bluuebunny/crossref_metadata_2025 huggingface dataset:
The Role of Non-Specific Interactions in Canonical and ALT-Associated PML-Bodies Formation and Dynamics
Authors: Fonin Alexander V. , Silonov Sergey A. , Shpironok Olesya G. , Antifeeva Iuliia A. , Petukhov Alexey V. , Romanovich Anna E. , Kuznetsova Irina M. , Uversky Vladimir N. , Turoverov Konstantin K.
Published: May 2021

In this work, we put forward a hypothesis about the decisive role of multivalent nonspecific interactions in the early stages of PML body formation. Our analysis of the PML isoform sequences showed that some of the PML isoforms, primarily PML-II, are prone to phase separation due to their polyampholytic properties and the disordered structure of their C-terminal domains. The similarity of the charge properties of the C-terminal domains of PML-II and PML-VI isoforms made it possible for the first time to detect migration of PML-VI from PML bodies to the periphery of the cell nucleus, similar to the migration of PML-II isoforms. We found a population of “small” (area less than 1 µm2) spherical PML bodies with high dynamics of PML isoforms exchange with nucleoplasm and a low fraction of immobilized proteins, which indicates their liquid state properties. Such structures can act as “seeds” of functionally active PML bodies, providing the necessary concentration of PML isoforms for the formation of intermolecular disulfide bonds between PML monomers. FRAP analysis of larger bodies of toroidal topology showed the existence of an insoluble scaffold in their structure. The hypothesis about the role of nonspecific multiple weak interactions in the formation of PML bodies is further supported by the change in the composition of the scaffold proteins of PML bodies, but not their solidification, under conditions of induction of dimerization of PML isoforms under oxidative stress. Using the colocalization of ALT-associated PML bodies (APBs) with TRF1, we identified APBs and showed the difference in the dynamic properties of APBs and canonical PML bodies.
The Role of Glycosyltransferases in Colorectal Cancer
Authors: Fernández-Ponce Cecilia , Geribaldi-Doldán Noelia , Sánchez-Gomar Ismael , Navarro Quiroz Roberto , Atencio Ibarra Linda , Gomez Escorcia Lorena , Fernández-Cisnal Ricardo , Aroca Martinez Gustavo , García-Cózar Francisco , Navarro Quiroz Elkin
Published: May 2021

Colorectal cancer (CRC) is one of the main causes of cancer death in the world. Post-translational modifications (PTMs) have been extensively studied in malignancies due to its relevance in tumor pathogenesis and therapy. This review is focused on the dysregulation of glycosyltransferase expression in CRC and its impact in cell function and in several biological pathways associated with CRC pathogenesis, prognosis and therapeutic approaches. Glycan structures act as interface molecules between cells and their environment and in several cases facilitate molecule function. CRC tissue shows alterations in glycan structures decorating molecules, such as annexin-1, mucins, heat shock protein 90 (Hsp90), β1 integrin, carcinoembryonic antigen (CEA), epidermal growth factor receptor (EGFR), insulin-like growth factor-binding protein 3 (IGFBP3), transforming growth factor beta (TGF-β) receptors, Fas (CD95), PD-L1, decorin, sorbin and SH3 domain-containing protein 1 (SORBS1), CD147 and glycosphingolipids. All of these are described as key molecules in oncogenesis and metastasis. Therefore, glycosylation in CRC can affect cell migration, cell–cell adhesion, actin polymerization, mitosis, cell membrane repair, apoptosis, cell differentiation, stemness regulation, intestinal mucosal barrier integrity, immune system regulation, T cell polarization and gut microbiota composition; all such functions are associated with the prognosis and evolution of the disease. According to these findings, multiple strategies have been evaluated to alter oligosaccharide processing and to modify glycoconjugate structures in order to control CRC progression and prevent metastasis. Additionally, immunotherapy approaches have contemplated the use of neo-antigens, generated by altered glycosylation, as targets for tumor-specific T cells or engineered CAR (Chimeric antigen receptors) T cells.
Heat Priming of Lentil (Lens culinaris Medik.) Seeds and Foliar Treatment with γ-Aminobutyric Acid (GABA), Confers Protection to Reproductive Function and Yield Traits under High-Temperature Stress Environments
Authors: Bhardwaj Anjali , Sita Kumari , Sehgal Akanksha , Bhandari Kalpna , Kumar Shiv , Prasad P. V. Vara , Jha Uday , Kumar Jitendra , Siddique Kadambot H. M. , Nayyar Harsh
Published: May 2021

