurological disorders. Exosomal miRNAs released by astrocytic response to neuroinflammation is not yet studied. In this study, primary human astrocytes were activated by IL-1β stimulation and we examined astrocytic exosomal miRNA cargo released in a neuroinflammatory stress model. Results indicate that acute neuroinflammation and oxidative stress induced by IL-1β generates the release of a specific subset of miRNAs via exosomes, which may have a potential role in regulating the inflammatory response. Additionally, these miRNAs may serve as potential biomarkers of neuroinflammation associated with neurological disorders and injuries.
Lessons Learned from Somatic Cell Nuclear Transfer
Authors: Gouveia Chantel , Huyser Carin , Egli Dieter , Pepper Michael S.
Published: April 2020

Somatic cell nuclear transfer (SCNT) has been an area of interest in the field of stem cell research and regenerative medicine for the past 20 years. The main biological goal of SCNT is to reverse the differentiated state of a somatic cell, for the purpose of creating blastocysts from which embryonic stem cells (ESCs) can be derived for therapeutic cloning, or for the purpose of reproductive cloning. However, the consensus is that the low efficiency in creating normal viable offspring in animals by SCNT (1–5%) and the high number of abnormalities seen in these cloned animals is due to epigenetic reprogramming failure. In this review we provide an overview of the current literature on SCNT, focusing on protocol development, which includes early SCNT protocol deficiencies and optimizations along with donor cell type and cell cycle synchrony; epigenetic reprogramming in SCNT; current protocol optimizations such as nuclear reprogramming strategies that can be applied to improve epigenetic reprogramming by SCNT; applications of SCNT; the ethical and legal implications of SCNT in humans; and specific lessons learned for establishing an optimized SCNT protocol using a mouse model.
Enhancement of Biomimetic Enzymatic Mineralization of Gellan Gum Polysaccharide Hydrogels by Plant-Derived Gallotannins
Authors: Douglas Timothy E. L. , Keppler Julia K. , Vandrovcová Marta , Plencner Martin , Beranová Jana , Feuereisen Michelle , Parakhonskiy Bogdan V. , Svenskaya Yulia , Atkin Vsevolod , Ivanova Anna , Ricquier Patrick , Balcaen Lieve , Vanhaecke Frank , Schieber Andreas , Bačáková Lucie , Skirtach Andre G.
Published: April 2020

Mineralization of hydrogel biomaterials with calcium phosphate (CaP) is considered advantageous for bone regeneration. Mineralization can be both induced by the enzyme alkaline phosphatase (ALP) and promoted by calcium-binding biomolecules, such as plant-derived polyphenols. In this study, ALP-loaded gellan gum (GG) hydrogels were enriched with gallotannins, a subclass of polyphenols. Five preparations were compared, namely three tannic acids of differing molecular weight (MW), pentagalloyl glucose (PGG), and a gallotannin-rich extract from mango kernel (Mangifera indica L.). Certain gallotannin preparations promoted mineralization to a greater degree than others. The various gallotannin preparations bound differently to ALP and influenced the size of aggregates of ALP, which may be related to ability to promote mineralization. Human osteoblast-like Saos-2 cells grew in eluate from mineralized hydrogels. Gallotannin incorporation impeded cell growth on hydrogels and did not impart antibacterial activity. In conclusion, gallotannin incorporation aided mineralization but reduced cytocompatibility.
Mimicking Sampson’s Retrograde Menstrual Theory in Rats: A New Rat Model for Ongoing Endometriosis-Associated Pain
Authors: Persoons Eleonora , De Clercq Katrien , Van den Eynde Charlotte , Pinto Sílvia João Poseiro coutinho , Luyten Katrien , Van Bree Rita , Tomassetti Carla , Voets Thomas , Vriens Joris
Published: April 2020

Endometriosis is a prevalent gynecologic disease, defined by dysfunctional endometrium-like lesions outside of the uterine cavity. These lesions are presumably established via retrograde menstruation, i.e., endometrial tissue that flows backwards during menses into the abdomen and deposits on the organs. As ongoing pain is one of the main pain symptoms of patients, an animal model that illuminates this problem is highly anticipated. In the present study, we developed and validated a rat model for ongoing endometriosis-associated pain. First, menstrual endometrial tissue was successfully generated in donor rats, as validated by gross examination, histology and qPCR. Next, endometriosis was induced in recipient animals by intraperitoneal injection of menstrual tissue. This resulted in neuro-angiogenesis as well as established endometriosis lesions, which were similar to their human counterparts, since epithelial and stromal cells were observed. Furthermore, significant differences were noted between control and endometriosis animals concerning bodyweight and posture changes, indicating the presence of ongoing pain in animals with endometriosis. In summary, a rat model for endometriosis was established that reliably mimics the human pathophysiology of endometriosis and in which signs of ongoing pain were detected, thus providing a new research tool for therapy development.
Calcium Channels as Novel Therapeutic Targets for Ovarian Cancer Stem Cells
Authors: Lee Heejin , Kim Jun Woo , Kim Dae Kyung , Choi Dong Kyu , Lee Seul , Yu Ji Hoon , Kwon Oh-Bin , Lee Jungsul , Lee Dong-Seok , Kim Jae Ho , Min Sang-Hyun
Published: April 2020

