bluuebunny/crossref_metadata_2025 huggingface dataset:
Virulence Factors of Meningitis-Causing Bacteria: Enabling Brain Entry across the Blood–Brain Barrier
Authors: Herold Rosanna , Schroten Horst , Schwerk Christian
Published: October 2019

Infections of the central nervous system (CNS) are still a major cause of morbidity and mortality worldwide. Traversal of the barriers protecting the brain by pathogens is a prerequisite for the development of meningitis. Bacteria have developed a variety of different strategies to cross these barriers and reach the CNS. To this end, they use a variety of different virulence factors that enable them to attach to and traverse these barriers. These virulence factors mediate adhesion to and invasion into host cells, intracellular survival, induction of host cell signaling and inflammatory response, and affect barrier function. While some of these mechanisms differ, others are shared by multiple pathogens. Further understanding of these processes, with special emphasis on the difference between the blood–brain barrier and the blood–cerebrospinal fluid barrier, as well as virulence factors used by the pathogens, is still needed.
In Vitro and In Vivo Pipeline for Validation of Disease-Modifying Effects of Systems Biology-Derived Network Treatments for Traumatic Brain Injury—Lessons Learned
Authors: Lipponen Anssi , Natunen Teemu , Hujo Mika , Ciszek Robert , Hämäläinen Elina , Tohka Jussi , Hiltunen Mikko , Paananen Jussi , Poulsen David , Kansanen Emilia , Ekolle Ndode-Ekane Xavier , Levonen Anna-Liisa , Pitkänen Asla
Published: October 2019

We developed a pipeline for the discovery of transcriptomics-derived disease-modifying therapies and used it to validate treatments in vitro and in vivo that could be repurposed for TBI treatment. Desmethylclomipramine, ionomycin, sirolimus and trimipramine, identified by in silico LINCS analysis as candidate treatments modulating the TBI-induced transcriptomics networks, were tested in neuron-BV2 microglial co-cultures, using tumour necrosis factor α as a monitoring biomarker for neuroinflammation, nitrite for nitric oxide-mediated neurotoxicity and microtubule associated protein 2-based immunostaining for neuronal survival. Based on (a) therapeutic time window in silico, (b) blood-brain barrier penetration and water solubility, (c) anti-inflammatory and neuroprotective effects in vitro (p < 0.05) and (d) target engagement of Nrf2 target genes (p < 0.05), desmethylclomipramine was validated in a lateral fluid-percussion model of TBI in rats. Despite the favourable in silico and in vitro outcomes, in vivo assessment of clomipramine, which metabolizes to desmethylclomipramine, failed to demonstrate favourable effects on motor and memory tests. In fact, clomipramine treatment worsened the composite neuroscore (p < 0.05). Weight loss (p < 0.05) and prolonged upregulation of plasma cytokines (p < 0.05) may have contributed to the worsened somatomotor outcome. Our pipeline provides a rational stepwise procedure for evaluating favourable and unfavourable effects of systems-biology discovered compounds that modulate post-TBI transcriptomics.
A Novel Claudinopathy Based on Claudin-10 Mutations
Authors: Milatz Susanne
Published: October 2019

Claudins are key components of the tight junction, sealing the paracellular cleft or composing size-, charge- and water-selective paracellular channels. Claudin-10 occurs in two major isoforms, claudin-10a and claudin-10b, which constitute paracellular anion or cation channels, respectively. For several years after the discovery of claudin-10, its functional relevance in men has remained elusive. Within the past two years, several studies appeared, describing patients with different pathogenic variants of the CLDN10 gene. Patients presented with dysfunction of kidney, exocrine glands and skin. This review summarizes and compares the recently published studies reporting on a novel autosomal-recessive disorder based on claudin-10 mutations.
Inverse Regulation of Lipocalin-2/24p3 Receptor/SLC22A17 and Lipocalin-2 Expression by Tonicity, NFAT5/TonEBP and Arginine Vasopressin in Mouse Cortical Collecting Duct Cells mCCD(cl.1): Implications for Osmotolerance
Authors: Probst Stephanie , Scharner Bettina , McErlean Ruairi , Lee Wing-Kee , Thévenod Frank
Published: October 2019

The rodent collecting duct (CD) expresses a 24p3/NGAL/lipocalin-2 (LCN2) receptor (SLC22A17) apically, possibly to mediate high-affinity reabsorption of filtered proteins by endocytosis, although its functions remain uncertain. Recently, we showed that hyperosmolarity/-tonicity upregulates SLC22A17 in cultured mouse inner-medullary CD cells, whereas activation of toll-like receptor 4 (TLR4), via bacterial lipopolysaccharides (LPS), downregulates SLC22A17. This is similar to the upregulation of Aqp2 by hyperosmolarity/-tonicity and arginine vasopressin (AVP), and downregulation by TLR4 signaling, which occur via the transcription factors NFAT5 (TonEBP or OREBP), cAMP-responsive element binding protein (CREB), and nuclear factor-kappa B, respectively. The aim of the study was to determine the effects of osmolarity/tonicity and AVP, and their associated signaling pathways, on the expression of SLC22A17 and its ligand, LCN2, in the mouse (m) cortical collecting duct cell line mCCD(cl.1). Normosmolarity/-tonicity corresponded to 300 mosmol/L, whereas the addition of 50–100 mmol/L NaCl for up to 72 h induced hyperosmolarity/-tonicity (400–500 mosmol/L). RT-PCR, qPCR, immunoblotting and immunofluorescence microscopy detected Slc22a17/SLC22A17 and Lcn2/LCN2 expression. RNAi silenced Nfat5, and the pharmacological agent 666-15 blocked CREB. Activation of TLR4 was induced with LPS. Similar to Aqp2, hyperosmotic/-tonic media and AVP upregulated Slc22a17/SLC22A17, via activation of NFAT5 and CREB, respectively, and LPS/TLR4 signaling downregulated Slc22a17/SLC22A17. Conversely, though NFAT5 mediated the hyperosmolarity/-tonicity induced downregulation of Lcn2/LCN2 expression, AVP reduced Lcn2/LCN2 expression and predominantly apical LCN2 secretion, evoked by LPS, through a posttranslational mode of action that was independent of CREB signaling. In conclusion, the hyperosmotic/-tonic upregulation of SLC22A17 in mCCD(cl.1) cells, via NFAT5, and by AVP, via CREB, suggests that SLC22A17 contributes to adaptive osmotolerance, whereas LCN2 downregulation could counteract increased proliferation and permanent damage of osmotically stressed cells.
Immunotherapies and Future Combination Strategies for Head and Neck Squamous Cell Carcinoma
Authors: Cristina Valerie , Herrera-Gómez Ruth Gabriela , Szturz Petr , Espeli Vittoria , Siano Marco
Published: October 2019