Gradually increasing temperatures at global and local scales are causing heat stress for cool and summer-season food legumes, such as lentil (Lens culinaris Medik.), which is highly susceptible to heat stress, especially during its reproductive stages of development. Hence, suitable strategies are needed to develop heat tolerance in this legume. In the present study, we tested the effectiveness of heat priming (HPr; 6 h at 35 °C) the lentil seeds and a foliar treatment of γ-aminobutyric acid (GABA; 1 mM; applied twice at different times), singly or in combination (HPr+GABA), under heat stress (32/20 °C) in two heat-tolerant (HT; IG2507, IG3263) and two heat-sensitive (HS; IG2821, IG2849) genotypes to mitigate heat stress. The three treatments significantly reduced heat injury to leaves and flowers, particularly when applied in combination, including leaf damage assessed as membrane injury, cellular oxidizing ability, leaf water status, and stomatal conductance. The combined HPr+GABA treatment significantly improved the photosynthetic function, measured as photosynthetic efficiency, chlorophyll concentration, and sucrose synthesis; and significantly reduced the oxidative damage, which was associated with a marked up-regulation in the activities of enzymatic antioxidants. The combined treatment also facilitated the synthesis of osmolytes, such as proline and glycine betaine, by upregulating the expression of their biosynthesizing enzymes (pyrroline-5-carboxylate synthase; betaine aldehyde dehydrogenase) under heat stress. The HPr+GABA treatment caused a considerable enhancement in endogenous levels of GABA in leaves, more so in the two heat-sensitive genotypes. The reproductive function, measured as germination and viability of pollen grains, receptivity of stigma, and viability of ovules, was significantly improved with combined treatment, resulting in enhanced pod number (21–23% in HT and 35–38% in HS genotypes, compared to heat stress alone) and seed yield per plant (22–24% in HT and 37–40% in HS genotypes, in comparison to heat stress alone). The combined treatment (HPr+GABA) was more effective and pronounced in heat-sensitive than heat-tolerant genotypes for all the traits tested. This study offers a potential solution for tackling and protecting heat stress injury in lentil plants.
The Co-Culture of Staphylococcal Biofilm and Fibroblast Cell Line: The Correlation of Biological Phenomena with Metabolic NMR1 Footprint
Authors: Czajkowska Joanna , Junka Adam , Hoppe Jakub , Toporkiewicz Monika , Pawlak Andrzej , Migdał Paweł , Oleksy-Wawrzyniak Monika , Fijałkowski Karol , Śmiglak Marcin , Markowska-Szczupak Agata
Published: May 2021

Staphylococcus aureus is one of the most prevalent pathogens associated with several types of biofilm-based infections, including infections of chronic wounds. Mature staphylococcal biofilm is extremely hard to eradicate from a wound and displays a high tendency to induce recurring infections. Therefore, in the present study, we aimed to investigate in vitro the interaction between S. aureus biofilm and fibroblast cells searching for metabolites that could be considered as potential biomarkers of critical colonization and infection. Utilizing advanced microscopy and microbiological methods to examine biofilm formation and the staphylococcal infection process, we were able to distinguish 4 phases of biofilm development. The analysis of staphylococcal biofilm influence on the viability of fibroblasts allowed us to pinpoint the moment of critical colonization—12 h post contamination. Based on the obtained model we performed a metabolomics analysis by 1H NMR spectroscopy to provide new insights into the pathophysiology of infection. We identified a set of metabolites related to the switch to anaerobic metabolism that was characteristic for staphylococcal biofilm co-cultured with fibroblast cells. The data presented in this study may be thus considered a noteworthy but preliminary step in the direction of developing a new, NMR-based tool for rapid diagnosing of infection in a chronic wound.
Tenascin-C in Heart Diseases—The Role of Inflammation
Authors: Imanaka-Yoshida Kyoko
Published: May 2021