Drug resistance in epithelial ovarian cancer (EOC) is reportedly attributed to the existence of cancer stem cells (CSC), because in most cancers, CSCs still remain after chemotherapy. To overcome this limitation, novel therapeutic strategies are required to prevent cancer recurrence and chemotherapy-resistant cancers by targeting cancer stem cells (CSCs). We screened an FDA-approved compound library and found four voltage-gated calcium channel blockers (manidipine, lacidipine, benidipine, and lomerizine) that target ovarian CSCs. Four calcium channel blockers (CCBs) decreased sphere formation, viability, and proliferation, and induced apoptosis in ovarian CSCs. CCBs destroyed stemness and inhibited the AKT and ERK signaling pathway in ovarian CSCs. Among calcium channel subunit genes, three L- and T-type calcium channel genes were overexpressed in ovarian CSCs, and downregulation of calcium channel genes reduced the stem-cell-like properties of ovarian CSCs. Expressions of these three genes are negatively correlated with the survival rate of patient groups. In combination therapy with cisplatin, synergistic effect was shown in inhibiting the viability and proliferation of ovarian CSCs. Moreover, combinatorial usage of manidipine and paclitaxel showed enhanced effect in ovarian CSCs xenograft mouse models. Our results suggested that four CCBs may be potential therapeutic drugs for preventing ovarian cancer recurrence.
Radiation Exposure of Peripheral Mononuclear Blood Cells Alters the Composition and Function of Secreted Extracellular Vesicles
Authors: Moertl Simone , Buschmann Dominik , Azimzadeh Omid , Schneider Michael , Kell Rosemarie , Winkler Klaudia , Tapio Soile , Hornhardt Sabine , Merl-Pham Juliane , Pfaffl Michael W. , Atkinson Michael J.
Published: April 2020

Normal tissue toxicity is a dose-limiting factor in radiation therapy. Therefore, a detailed understanding of the normal tissue response to radiation is necessary to predict the risk of normal tissue toxicity and to development strategies for tissue protection. One component of normal tissue that is continuously exposed during therapeutic irradiation is the circulating population of peripheral blood mononuclear cells (PBMC). PBMCs are highly sensitive to ionizing radiation (IR); however, little is known about how IR affects the PBMC response on a systemic level. It was the aim of this study to investigate whether IR was capable to induce changes in the composition and function of extracellular vesicles (EVs) secreted from PBMCs after radiation exposure to different doses. Therefore, whole blood samples from healthy donors were exposed to X-ray radiation in the clinically relevant doses of 0, 0.1, 2 or 6 Gy and PBMC-secreted EVs were isolated 72 h later. Proteome and miRNome analysis of EVs as well as functional studies were performed. Secreted EVs showed a dose-dependent increase in the number of significantly deregulated proteins and microRNAs. For both, proteome and microRNA data, principal component analysis showed a dose-dependent separation of control and exposed groups. Integrated pathway analysis of the radiation-regulated EV proteins and microRNAs consistently predicted an association of deregulated molecules with apoptosis, cell death and survival. Functional studies identified endothelial cells as an efficient EV recipient system, in which irradiation of recipient cells further increased the uptake. Furthermore an apoptosis suppressive effect of EVs from irradiated PBMCs in endothelial recipient cells was detected. In summary, this study demonstrates that IR modifies the communication between PBMCs and endothelial cells. EVs from irradiated PBMC donors were identified as transmitters of protective signals to irradiated endothelial cells. Thus, these data may lead to the discovery of biomarker candidates for radiation dosimetry and even more importantly, they suggest EVs as a novel systemic communication pathway between irradiated normal, non-cancer tissues.
Brain–Heart Axis and Biomarkers of Cardiac Damage and Dysfunction after Stroke: A Systematic Review and Meta-Analysis
Authors: Xu Chengyang , Zheng Ang , He Tianyi , Cao Zhipeng
Published: April 2020