Head and neck squamous cell carcinoma (HNSCC) is often diagnosed at an advanced stage and has a dismal prognosis. Nearly 10 years after the approval of cetuximab, anti-PD1/PD-L1 checkpoint inhibitors are the first drugs that have shown any survival benefit for the treatment on platinum-refractory recurrent/metastatic (R/M) HNSCC. Furthermore, checkpoint inhibitors are better tolerated than chemotherapy. The state of the art in the treatment of R/M HNSCC is changing, thanks to improved results for checkpoint inhibitors. Results for these treatments are also awaited in curative settings and for locally advanced HNSCC. Unfortunately, the response rate of immunotherapy is low. Therefore, the identification of predictive biomarkers of response and resistance to anti-PD1/PD-L1 is a key point for better selecting patients that would benefit the most from immunotherapy. Furthermore, the combination of checkpoint inhibitors with various agents is being currently evaluated to improve the response rate, prolong response duration, and even increase the chances for a cure. In this review, we summarize the most important results regarding immune targeting agents for HNSCC, predictive biomarkers for resistance to immune therapies, and future perspectives.
Soyuretox, an Intrinsically Disordered Polypeptide Derived from Soybean (Glycine Max) Ubiquitous Urease with Potential Use as a Biopesticide
Authors: Kappaun Karine , Martinelli Anne H. S. , Broll Valquiria , Zambelli Barbara , Lopes Fernanda C. , Ligabue-Braun Rodrigo , Fruttero Leonardo L. , Moyetta Natalia R. , Bonan Carla D. , Carlini Celia R. , Ciurli Stefano
Published: October 2019

Ureases from different biological sources display non-ureolytic properties that contribute to plant defense, in addition to their classical enzymatic urea hydrolysis. Antifungal and entomotoxic effects were demonstrated for Jaburetox, an intrinsically disordered polypeptide derived from jack bean (Canavalia ensiformis) urease. Here we describe the properties of Soyuretox, a polypeptide derived from soybean (Glycine max) ubiquitous urease. Soyuretox was fungitoxic to Candida albicans, leading to the production of reactive oxygen species. Soyuretox further induced aggregation of Rhodnius prolixus hemocytes, indicating an interference on the insect immune response. No relevant toxicity of Soyuretox to zebrafish larvae was observed. These data suggest the presence of antifungal and entomotoxic portions of the amino acid sequences encompassing both Soyuretox and Jaburetox, despite their small sequence identity. Nuclear Magnetic Resonance (NMR) and circular dichroism (CD) spectroscopic data revealed that Soyuretox, in analogy with Jaburetox, possesses an intrinsic and largely disordered nature. Some folding is observed upon interaction of Soyuretox with sodium dodecyl sulfate (SDS) micelles, taken here as models for membranes. This observation suggests the possibility for this protein to modify its secondary structure upon interaction with the cells of the affected organisms, leading to alterations of membrane integrity. Altogether, Soyuretox can be considered a promising biopesticide for use in plant protection.
Meta-Analysis of Gene Expression Changes in the Blood of Patients with Mild Cognitive Impairment and Alzheimer’s Disease Dementia
Authors: Bottero Virginie , Potashkin Judith A.
Published: October 2019

Background: Dementia is a major public health concern affecting approximately 47 million people worldwide. Mild cognitive impairment (MCI) is one form of dementia that affects an individual’s memory with or without affecting their daily life. Alzheimer’s disease dementia (ADD) is a more severe form of dementia that usually affects elderly individuals. It remains unclear whether MCI is a distinct disorder from or an early stage of ADD. Methods: Gene expression data from blood were analyzed to identify potential biomarkers that may be useful for distinguishing between these two forms of dementia. Results: A meta-analysis revealed 91 genes dysregulated in individuals with MCI and 387 genes dysregulated in ADD. Pathway analysis identified seven pathways shared between MCI and ADD and nine ADD-specific pathways. Fifteen transcription factors were associated with MCI and ADD, whereas seven transcription factors were specific for ADD. Mir-335-5p was specific for ADD, suggesting that it may be useful as a biomarker. Diseases that are associated with MCI and ADD included developmental delays, cognition impairment, and movement disorders. Conclusion: These results provide a better molecular understanding of peripheral changes that occur in MCI and ADD patients and may be useful in the identification of diagnostic and prognostic biomarkers.
Modelling Neurotropic Flavivirus Infection in Human Induced Pluripotent Stem Cell-Derived Systems
Authors: Desole Giovanna , Sinigaglia Alessandro , Riccetti Silvia , Masi Giulia , Pacenti Monia , Trevisan Marta , Barzon Luisa
Published: October 2019

Generation of human induced pluripotent stem cells (hiPSCs) and their differentiation into a variety of cells and organoids have allowed setting up versatile, non-invasive, ethically sustainable, and patient-specific models for the investigation of the mechanisms of human diseases, including viral infections and host–pathogen interactions. In this study, we investigated and compared the infectivity and replication kinetics in hiPSCs, hiPSC-derived neural stem cells (NSCs) and undifferentiated neurons, and the effect of viral infection on host innate antiviral responses of representative flaviviruses associated with diverse neurological diseases, i.e., Zika virus (ZIKV), West Nile virus (WNV), and dengue virus (DENV). In addition, we exploited hiPSCs to model ZIKV infection in the embryo and during neurogenesis. The results of this study confirmed the tropism of ZIKV for NSCs, but showed that WNV replicated in these cells with much higher efficiency than ZIKV and DENV, inducing massive cell death. Although with lower efficiency, all flaviviruses could also infect pluripotent stem cells and neurons, inducing similar patterns of antiviral innate immune response gene expression. While showing the usefulness of hiPSC-based infection models, these findings suggest that additional virus-specific mechanisms, beyond neural tropism, are responsible for the peculiarities of disease phenotype in humans.
Spike Activator 1, Encoding a bHLH, Mediates Axillary Bud Development and Spike Initiation in Phalaenopsis aphrodite
Authors: Lin Yi-Jyun , Li Min-Jeng , Hsing Hung-Chien , Chen Tien-Kuan , Yang Ting-Ting , Ko Swee-Suak
Published: October 2019

Double-spikes Phalaenopsis orchids have greater market value than those with single-spike. In this study, a gene designated as Spike Activator 1 (SPK1), which encodes a basic helix-loop-helix (bHLH) transcription factor, was isolated and characterized from Phalaenopsis aphrodite (moth orchid). SPK1 was highly expressed in the meristematic tissues. In the axillary bud, SPK1 was highly upregulated by a moderately low temperature of 20 °C but downregulated by a spike inhibition temperature of 30 °C. SPK1 protein is localized in the nucleus. Another bHLH, bHLH35, which is also highly expressed in young tissues in the same way as SPK1 was also identified. In contrast to SPK1, bHLH35 transcripts are downregulated at 20 °C but upregulated at 30 °C. Bimolecular florescence complementation assay and yeast two-hybrid assays indicated that SPK1 interacts with bHLH35 and forms a heterodimer. Virus-induced gene silencing (VIGS) showed that 7 out of 15 vector control plants produced double spikes but that only 1 out of 15 VIGS-spk1 plants produced double spikes. RT-qPCR results indicated that VIGS-spk1 downregulated gene expression levels of SPK1, FT, CYCB, and EXPA8. Overall, we propose that SPK1 plays an essential role in early axillary bud development and spike initiation of P. aphrodite.
Insights into the Interaction Mechanisms of the Proviral Integration Site of Moloney Murine Leukemia Virus (Pim) Kinases with Pan-Pim Inhibitors PIM447 and AZD1208: A Molecular Dynamics Simulation and MM/GBSA Calculation Study
Authors: Chen Qingqing , Wang Yan , Shi Shanshan , Li Kaihang , Zhang Ling , Gao Jian
Published: October 2019

Based on the up-regulation of the proviral integration site of the Moloney murine leukemia virus (Pim) kinase family (Pim1, 2, and 3) observed in several types of leukemias and lymphomas, the development of pan-Pim inhibitors is an attractive therapeutic strategy. While only PIM447 and AZD1208 have entered the clinical stages. To elucidate the interaction mechanisms of three Pim kinases with PIM447 and AZD1208, six Pim/ligand systems were studied by homology modeling, molecular docking, molecular dynamics (MD) simulation and molecular mechanics/generalized Born surface area (MM/GBSA) binding free energy calculation. The residues of the top group (Leu44, Val52, Ala65, Lys67, and Leu120 in Pim1) dominated the pan-Pim inhibitors binding to Pim kinases. The residues of the bottom group (Gln127, Asp128, and Leu174 in Pim1) were crucial for Pims/PIM447 systems, while the contributions of these residues were decreased sharply for Pims/AZD1208 systems. It is likely that the more potent pan-Pim inhibitors should be bound strongly to the top and bottom groups. The residues of the left, right and loop groups were located in the loop regions of the binding pocket, however, the flexibility of these regions triggered the protein interacting with diverse pan-Pim inhibitors efficiently. We hope this work can provide valuable information for the design of novel pan-Pim inhibitors in the future.
Dynamics of Plant Metabolism during Cold Acclimation
Authors: Fürtauer Lisa , Weiszmann Jakob , Weckwerth Wolfram , Nägele Thomas
Published: October 2019

Plants have evolved strategies to tightly regulate metabolism during acclimation to a changing environment. Low temperature significantly constrains distribution, growth and yield of many temperate plant species. Exposing plants to low but non-freezing temperature induces a multigenic processes termed cold acclimation, which eventually results in an increased freezing tolerance. Cold acclimation comprises reprogramming of the transcriptome, proteome and metabolome and affects communication and signaling between subcellular organelles. Carbohydrates play a central role in this metabolic reprogramming. This review summarizes current knowledge about the role of carbohydrate metabolism in plant cold acclimation with a focus on subcellular metabolic reprogramming, its thermodynamic constraints under low temperature and mathematical modelling of metabolism.
Analysis of Transcriptome, Selected Intracellular Signaling Pathways, Proliferation and Apoptosis of LNCaP Cells Exposed to High Leptin Concentrations
Authors: Szyszka Marta , Paschke Lukasz , Tyczewska Marianna , Jopek Karol , Celichowski Piotr , Milecka Paulina , Sultanova Gulnara , Stelcer Ewelina , Malinska Agnieszka , Malendowicz Ludwik K. , Rucinski Marcin
Published: October 2019