Background: Cardiac complications after a stroke are the second leading cause of death worldwide, affecting the treatment and outcomes of stroke patients. Cardiac biomarkers such as cardiac troponin (cTn), brain natriuretic peptide (BNP), and N-terminal pro-brain natriuretic peptide (NT-proBNP) have been frequently reported in patients undergoing a stroke. The aim of the present study is to meta-analyze the relationship between changes in such cardiac biomarkers and stroke and to present a systematic review of the previous literature, so as to explore the brain–heart axis. Methods: We searched four online databases pertinent to the literature, including PubMed, Embase, the Cochrane Library, and the Web of Science. Then, we performed a meta-analysis to investigate changes in cTn, BNP, and NT-proBNP associated with different types of stroke. Results and Conclusions: A significant increase in cTnI concentration was found in patients exhibiting a brain hemorrhage. BNP increased in cases of brain infarction, while the NT-proBNP concentration was significantly elevated in patients suffering an acute ischemic stroke and brain hemorrhage, indicating cardiac damage and dysfunction after a stroke. Our analysis suggests that several potential mechanisms may be involved in the brain–heart axis. Finally, clinicians should pay careful attention to monitoring cardiac function in the treatment of cerebrovascular diseases in order to provide a timely and more accurate treatment.
Efficient Cardiac Differentiation of Human Amniotic Fluid-Derived Stem Cells into Induced Pluripotent Stem Cells and Their Potential Immune Privilege
Authors: Fang Yi-Hsien , Wang Saprina P.H. , Gao Zi-Han , Wu Sheng-Nan , Chang Hsien-Yuan , Yang Pei-Jung , Liu Ping-Yen , Liu Yen-Wen
Published: April 2020

Mature mammalian hearts possess very limited regenerative potential. The irreversible cardiomyocyte loss after heart injury can lead to heart failure and death. Pluripotent stem cells (PSCs) can differentiate into cardiomyocytes for cardiac repair, but there are obstacles to their clinical application. Among these obstacles is their potential for post-transplant rejection. Although human amniotic fluid-derived stem cells (hAFSCs) are immune privileged, they cannot induce cardiac differentiation. Thus, we generated hAFSC-derived induced PSCs (hAFSC-iPSCs) and used a Wnt-modulating differentiation protocol for the cardiac differentiation of hAFSC-iPSCs. In vitro studies using flow cytometry, immunofluorescence staining, and patch-clamp electrophysiological study, were performed to identify the characteristics of hAFSC-iPSC-derived cardiomyocytes (hAFSC-iPSC-CMs). We injected hAFSC-iPSC-CMs intramuscularly into rat infarcted hearts to evaluate the therapeutic potential of hAFSC-iPSC-CM transplantation. At day 21 of differentiation, the hAFSC-iPSC-CMs expressed cardiac-specific marker (cardiac troponin T), presented cardiomyocyte-specific electrophysiological properties, and contracted spontaneously. Importantly, these hAFSC-iPSC-CMs demonstrated low major histocompatibility complex (MHC) class I antigen expression and the absence of MHC class II antigens, indicating their low immunogenicity. The intramyocardial transplantation of hAFSC-iPSC-CMs restored cardiac function, partially remuscularized the injured region, and reduced fibrosis in the rat infarcted hearts. Therefore, hAFSC-iPSCs are potential candidates for the repair of infarcted myocardium.
Overexpression of Platelet-Derived Growth Factor and Its Receptor Are Correlated with Oral Tumorigenesis and Poor Prognosis in Oral Squamous Cell Carcinoma
Authors: Lin Li-Han , Lin Jiun-Sheng , Yang Cheng-Chieh , Cheng Hui-Wen , Chang Kuo-Wei , Liu Chung-Ji
Published: April 2020

Oral squamous cell carcinoma (OSCC) is a cancerous disease with poor prognosis. According to the statistics, the 5-year survival rate has not improved significantly over the past 20 years. The platelet-derived growth factor (PDGF) and its signaling pathway is a key regulator of angiogenesis and tumorigenesis. High level of PDGF and its receptor (PDGFR) have been reported in several types of malignancies. In this study, we investigated the relationship of the molecular expression levels of PDGF and PDGFR with clinicopathological parameters in OSCC. To this end, we measured the mRNA and protein levels of PDGF and PDGFR by real-time quantitative PCR (qRT-PCR), immunohistochemistry, and enzyme-linked immunosorbent assay (ELISA), respectively. We found positive correlations of the mRNA levels of PDGFA, PDGFB, and PDGFRB with lymph node metastasis and poor overall survival (OS). High expression of PDGF, PDGFRA, and PDGFRB were remarkably associated with lymph node metastasis and poor OS, as determined by immunohistochemistry. Preoperative serum levels of PDGF-AA and PDGF-BB had a positive correlation with preoperative platelet count. Elevated serum levels of PDGF-AA. PDGF-BB, and platelet count correlated with lymph node metastasis and an unfavorable outcome. In multivariate Cox regression analysis, PDGFA mRNA, PDGFB mRNA, PDGFRB mRNA, PDGF immunoexpression, PDGFRB immunoexpression, serum PDGF-AA, serum PDGF-BB, and platelet count emerged as significant independent prognostic factors for OS. In vitro, we found that elevated PDGF promotes colony formation, migration, and invasiveness of SAS and OECM-1 cancer cell lines. Our results suggest that the expression level of serum PDGF has the potential to become a useful diagnostic marker for the prognosis of OSCC. In addition, PDGFR should be considered as a potential therapeutic target for OSCC. Furthermore, research should be undertaken to elucidate the role of PDGF and PDGFR regarding the behavior of tumor cells in OSCC.
Proteomic Analysis of Renal Biomarkers of Kidney Allograft Fibrosis—A Study in Renal Transplant Patients
Authors: Mortensen Line Aas , Svane Anne Marie , Burton Mark , Bistrup Claus , Thiesson Helle Charlotte , Marcussen Niels , Beck Hans Christian
Published: April 2020