Leptin, the first discovered adipokine, has been connected to various physiological and pathophysiological processes, including cancerogenesis. Increasing evidence confirms its influence on prostate cancer cells. However, studies on the effects of leptin on the proliferation and apoptosis of the androgen-sensitive LNCaP line of prostate cancer cells brought conflicting results. Therefore, we performed studies on the effects of high LEP concentration (1 × 10−6 M) on gene expression profile, change of selected signaling pathways, proliferation and apoptosis of LNCaP cells. RTCA (real-time cell analyzer) revealed inhibitory effect of LEP on cell proliferation, but lower LEP concentrations (10−8 and 10−10 M) did not affect cell division. Moreover, flow cytometry with a specific antibody for Cleaved PARP-1, an apoptosis marker, confirmed the activation of apoptosis in leptin-exposed LNCaP line of prostate cancer cells. Within 24 h LEP (10−6 M) increases expression of 297 genes and decreases expression of 119 genes. Differentially expressed genes (DEGs) were subjected to functional annotation and clusterization using the DAVID bioinformatics tools. Most ontological groups are associated with proliferation and apoptosis (seven groups), immune response (six) and extracellular matrix (two). These results were confirmed by the Gene Set Enrichment Analysis (GSEA). The leptin’s effect on apoptosis stimulation was also confirmed using Pathview library. These results were also confirmed by qPCR method. The results of Western Blot analysis (exposure to LEP 10 min, 1, 2, 4 and 24 h) suggest (after 24 h) decrease of p38 MAPK, p44-42 mitogen-activated protein kinase and Bcl-2 phosphorylated at threonine 56. Moreover, exposure of LNCaP cells to LEP significantly stimulates the secretion of matrix metallopeptidase 7 (MMP7). Obtained results suggest activation of apoptotic processes in LNCaP cells cultured at high LEP concentration. At the same time, this activation is accompanied by inhibition of proliferation of the tested cells.
Gender Differences in the Pharmacological Actions of Pegylated Glucagon-Like Peptide-1 on Endothelial Progenitor Cells and Angiogenic Precursor Cells in a Combination of Metabolic Disorders and Lung Emphysema
Authors: Pershina Olga Victorovna , Pakhomova Angelina Vladimirovna , Widera Darius , Ermakova Natalia Nicolaevna , Epanchintsev Anton Alexandrovich , Pan Edgar Sergeevich , Krupin Vyacheslav Andreevich , Vaizova Olga Evgenevna , Putrova Olesia Dmitrievna , Sandrikina Lubov Alexandrovna , Kurochkina Irina Vitalevna , Morozov Sergey Georgievich , Kubatiev Aslan Amirkhanovich , Dygai Alexander Mikhaylovich , Skurikhin Evgenii Germanovich
Published: October 2019

In clinical practice, the metabolic syndrome (MetS) is often associated with chronic obstructive pulmonary disease (COPD). Although gender differences in MetS are well documented, little is known about sex-specific differences in the pathogenesis of COPD, especially when combined with MetS. Consequently, it is not clear whether the same treatment regime has comparable efficacy in men and women diagnosed with MetS and COPD. In the present study, using sodium glutamate, lipopolysaccharide, and cigarette smoke extract, we simulated lipid metabolism disorders, obesity, hyperglycemia, and pulmonary emphysema (comorbidity) in male and female C57BL/6 mice. We assessed the gender-specific impact of lipid metabolism disorders and pulmonary emphysema on angiogenic precursor cells (endothelial progenitor cells (EPC), pericytes, vascular smooth muscle cells, cells of the lumen of the nascent vessel), as well as the biological effects of pegylated glucagon-like peptide 1 (pegGLP-1) in this experimental paradigm. Simulation of MetS/COPD comorbidity caused an accumulation of EPC (CD45−CD31+CD34+), pericytes, and vascular smooth muscle cells in the lungs of female mice. In contrast, the number of cells involved in the angiogenesis decreased in the lungs of male animals. PegGLP-1 had a positive effect on lipids and area under the curve (AUC), obesity, and prevented the development of pulmonary emphysema. The severity of these effects was stronger in males than in females. Furthermore, PegGLP-1 stimulated regeneration of pulmonary endothelium. At the same time, PegGLP-1 administration caused a mobilization of EPC (CD45−CD31+CD34+) into the bloodstream in females and migration of precursors of angiogenesis and vascular smooth muscle cells to the lungs in male animals. Gender differences in stimulatory action of pegGLP-1 on CD31+ endothelial lung cells in vitro were not observed. Based on these findings, we postulated that the cellular mechanism of in vivo regeneration of lung epithelium was at least partly gender-specific. Thus, we concluded that a pegGLP-1-based treatment regime for metabolic disorder and COPD should be further developed primarily for male patients.
PFKFB3 Inhibition Attenuates Oxaliplatin-Induced Autophagy and Enhances Its Cytotoxicity in Colon Cancer Cells
Authors: Yan Siyuan , Zhou Nan , Zhang Deru , Zhang Kaile , Zheng Wenao , Bao Yonghua , Yang Wancai
Published: October 2019

6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase isoform 3 (PFKFB3), a glycolytic enzyme highly expressed in cancer cells, has been reported to participate in regulating metabolism, angiogenesis, and autophagy. Although anti-cancer drug oxaliplatin (Oxa) effectively inhibits cell proliferation and induces apoptosis, the growing resistance and side-effects make it urgent to improve the therapeutic strategy of Oxa. Although Oxa induces the autophagy process, the role of PFKFB3 in this process remains unknown. In addition, whether PFKFB3 affects the cytotoxicity of Oxa has not been investigated. Here, we show that Oxa-inhibited cell proliferation and migration concomitant with the induction of apoptosis and autophagy in SW480 cells. Both inhibition of autophagy by small molecule inhibitors and siRNA modification decreased the cell viability loss and apoptosis induced by Oxa. Utilizing quantitative PCR and immunoblotting, we observed that Oxa increased PFKFB3 expression in a time- and dose-dependent manner. Meanwhile, suppression of PFKFB3 attenuated both the basal and Oxa-induced autophagy, by monitoring the autophagic flux and phosphorylated-Ulk1, which play essential roles in autophagy initiation. Moreover, PFKFB3 inhibition further inhibited the cell proliferation/migration, and cell viability decreased by Oxa. Collectively, the presented data demonstrated that PFKFB3 inhibition attenuated Oxa-induced autophagy and enhanced its cytotoxicity in colorectal cancer cells.
Methods and Guidelines for Measurement of Glucagon in Plasma
Authors: Holst Jens J. , Wewer Albrechtsen Nicolai J.
Published: October 2019