Renal transplantation is the preferred treatment of end stage renal disease, but allograft survival is limited by the development of interstitial fibrosis and tubular atrophy in response to various stimuli. Much effort has been put into identifying new protein markers of fibrosis to support the diagnosis. In the present work, we performed an in-depth quantitative proteomics analysis of allograft biopsies from 31 prevalent renal transplant patients and correlated the quantified proteins with the volume fraction of fibrosis as determined by a morphometric method. Linear regression analysis identified four proteins that were highly associated with the degree of interstitial fibrosis, namely Coagulation Factor XIII A chain (estimate 18.7, adjusted p < 0.03), Uridine Phosphorylase 1 (estimate 19.4, adjusted p < 0.001), Actin-related protein 2/3 subunit 2 (estimate 34.2, adjusted p < 0.05) and Cytochrome C Oxidase Assembly Factor 6 homolog (estimate −44.9, adjusted p < 0.002), even after multiple testing. Proteins that were negatively associated with fibrosis (p < 0.005) were primarily related to normal metabolic processes and respiration, whereas proteins that were positively associated with fibrosis (p < 0.005) were involved in catabolic processes, cytoskeleton organization and the immune response. The identified proteins may be candidates for further validation with regards to renal fibrosis. The results support the notion that cytoskeleton organization and immune responses are prevalent processes in renal allograft fibrosis.
Mapping of QTLs Associated with Yield and Yield Related Traits in Durum Wheat (Triticum durum Desf.) Under Irrigated and Drought Conditions
Authors: Rehman Arif Mian Abdur , Attaria Fauzia , Shokat Sajid , Akram Saba , Waheed Muhammad Qandeel , Arif Anjuman , Börner Andreas
Published: April 2020

Global durum wheat consumption (Triticum durum Desf.) is ahead of its production. One reason for this is abiotic stress, e.g., drought. Breeding for resistance to drought is complicated by the lack of fast, reproducible screening techniques and the inability to routinely create defined and repeatable water stress conditions. Here, we report the first analysis of dissection of yield and yield-related traits in durum wheat in Pakistan, seeking to elucidate the genetic components of yield and agronomic traits. Analysis of several traits revealed a total of 221 (160 with logarithm of odds (LOD) > 2 ≤ 3 and 61 with LOD > 3) quantitative trait loci (QTLs) distributed on all fourteen durum wheat chromosomes, of which 109 (78 with LOD > 2 ≤ 3 and 31 with LOD > 3) were observed in 2016-17 (S1) and 112 (82 with LOD > 2 ≤ 3 and 30 with LOD > 3) were observed in 2017-18 (S2). Allelic profiles of yield QTLs on chromosome 2A and 7B indicate that allele A of Xgwm895 and allele B of Xbarc276 can enhance the Yd up to 6.16% in control and 5.27% under drought. Moreover, if combined, a yield gain of up to 11% would be possible.
Aryl Hydrocarbon Receptor Activation and Tissue Factor Induction by Fluid Shear Stress and Indoxyl Sulfate in Endothelial Cells
Authors: Lano Guillaume , Laforêt Manon , Von Kotze Clarissa , Perrin Justine , Addi Tawfik , Brunet Philippe , Poitevin Stéphane , Burtey Stéphane , Dou Laetitia
Published: April 2020

Endogenous agonists of the transcription factor aryl hydrocarbon receptor (AHR) such as the indolic uremic toxin, indoxyl sulfate (IS), accumulate in patients with chronic kidney disease. AHR activation by indolic toxins has prothrombotic effects on the endothelium, especially via tissue factor (TF) induction. In contrast, physiological AHR activation by laminar shear stress (SS) is atheroprotective. We studied the activation of AHR and the regulation of TF by IS in cultured human umbilical vein endothelial cells subjected to laminar fluid SS (5 dynes/cm2). SS and IS markedly increased the expression of AHR target genes PTGS2 (encoding for COX2), AHRR, CYP1A1, and CYP1B1, as well as F3 (encoding for TF), in an AHR-dependent way. IS amplified SS-induced TF mRNA and protein expression and upregulation of AHR target genes. Interestingly, tyrosine kinase inhibition by genistein decreased SS- but not IS-induced TF expression. Finally, the increase in TF expression induced by laminar SS was not associated with increased TF activity. In contrast, IS increased TF activity, even under antithrombotic SS conditions. In conclusion, IS and SS induce AHR activation and AHR-dependent TF upregulation by different mechanisms. Impairment of the antithrombotic properties of shear stressed endothelium by toxic AHR agonists could favor cardiovascular diseases in CKD.
Calebin A Potentiates the Effect of 5-FU and TNF-β (Lymphotoxin α) against Human Colorectal Cancer Cells: Potential Role of NF-κB
Authors: Buhrmann Constanze , Kunnumakkara Ajaikumar , Popper Bastian , Majeed Muhammed , Aggarwal Bharat , Shakibaei Mehdi
Published: April 2020