Glucagon circulates in concentrations in the low picomolar range, which is demanding regarding the sensitivity of the methods for quantification applied. In addition, the differential and tissue specific proteolytic processing of the glucagon precursor and the presence in of several glucagon-like sequences, not only in the precursor of glucagon, but also in a number of other peptides of the glucagon-secretin family of peptides, put special demands on the specificity of the assays. Finally, experience has shown that unspecific interference of plasma components has presented additional problems. All of these problems have resulted in a lot of diverging results concerning measured and reported glucagon responses in both humans and experimental animals that have and still are causing considerable debate and controversy. There is very solid evidence that glucagon is an important hormone in human and mammalian metabolism, but its precise physiological role in glucose and lipid metabolism and in metabolic disease has been difficult to establish, not least because of these difficulties. It was our purpose with this review to discuss the methods of glucagon quantification and discuss pitfalls and sources of error. We also reviewed some of the dogmas regarding glucagon secretion in the light of the methodological difficulties.
Novel Mutations Segregating with Complete Androgen Insensitivity Syndrome and Their Molecular Characteristics
Authors: Malcher Agnieszka , Jedrzejczak Piotr , Stokowy Tomasz , Monem Soroosh , Nowicka-Bauer Karolina , Zimna Agnieszka , Czyzyk Adam , Maciejewska-Jeske Marzena , Meczekalski Blazej , Bednarek-Rajewska Katarzyna , Wozniak Aldona , Rozwadowska Natalia , Kurpisz Maciej
Published: October 2019

We analyzed three cases of Complete Androgen Insensitivity Syndrome (CAIS) and report three hitherto undisclosed causes of the disease. RNA-Seq, Real-timePCR, Western immunoblotting, and immunohistochemistry were performed with the aim of characterizing the disease-causing variants. In case No.1, we have identified a novel androgen receptor (AR) mutation (c.840delT) within the first exon in the N-terminal transactivation domain. This thymine deletion resulted in a frameshift and thus introduced a premature stop codon at amino acid 282. In case No.2, we observed a nonsynonymous mutation in the ligand-binding domain (c.2491C>T). Case No.3 did not reveal AR mutation; however, we have found a heterozygous mutation in CYP11A1 gene, which has a role in steroid hormone biosynthesis. Comparative RNA-Seq analysis of CAIS and control revealed 4293 significantly deregulated genes. In patients with CAIS, we observed a significant increase in the expression levels of PLCXD3, TM4SF18, CFI, GPX8, and SFRP4, and a significant decrease in the expression of SPATA16, TSACC, TCP10L, and DPY19L2 genes (more than 10-fold, p < 0.05). Our findings will be helpful in molecular diagnostics of patients with CAIS, as well as the identified genes could be also potential biomarkers for the germ cells differentiation process.
Lifestyle and Food Habits Impact on Chronic Diseases: Roles of PPARs
Authors: d’Angelo Michele , Castelli Vanessa , Tupone Maria Grazia , Catanesi Mariano , Antonosante Andrea , Dominguez-Benot Reyes , Ippoliti Rodolfo , Cimini Anna Maria , Benedetti Elisabetta
Published: October 2019

Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors that exert important functions in mediating the pleiotropic effects of diverse exogenous factors such as physical exercise and food components. Particularly, PPARs act as transcription factors that control the expression of genes implicated in lipid and glucose metabolism, and cellular proliferation and differentiation. In this review, we aim to summarize the recent advancements reported on the effects of lifestyle and food habits on PPAR transcriptional activity in chronic disease.
The Role of MicroRNAs in Diabetes-Related Oxidative Stress
Authors: Qadir Mirza Muhammad Fahd , Klein Dagmar , Álvarez-Cubela Silvia , Domínguez-Bendala Juan , Pastori Ricardo Luis
Published: October 2019

Cellular stress, combined with dysfunctional, inadequate mitochondrial phosphorylation, produces an excessive amount of reactive oxygen species (ROS) and an increased level of ROS in cells, which leads to oxidation and subsequent cellular damage. Because of its cell damaging action, an association between anomalous ROS production and disease such as Type 1 (T1D) and Type 2 (T2D) diabetes, as well as their complications, has been well established. However, there is a lack of understanding about genome-driven responses to ROS-mediated cellular stress. Over the last decade, multiple studies have suggested a link between oxidative stress and microRNAs (miRNAs). The miRNAs are small non-coding RNAs that mostly suppress expression of the target gene by interaction with its 3’untranslated region (3′UTR). In this paper, we review the recent progress in the field, focusing on the association between miRNAs and oxidative stress during the progression of diabetes.
Aryl Hydrocarbon Receptor in Atopic Dermatitis and Psoriasis
Authors: Furue Masutaka , Hashimoto-Hachiya Akiko , Tsuji Gaku
Published: October 2019