Objective: The majority of chemotherapeutic agents stimulate NF-κB signaling that mediates cell survival, proliferation and metastasis. The natural turmeric non-curcuminoid derivate Calebin A has been shown to suppress cell growth, invasion and colony formation in colorectal cancer cells (CRC) by suppression of NF-κB signaling. Therefore, we hypothesized here that Calebin A might chemosensitize the TNF-β-treated tumor cells and potentiates the effect of 5-Fluorouracil (5-FU) in advanced CRC. Materials and Methods: CRC cells (HCT116) and their clonogenic 5-FU chemoresistant counterparts (HCT116R) were cultured in monolayer or alginate-based 3D tumor environment culture and were treated with/without Calebin A, TNF-β, 5-FU, BMS-345541 and DTT (dithiothreitol). Results: The results showed that TNF-β increased proliferation, invasion and resistance to apoptosis in chemoresistant CRC cells. Pretreatment with Calebin A significantly chemosensitized HCT116R to 5-FU and inhibited the TNF-β-induced enhanced efforts for survival, invasion and anti-apoptotic effects. We found further that Calebin A significantly suppressed TNF-β-induced phosphorylation and nuclear translocation of p65-NF-κB, similar to BMS-345541 (specific IKK inhibitor) and NF-κB-induced tumor-promoting biomarkers (NF-κB, β1-Integrin, MMP-9, CXCR4, Ki67). This was associated with increased apoptosis in HCT116 and HCT116R cells. Furthermore, blocking of p65-NF-κB stimulation by Calebin A was imparted through the downmodulation of p65-NF-κB binding to the DNA and this suppression was turned by DTT. Conclusion: Our findings indicate, for the first time, that Calebin A chemosensitizes human CRC cells to chemotherapy by targeting of the p65-NF-κB signaling pathway.
Cytotoxic Mechanism of Excess Polyamines Functions through Translational Repression of Specific Proteins Encoded by Polyamine Modulon
Authors: Sakamoto Akihiko , Sahara Junpei , Kawai Gota , Yamamoto Kaneyoshi , Ishihama Akira , Uemura Takeshi , Igarashi Kazuei , Kashiwagi Keiko , Terui Yusuke
Published: April 2020

Excessive accumulation of polyamines causes cytotoxicity, including inhibition of cell growth and a decrease in viability. We investigated the mechanism of cytotoxicity caused by spermidine accumulation under various conditions using an Escherichia coli strain deficient in spermidine acetyltransferase (SAT), a key catabolic enzyme in controlling polyamine levels. Due to the excessive accumulation of polyamines by the addition of exogenous spermidine to the growth medium, cell growth and viability were markedly decreased through translational repression of specific proteins [RMF (ribosome modulation factor) and Fis (rRNA transcription factor) etc.] encoded by members of polyamine modulon, which are essential for cell growth and viability. In particular, synthesis of proteins that have unusual locations of the Shine–Dalgarno (SD) sequence in their mRNAs was inhibited. In order to elucidate the molecular mechanism of cytotoxicity by the excessive accumulation of spermidine, the spermidine-dependent structural change of the bulged-out region in the mRNA at the initiation site of the rmf mRNA was examined using NMR analysis. It was suggested that the structure of the mRNA bulged-out region is affected by excess spermidine, so the SD sequence of the rmf mRNA cannot approach initiation codon AUG.
Chronic Regulation of miR-124-3p in the Perilesional Cortex after Experimental and Human TBI
Authors: Vuokila Niina , Aronica Eleonora , Korotkov Anatoly , van Vliet Erwin Alexander , Nuzhat Salma , Puhakka Noora , Pitkänen Asla
Published: April 2020

Traumatic brain injury (TBI) dysregulates microRNAs, which are the master regulators of gene expression. Here we investigated the changes in a brain-enriched miR-124-3p, which is known to associate with major post-injury pathologies, such as neuroinflammation. RT-qPCR of the rat tissue sampled at 7 d and 3 months in the perilesional cortex adjacent to the necrotic lesion core (aPeCx) revealed downregulation of miR-124-3p at 7 d (fold-change (FC) 0.13, p < 0.05 compared with control) and 3 months (FC 0.40, p < 0.05) post-TBI. In situ hybridization confirmed the downregulation of miR-124-3p at 7 d and 3 months post-TBI in the aPeCx (both p < 0.01). RT-qPCR confirmed the upregulation of the miR-124-3p target Stat3 in the aPeCx at 7 d post-TBI (7-fold, p < 0.05). mRNA-Seq revealed 312 downregulated and 311 upregulated miR-124 targets (p < 0.05). To investigate whether experimental findings translated to humans, we performed in situ hybridization of miR-124-3p in temporal lobe autopsy samples of TBI patients. Our data revealed downregulation of miR-124-3p in individual neurons of cortical layer III. These findings indicate a persistent downregulation of miR-124-3p in the perilesional cortex that might contribute to post-injury neurodegeneration and inflammation.
miR-1285-3p Controls Colorectal Cancer Proliferation and Escape from Apoptosis through DAPK2
Authors: Villanova Lidia , Barbini Chiara , Piccolo Cristina , Boe Alessandra , De Maria Ruggero , Fiori Micol Eleonora
Published: April 2020