The aryl hydrocarbon receptor (AHR)/AHR-nuclear translocator (ARNT) system is a sensitive sensor for small molecular, xenobiotic chemicals of exogenous and endogenous origin, including dioxins, phytochemicals, microbial bioproducts, and tryptophan photoproducts. AHR/ARNT are abundantly expressed in the skin. Once activated, the AHR/ARNT axis strengthens skin barrier functions and accelerates epidermal terminal differentiation by upregulating filaggrin expression. In addition, AHR activation induces oxidative stress. However, some AHR ligands simultaneously activate the nuclear factor-erythroid 2-related factor-2 (NRF2) transcription factor, which is a master switch of antioxidative enzymes that neutralizes oxidative stress. The immunoregulatory system governing T-helper 17/22 (Th17/22) and T regulatory cells (Treg) is also regulated by the AHR system. Notably, AHR agonists, such as tapinarof, are currently used as therapeutic agents in psoriasis and atopic dermatitis. In this review, we summarize recent topics on AHR related to atopic dermatitis and psoriasis.
Generation of Mouse Parthenogenetic Epiblast Stem Cells and Their Imprinting Patterns
Authors: Seo Bong Jong , Jang Hyun Sik , Song Hyuk , Park Chankyu , Hong Kwonho , Lee Jeong Woong , Do Jeong Tae
Published: October 2019

Pluripotent stem cells can be established from parthenogenetic embryos, which only possess maternal alleles with maternal-specific imprinting patterns. Previously, we and others showed that parthenogenetic embryonic stem cells (pESCs) and parthenogenetic induced pluripotent stem cells (piPSCs) progressively lose the bimaternal imprinting patterns. As ESCs and iPSCs are naïve pluripotent stem cells, parthenogenetic primed pluripotent stem cells have not yet been established, and thus, their imprinting patterns have not been studied. Here, we first established parthenogenetic epiblast stem cells (pEpiSCs) from 7.5 dpc parthenogenetic implantation embryos and compared the expression patterns and DNA methylation status of the representative imprinted genes with biparental EpiSCs. We found that there were no striking differences between pEpiSCs and biparental EpiSCs with respect to morphology, pluripotency gene expression, and differentiation potential, but there were differences in the expression and DNA methylation status of imprinted genes (H19, Igf2, Peg1, and Peg3). Moreover, pEpiSCs displayed a different DNA methylation pattern compared with that of parthenogenetic neural stem cells (pNSCs), which showed a typical bimaternal imprinting pattern. These results suggest that both naïve pluripotent stem cells and primed pluripotent stem cells have an unstable imprinting status.
Dp71 Expression in Human Glioblastoma
Authors: Ruggieri Simona , De Giorgis Michelina , Annese Tiziana , Tamma Roberto , Notarangelo Angelo , Marzullo Andrea , Senetta Rebecca , Cassoni Paola , Notarangelo Michela , Ribatti Domenico , Nico Beatrice
Published: October 2019

Background: Dp71 is the most abundant dystrophin (DMD) gene product in the nervous system. Mutation in the Dp71 coding region is associated with cognitive disturbances in Duchenne muscular dystrophy (DMD) patients, but the function of dystrophin Dp71 in tumor progression remains to be established. This study investigated Dp71 expression in glioblastoma, the most common and aggressive primary tumor of the central nervous system (CNS). Methods: Dp71 expression was analyzed by immunofluorescence, immunohistochemistry, RT-PCR, and immunoblotting in glioblastoma cell lines and cells isolated from human glioblastoma multiforme (GBM) bioptic specimens. Results: Dp71 isoform was expressed in normal human astrocytes (NHA) cell lines and decreased in glioblastoma cell lines and cells isolated from human glioblastoma multiforme bioptic specimens. Moreover, Dp71 was localized in the nucleus in normal cells, while it was localized into the cytoplasm of glioblastoma cells organized in clusters. We have shown, by double labeling, that Dp71 colocalizes with lamin B in normal astrocytes cells, confirming the roles of Dp71 and lamin B in maintaining nuclear architecture. Finally, we demonstrated that decreased Dp71 protein in cells isolated from human bioptic specimens was inversely correlated with the Ki-67 tumor proliferative index. Conclusion: A decreased Dp71 expression is associated with cancer proliferation and poor prognosis in glioblastoma.
Dasatinib Inhibits Procoagulant and Clot Retracting Activities of Human Platelets
Authors: Beke Debreceni Ildikó , Mezei Gabriella , Batár Péter , Illés Árpád , Kappelmayer János
Published: October 2019

Tyrosine kinase inhibitors (TKI) such as the BCR-ABL inhibitor dasatinib and nilotinib are highly effective therapies for chronic myeloid leukemia (CML). However, several lines of evidence suggest that dasatinib can induce bleeding which may be due to impaired collagen-induced platelet adhesion, aggregation, and secretion. Sarcoma family kinases (SFK) play central role in the GPVI-induced signaling pathway. We aimed to investigate whether and how dasatinib can modulate SFK-mediated platelet procoagulant activity in a purified system and in dasatinib/nilotinib treated CML patients. In platelet rich plasmas of healthy volunteers, dasatinib dose-dependently reduced convulxin-induced phosphatidylserine exposure and attenuated thrombin formation. Similarly to these changes, integrin activation and clot retraction were also significantly inhibited by 100 nM dasatinib. Platelets isolated from dasatinib treated patients showed a significantly lower phosphatidylserine expression upon convulxin activation compared to premedication levels. In these samples, thrombin generation was significantly slower, and the quantity of formed thrombin was less compared to the trough sample. Western blot analyses showed decreased phosphorylation levels of the C-terminal tail and the activation loop of SFKs upon dasatinib administration. Taken together, these results suggest that dasatinib inhibits the formation of procoagulant platelets via the GPVI receptor by inhibiting phosphorylation of SFKs.
Neutral Sphingomyelinase Modulation in the Protective/Preventive Role of rMnSOD from Radiation-Induced Damage in the Brain
Authors: Cataldi Samuela , Borrelli Antonella , Ceccarini Maria Rachele , Nakashidze Irina , Codini Michela , Belov Oleg , Ivanov Alexander , Krasavin Eugene , Ferri Ivana , Conte Carmela , Patria Federica Filomena , Traina Giovanna , Beccari Tommaso , Mancini Aldo , Curcio Francesco , Ambesi-Impiombato Francesco Saverio , Albi Elisabetta
Published: October 2019