MicroRNAs are tiny but powerful regulators of gene expression at the post-transcriptional level. Aberrant expression of oncogenic and tumor-suppressor microRNAs has been recognized as a common feature of human cancers. Colorectal cancer represents a major clinical challenge in the developed world and the design of innovative therapeutic approaches relies on the identification of novel biological targets. Here, we perform a functional screening in colorectal cancer cells using a library of locked nucleic acid (LNA)-modified anti-miRs in order to unveil putative oncogenic microRNAs whose inhibition yields a cytotoxic effect. We identify miR-1285-3p and further explore the effect of its targeting in both commercial cell lines and primary colorectal cancer stem cells, finding induction of cell cycle arrest and apoptosis. We show that DAPK2, a known tumor-suppressor, is a novel miR-1285 target and mediates both the anti-proliferative and the pro-apoptotic effects of miR-1285 depletion. Altogether, our findings uncover a novel oncogenic microRNA in colorectal cancer and lay the foundation for further studies aiming at the development of possible therapeutic strategies based on miR-1285 targeting.
Functional Study of the Retrotransposon-Derived Human PEG10 Protease
Authors: Golda Mária , Mótyán János András , Mahdi Mohamed , Tőzsér József
Published: April 2020

Paternally expressed gene 10 (PEG10) is a human retrotransposon-derived imprinted gene. The mRNA of PEG10 encodes two protein isoforms: the Gag-like protein (RF1PEG10) is coded by reading frame 1, while the Gag-Pol-like polyprotein (RF1/RF2PEG10) is coded by reading frames 1 and 2. The proteins are translated by a typical retroviral frameshift mechanism. The protease (PR) domain of RF2PEG10 contains an -Asp-Ser-Gly- sequence, which corresponds to the consensus -Asp-Ser/Thr-Gly- active-site motif of retroviral aspartic proteases. The function of the aspartic protease domain of RF2PEG10 remains unclear. To elucidate the function of PEG10 protease (PRPEG10), we designed a frameshift mutant (fsRF1/RF2PEG10) for comparison with the RF1/RF2PEG10 form. To study the effects of PRPEG10 on cellular proliferation and viability, mammalian HEK293T and HaCaT cells were transfected with plasmids coding for either RF1/RF2PEG10, the frameshift mutant (fsRF1/RF2PEG10), or a PR active-site (D370A) mutant fsRF1/RF2PEG10. Our results indicate that fsRF1/RF2PEG10 overexpression results in increased cellular proliferation. Remarkably, transfection with fsRF1/RF2PEG10 had a detrimental effect on cell viability. We hypothesize that PRPEG10 plays an important role in the function of this retroviral remnant, mediating the proliferation of cells and possibly implicating it in the inhibition of apoptosis.
Zinc Differentially Modulates the Assembly of Soluble and Polymerized Vimentin
Authors: Mónico Andreia , Zorrilla Silvia , Rivas Germán , Pérez-Sala Dolores
Published: April 2020

The intermediate filament protein vimentin constitutes a critical sensor for electrophilic and oxidative stress. We previously showed that vimentin interacts with zinc, which affects its assembly and redox sensing. Here, we used vimentin wt and C328S, an oxidation-resistant mutant showing improved NaCl-induced polymerization, to assess the impact of zinc on soluble and polymerized vimentin by light scattering and electron microscopy. Zinc acts as a switch, reversibly inducing the formation of vimentin oligomeric species. High zinc concentrations elicit optically-detectable vimentin structures with a characteristic morphology depending on the support. These effects also occur in vimentin C328S, but are not mimicked by magnesium. Treatment of vimentin with micromolar ZnCl2 induces fibril-like particles that do not assemble into filaments, but form aggregates upon subsequent addition of NaCl. In contrast, when added to NaCl-polymerized vimentin, zinc increases the diameter or induces lateral association of vimentin wt filaments. Remarkably, these effects are absent or attenuated in vimentin C328S filaments. Therefore, the zinc-vimentin interaction depends on the chemical environment and on the assembly state of the protein, leading to atypical polymerization of soluble vimentin, likely through electrostatic interactions, or to broadening and lateral association of preformed filaments through mechanisms requiring the cysteine residue. Thus, the impact of zinc on vimentin assembly and redox regulation is envisaged.
Amination of Graphene Oxide Leads to Increased Cytotoxicity in Hepatocellular Carcinoma Cells
Authors: Georgieva Milena , Vasileva Bela , Speranza Giorgio , Wang Dayong , Stoyanov Kalin , Draganova-Filipova Milena , Zagorchev Plamen , Sarafian Victoria , Miloshev George , Krasteva Natalia
Published: April 2020