Studies on the relationship between reactive oxygen species (ROS)/manganese superoxide dismutase (MnSOD) and sphingomyelinase (SMase) are controversial. It has been demonstrated that SMase increases the intracellular ROS level and induces gene expression for MnSOD protein. On the other hand, some authors showed that ROS modulate the activation of SMase. The human recombinant manganese superoxide dismutase (rMnSOD) exerting a radioprotective effect on normal cells, qualifies as a possible pharmaceutical tool to prevent and/or cure damages derived from accidental exposure to ionizing radiation. This study aimed to identify neutral SMase (nSMase) as novel molecule connecting rMnSOD to its radiation protective effects. We used a new, and to this date, unique, experimental model to assess the effect of both radiation and rMnSOD in the brain of mice, within a collaborative project among Italian research groups and the Joint Institute for Nuclear Research, Dubna (Russia). Mice were exposed to a set of minor γ radiation and neutrons and a spectrum of neutrons, simulating the radiation levels to which cosmonauts will be exposed during deep-space, long-term missions. Groups of mice were treated or not-treated (controls) with daily subcutaneous injections of rMnSOD during a period of 10 days. An additional group of mice was also pretreated with rMnSOD for three days before irradiation, as a model for preventive measures. We demonstrate that rMnSOD significantly protects the midbrain cells from radiation-induced damage, inducing a strong upregulation of nSMase gene and protein expression. Pretreatment with rMnSOD before irradiation protects the brain with a value of very high nSMase activity, indicating that high levels of activity might be sufficient to exert the rMnSOD preventive role. In conclusion, the protective effect of rMnSOD from radiation-induced brain damage may require nSMase enzyme.
Removal of the Polyglutamine Repeat of Ataxin-3 by Redirecting pre-mRNA Processing
Authors: McIntosh Craig S. , Aung-Htut May Thandar , Fletcher Sue , Wilton Steve D.
Published: October 2019

Spinocerebellar ataxia type 3 (SCA3) is a devastating neurodegenerative disease for which there is currently no cure, nor effective treatment strategy. One of nine polyglutamine disorders known to date, SCA3 is clinically heterogeneous and the main feature is progressive ataxia, which in turn affects speech, balance and gait of the affected individual. SCA3 is caused by an expanded polyglutamine tract in the ataxin-3 protein, resulting in conformational changes that lead to toxic gain of function. The expanded glutamine tract is located at the 5′ end of the penultimate exon (exon 10) of ATXN3 gene transcript. Other studies reported removal of the expanded glutamine tract using splice switching antisense oligonucleotides. Here, we describe improved efficiency in the removal of the toxic polyglutamine tract of ataxin-3 in vitro using phosphorodiamidate morpholino oligomers, when compared to antisense oligonucleotides composed of 2′-O-methyl modified bases on a phosphorothioate backbone. Significant downregulation of both the expanded and non-expanded protein was induced by the morpholino antisense oligomer, with a greater proportion of ataxin-3 protein missing the polyglutamine tract. With growing concerns over toxicity associated with long-term administration of phosphorothioate oligonucleotides, the use of a phosphorodiamidate morpholino oligomer may be preferable for clinical application. These results suggest that morpholino oligomers may provide greater therapeutic benefit for the treatment of spinocerebellar ataxia type 3, without toxic effects.
The Systemic Immune Response to Collagen-Induced Arthritis and the Impact of Bone Injury in Inflammatory Conditions
Authors: Teixeira José H. , Silva Andreia M. , Almeida Maria Inês , Bessa-Gonçalves Mafalda , Cunha Carla , Barbosa Mário A. , Santos Susana G.
Published: October 2019

Rheumatoid arthritis (RA) is a systemic disease that affects the osteoarticular system, associated with bone fragility and increased risk of fractures. Herein, we aimed to characterize the systemic impact of the rat collagen-induced arthritis (CIA) model and explore its combination with femoral bone defect (FD). The impact of CIA on endogenous mesenchymal stem/stromal cells (MSC) was also investigated. CIA induction led to enlarged, more proliferative, spleen and draining lymph nodes, with altered proportion of lymphoid populations. Upon FD, CIA animals increased the systemic myeloid cell proportions, and their expression of co-stimulatory molecules CD40 and CD86. Screening plasma cytokine/chemokine levels showed increased tumor necrosis factor-α (TNF-α), Interleukin (IL)-17, IL-4, IL-5, and IL-12 in CIA, and IL-2 and IL-6 increased in CIA and CIA+FD, while Fractalkine and Leptin were decreased in both groups. CIA-derived MSC showed lower metabolic activity and proliferation, and significantly increased osteogenic and chondrogenic differentiation markers. Exposure of control-MSC to TNF-α partially mimicked the CIA-MSC phenotype in vitro. In conclusion, inflammatory conditions of CIA led to alterations in systemic immune cell proportions, circulating mediators, and in endogenous MSC. CIA animals respond to FD, and the combined model can be used to study the mechanisms of bone repair in inflammatory conditions.
Silenced ZNF154 Is Associated with Longer Survival in Resectable Pancreatic Cancer
Authors: Wiesmueller Felix , Kopke Josephin , Aust Daniela , Roy Janine , Dahl Andreas , Pilarsky Christian , Grützmann Robert
Published: October 2019