Clinically, there is an urgent need to identify new therapeutic strategies for selectively treating cancer cells. One of the directions in this research is the development of biocompatible therapeutics that selectively target cancer cells. Here, we show that novel aminated graphene oxide (haGO-NH2) nanoparticles demonstrate increased toxicity towards human hepatocellular cancer cells compared to pristine graphene oxide(GO). The applied novel strategy for amination leads to a decrease in the size of haGO-NH2 and their zeta potential, thus, assuring easier penetration through the cell membrane. After characterization of the biological activities of pristine and aminated GO, we have demonstrated strong cytotoxicity of haGO-NH2 toward hepatic cancer cells—HepG2 cell line, in a dose-dependent manner. We have presented evidence that the cytotoxic effects of haGO-NH2 on hepatic cancer cells were due to cell membrane damage, mitochondrial dysfunction and increased reactive oxygen species (ROS) production. Intrinsically, our current study provides new rationale for exploiting aminated graphene oxide as an anticancer therapeutic.
Evaluation of the Potential Role of Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) in Niemann–Pick Disease, Type C1
Authors: Cawley Niamh X. , Lyons Anna T. , Abebe Daniel , Wassif Christopher A. , Porter Forbes D.
Published: April 2020

Niemann–Pick disease, type C1, is a cholesterol storage disease where unesterified cholesterol accumulates intracellularly. In the cerebellum this causes neurodegeneration of the Purkinje neurons that die in an anterior-to-posterior and time-dependent manner. This results in cerebellar ataxia as one of the major outcomes of the disease. Proprotein convertase subtilisin/kexin type 9 (PCSK9) plays a significant role in the regulation of serum cholesterol levels by modulating LDL receptor levels on peripheral tissues. In the central nervous system, PCSK9 may have a similar effect on the closely related VLDL and ApoE2 receptors to regulate brain cholesterol. In addition, regulation of VLDLR and ApoER2 by PCSK9 may contribute to neuronal apoptotic pathways through Reelin, the primary ligand of VLDLR and ApoER2. Defects in reelin signaling results in cerebellar dysfunction leading to ataxia as seen in the Reeler mouse. Our recent findings that Pcsk9 is expressed ~8-fold higher in the anterior lobules of the cerebellum compared to the posterior lobule X, which is resistant to neurodegeneration, prompted us to ask whether PCSK9 could play a role in NPC1 disease progression. We addressed this question genetically, by characterizing NPC1 disease in the presence or absence of PCSK9. Analysis of double mutant Pcsk9-/-/Npc1-/- mice by disease severity scoring, motor assessments, lifespan, and cerebellar Purkinje cell staining, showed no obvious difference in NPC1 disease progression with that of Npc1-/- mice. This suggests that PCSK9 does not play an apparent role in NPC1 disease progression.
Exploring the Etiological Links behind Neurodegenerative Diseases: Inflammatory Cytokines and Bioactive Kynurenines
Authors: Tanaka Masaru , Toldi József , Vécsei László
Published: April 2020

Alzheimer’s disease (AD) and Parkinson’s disease (PD) are the most common neurodegenerative diseases (NDs), presenting a broad range of symptoms from motor dysfunctions to psychobehavioral manifestations. A common clinical course is the proteinopathy-induced neural dysfunction leading to anatomically corresponding neuropathies. However, current diagnostic criteria based on pathology and symptomatology are of little value for the sake of disease prevention and drug development. Overviewing the pathomechanism of NDs, this review incorporates systematic reviews on inflammatory cytokines and tryptophan metabolites kynurenines (KYNs) of human samples, to present an inferential method to explore potential links behind NDs. The results revealed increases of pro-inflammatory cytokines and neurotoxic KYNs in NDs, increases of anti-inflammatory cytokines in AD, PD, Huntington’s disease (HD), Creutzfeldt–Jakob disease, and human immunodeficiency virus (HIV)-associated neurocognitive disorders, and decreases of neuromodulatory KYNs in AD, PD, and HD. The results reinforced a strong link between inflammation and neurotoxic KYNs, confirmed activation of adaptive immune response, and suggested a possible role in the decrease of neuromodulatory KYNs, all of which may contribute to the development of chronic low grade inflammation. Commonalities of multifactorial NDs were discussed to present a current limit of diagnostic criteria, a need for preclinical biomarkers, and an approach to search the initiation factors of NDs.
Molecular Characterization of Tc964, A Novel Antigenic Protein from Trypanosoma cruzi
Authors: Ruiz-Márvez Elizabeth , Ramírez César Augusto , Rodríguez Eliana Rocío , Flórez Magda Mellisa , Delgado Gabriela , Guzmán Fanny , Gómez-Puertas Paulino , Requena José María , Puerta Concepción J.
Published: April 2020

The Tc964 protein was initially identified by its presence in the interactome associated with the LYT1 mRNAs, which code for a virulence factor of Trypanosoma cruzi. Tc964 is annotated in the T. cruzi genome as a hypothetical protein. According to phylogenetic analysis, the protein is conserved in the different genera of the Trypanosomatidae family; however, recognizable orthologues were not identified in other groups of organisms. Therefore, as a first step, an in-depth molecular characterization of the Tc946 protein was carried out. Based on structural predictions and molecular dynamics studies, the Tc964 protein would belong to a particular class of GTPases. Subcellular fractionation analysis indicated that Tc964 is a nucleocytoplasmic protein. Additionally, the protein was expressed as a recombinant protein in order to analyze its antigenicity with sera from Chagas disease (CD) patients. Tc964 was found to be antigenic, and B-cell epitopes were mapped by the use of synthetic peptides. In parallel, the Leishmania major homologue (Lm964) was also expressed as recombinant protein and used for a preliminary evaluation of antigen cross-reactivity in CD patients. Interestingly, Tc964 was recognized by sera from Chronic CD (CCD) patients at different stages of disease severity, but no reactivity against this protein was observed when sera from Colombian patients with cutaneous leishmaniasis were analyzed. Therefore, Tc964 would be adequate for CD diagnosis in areas where both infections (CD and leishmaniasis) coexist, even though additional assays using larger collections of sera are needed in order to confirm its usefulness for differential serodiagnosis.
Human Melanoma-Cell Metabolic Profiling: Identification of Novel Biomarkers Indicating Metastasis
Authors: Kosmopoulou Mariangela , Giannopoulou Aikaterini F. , Iliou Aikaterini , Benaki Dimitra , Panagiotakis Aristeidis , Velentzas Athanassios D. , Konstantakou Eumorphia G. , Papassideri Issidora S. , Mikros Emmanuel , Stravopodis Dimitrios J. , Gikas Evagelos
Published: April 2020

Melanoma is the most aggressive type of skin cancer, leading to metabolic rewiring and enhancement of metastatic transformation. Efforts to improve its early and accurate diagnosis are largely based on preclinical models and especially cell lines. Hence, we herein present a combinational Nuclear Magnetic Resonance (NMR)- and Ultra High Performance Liquid Chromatography-High-Resolution Tandem Mass Spectrometry (UHPLC-HRMS/MS)-mediated untargeted metabolomic profiling of melanoma cells, to landscape metabolic alterations likely controlling metastasis. The cell lines WM115 and WM2664, which belong to the same patient, were examined, with WM115 being derived from a primary, pre-metastatic, tumor and WM2664 clonally expanded from lymph-node metastases. Metabolite samples were analyzed using NMR and UHPLC-HRMS. Multivariate statistical analysis of high resolution NMR and MS (positive and negative ionization) results was performed by Principal Component Analysis (PCA), Partial Least Squares-Discriminant Analysis (PLS-DA) and Orthogonal Partial Least Squares-Discriminant Analysis (OPLS-DA), while metastasis-related biomarkers were determined on the basis of VIP lists, S-plots and Student’s t-tests. Receiver Operating Characteristic (ROC) curves of NMR and MS data revealed significantly differentiated metabolite profiles for each cell line, with WM115 being mainly characterized by upregulated levels of phosphocholine, choline, guanosine and inosine. Interestingly, WM2664 showed notably increased contents of hypoxanthine, myo-inositol, glutamic acid, organic acids, purines, pyrimidines, AMP, ADP, ATP and UDP(s), thus indicating the critical roles of purine, pyrimidine and amino acid metabolism during human melanoma metastasis.
Structural and Magnetic Studies of Bulk Nanocomposite Magnets Derived from Rapidly Solidified Pr-(Fe,Co)-(Zr,Nb)-B Alloy
Authors: Pawlik Katarzyna , Pawlik Piotr , Wysłocki Jerzy J. , Kaszuwara Waldemar
Published: April 2020

In the present study, the phase constitution, microstructure and magnetic properties of the nanocrystalline magnets, derived from fully amorphous or partially crystalline samples by annealing, were analyzed and compared. The melt-spun ribbons (with a thickness of ~30 µm) and suction-cast 0.5 mm and 1 mm thick plates of the Pr9Fe50Co13Zr1Nb4B23 alloy were soft magnetic in the as-cast state. In order to modify their magnetic properties, the annealing process was carried out at various temperatures from 923K to 1033K for 5 min. The Rietveld refinement of X-ray diffraction patterns combined with the partial or no known crystal structures (PONKCS) method allowed one to quantify the component phases and calculate their crystalline grain sizes. It was shown that the volume fraction of constituent phases and their crystallite sizes for the samples annealed at a particular temperature, dependent on the rapid solidification conditions, and thus a presence or absence of the crystallization nuclei in the as-cast state. Additionally, a thermomagnetic analysis was used as a complementary method to confirm the phase constitution. The hysteresis loops have shown that most of the samples exhibit a remanence enhancement typical for the soft/hard magnetic nanocomposite. Moreover, for the plates annealed at the lowest temperatures, the highest coercivities up to ~1150 kA/m were measured.