Pancreatic cancer has become the third leading cause of cancer-related death in the Western world despite advances in therapy of other cancerous lesions. Late diagnosis due to a lack of symptoms during early disease allows metastatic spread of the tumor. Most patients are considered incurable because of metastasized disease. On a cellular level, pancreatic cancer proves to be rather resistant to chemotherapy. Hence, early detection and new therapeutic targets might improve outcomes. The detection of DNA promoter hypermethylation has been described as a method to identify putative genes of interest in cancer entities. These genes might serve as either biomarkers or might lead to a better understanding of the molecular mechanisms involved. We checked tumor specimens from 80 patients who had undergone pancreatic resection for promoter hypermethylation of the zinc finger protein ZNF154. Then, we further characterized the effects of ZNF154 on cell viability and gene expression by in vitro experiments. We found a significant association between ZNF154 hypermethylation and better survival in patients with resectable pancreatic cancer. Moreover, we suspect that the cell growth suppressor SLFN5 might be linked to a silenced ZNF154 in pancreatic cancer.
Deep Eutectic Solvents as New Reaction Media to Produce Alkyl-Glycosides Using Alpha-Amylase from Thermotoga maritima
Authors: Miranda-Molina Alfonso , Xolalpa Wendy , Strompen Simon , Arreola-Barroso Rodrigo , Olvera Leticia , López-Munguía Agustín , Castillo Edmundo , Saab-Rincon Gloria
Published: October 2019

Deep Eutectic Solvents (DES) were investigated as new reaction media for the synthesis of alkyl glycosides catalyzed by the thermostable α-amylase from Thermotoga maritima Amy A. The enzyme was almost completely deactivated when assayed in a series of pure DES, but as cosolvents, DES containing alcohols, sugars, and amides as hydrogen-bond donors (HBD) performed best. A choline chloride:urea based DES was further characterized for the alcoholysis reaction using methanol as a nucleophile. As a cosolvent, this DES increased the hydrolytic and alcoholytic activity of the enzyme at low methanol concentrations, even when both activities drastically dropped when methanol concentration was increased. To explain this phenomenon, variable-temperature, circular dichroism characterization of the protein was conducted, finding that above 60 °C, Amy A underwent large conformational changes not observed in aqueous medium. Thus, 60 °C was set as the temperature limit to carry out alcoholysis reactions. Higher DES contents at this temperature had a detrimental but differential effect on hydrolysis and alcoholysis reactions, thus increasing the alcoholyisis/hydrolysis ratio. To the best of our knowledge, this is the first report on the effect of DES and temperature on an enzyme in which structural studies made it possible to establish the temperature limit for a thermostable enzyme in DES.
Ultra-Rapid Glutathionylation of Ribonuclease: Is This the Real Incipit of Its Oxidative Folding?
Authors: Bocedi Alessio , Cattani Giada , Gambardella Giorgia , Ticconi Silvia , Cozzolino Flora , Di Fusco Ornella , Pucci Piero , Ricci Giorgio
Published: October 2019

Many details of oxidative folding of proteins remain obscure, in particular, the role of oxidized glutathione (GSSG). This study reveals some unknown aspects. When a reduced ribonuclease A refolds in the presence of GSSG, most of its eight cysteines accomplish a very fast glutathionylation. In particular, one single cysteine, identified as Cys95 by mass spectrometry, displays 3600 times higher reactivity when compared with an unperturbed protein cysteine. Furthermore, the other five cysteines show 40–50 times higher reactivity toward GSSG. This phenomenon is partially due to a low pKa value of most of these cysteines (average pKa = 7.9), but the occurrence of a reversible GSSG-ribonuclease complex (KD = 0.12 mM) is reasonably responsible for the extraordinary hyper-reactivity of Cys95. Neither hyper-reactivity nor some protein-disulfide complexes have been found by reacting a reduced ribonuclease with other natural disulfides i.e., cystine, cystamine, and homocystine. Hyper-reactivity of all cysteines was observed toward 5,5’-dithiobis-(2-nitrobenzoic acid). Given that GSSG is present in high concentrations in the endoplasmic reticulum, this property may shed light on the early step of its oxidative folding. The ultra-rapid glutathionylation of cysteines, only devoted to form disulfides, is a novel property of the molten globule status of the ribonuclease.
Identification of Rice Large Grain Gene GW2 by Whole-Genome Sequencing of a Large Grain-Isogenic Line Integrated with Japonica Native Gene and Its Linkage Relationship with the Co-integrated Semidwarf Gene d60 on Chromosome 2
Authors: Tomita Motonori , Yazawa Shiho , Uenishi Yoshimasa
Published: October 2019

Genetic analysis of “InochinoIchi,” an exceptionally large grain rice variety, was conducted through five continuous backcrosses with Koshihikari as a recurrent parent using the large grain F3 plant in Koshihikari × Inochinoichi as a nonrecurrent parent. Thorough the F2 and all BCnF2 generations, large, medium, and small grain segregated in a 1:2:1 ratio, indicating that the large grain is controlled by a single allele. Mapping by using simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) markers with small grain homozygous segregants in the F2 of Nipponbare × Inochinoichi, revealed linkage with around 7.7 Mb markers from the distal end of the short arm of chromosome 2. Whole-genome sequencing on a large grain isogenic Koshihikari (BC4F2) using next-generation sequencing (NGS) identified a single nucleotide deletion in GW2 gene, which is located 8.1 Mb from the end of chromosome 2, encoding a RING protein with E3 ubiquitin ligase activity. The GW2-integrated isogenic Koshihikari showed a 34% increase in thousand kernel weight compared to Koshihikari, while retaining a taste score of 80. We further developed a large grain/semi-dwarf isogenic Koshihikari integrated with GW2 and the semidwarfing gene d60, which was found to be localized on chromosome 2. The combined genotype secured high yielding while providing robustness to withstand climate change, which can contribute to the New Green Revolution.