Tenascin-C (TNC) is a large extracellular matrix (ECM) glycoprotein and an original member of the matricellular protein family. TNC is transiently expressed in the heart during embryonic development, but is rarely detected in normal adults; however, its expression is strongly up-regulated with inflammation. Although neither TNC-knockout nor -overexpressing mice show a distinct phenotype, disease models using genetically engineered mice combined with in vitro experiments have revealed multiple significant roles for TNC in responses to injury and myocardial repair, particularly in the regulation of inflammation. In most cases, TNC appears to deteriorate adverse ventricular remodeling by aggravating inflammation/fibrosis. Furthermore, accumulating clinical evidence has shown that high TNC levels predict adverse ventricular remodeling and a poor prognosis in patients with various heart diseases. Since the importance of inflammation has attracted attention in the pathophysiology of heart diseases, this review will focus on the roles of TNC in various types of inflammatory reactions, such as myocardial infarction, hypertensive fibrosis, myocarditis caused by viral infection or autoimmunity, and dilated cardiomyopathy. The utility of TNC as a biomarker for the stratification of myocardial disease conditions and the selection of appropriate therapies will also be discussed from a clinical viewpoint.
Detecting Variants in the NBN Gene While Testing for Hereditary Breast Cancer: What to Do Next?
Authors: Zuntini Roberta , Bonora Elena , Pradella Laura Maria , Amato Laura Benedetta , Vidone Michele , De Fanti Sara , Catucci Irene , Cortesi Laura , Medici Veronica , Ferrari Simona , Gasparre Giuseppe , Peterlongo Paolo , Sazzini Marco , Turchetti Daniela
Published: May 2021

The NBN gene has been included in breast cancer (BC) multigene panels based on early studies suggesting an increased BC risk for carriers, though not confirmed by recent research. To evaluate the impact of NBN analysis, we assessed the results of NBN sequencing in 116 BRCA-negative BC patients and reviewed the literature. Three patients (2.6%) carried potentially relevant variants: two, apparently unrelated, carried the frameshift variant c.156_157delTT and another one the c.628G>T variant. The latter was subsequently found in 4/1390 (0.3%) BC cases and 8/1580 (0.5%) controls in an independent sample, which, together with in silico predictions, provided evidence against its pathogenicity. Conversely, the rare c.156_157delTT variant was absent in the case-control set; moreover, a 50% reduction of NBN expression was demonstrated in one carrier. However, in one family it failed to co-segregate with BC, while the other carrier was found to harbor also a probably pathogenic TP53 variant that may explain her phenotype. Therefore, the c.156_157delTT, although functionally deleterious, was not supported as a cancer-predisposing defect. Pathogenic/likely pathogenic NBN variants were detected by multigene panels in 31/12314 (0.25%) patients included in 15 studies. The risk of misinterpretation of such findings is substantial and supports the exclusion of NBN from multigene panels.
Chemotactic and Angiogenic Potential of Mineralized Collagen Scaffolds Functionalized with Naturally Occurring Bioactive Factor Mixtures to Stimulate Bone Regeneration
Authors: Bretschneider Henriette , Quade Mandy , Lode Anja , Gelinsky Michael , Rammelt Stefan , Vater Corina
Published: May 2021

To develop cost-effective and efficient bone substitutes for improved regeneration of bone defects, heparin-modified mineralized collagen scaffolds were functionalized with concentrated, naturally occurring bioactive factor mixtures derived from adipose tissue, platelet-rich plasma and conditioned medium from a hypoxia-treated human bone marrow-derived mesenchymal stem cell line. Besides the analysis of the release kinetics of functionalized scaffolds, the bioactivity of the released bioactive factors was tested with regard to chemotaxis and angiogenic tube formation. Additionally, functionalized scaffolds were seeded with human bone marrow-derived mesenchymal stromal cells (hBM-MSC) and their osteogenic and angiogenic potential was investigated. The release of bioactive factors from the scaffolds was highest within the first 3 days. Bioactivity of the released factors could be confirmed for all bioactive factor mixtures by successful chemoattraction of hBM-MSC in a transwell assay as well as by the formation of prevascular structures in a 2D co-culture system of hBM-MSC and human umbilical vein endothelial cells. The cells seeded directly onto the functionalized scaffolds were able to express osteogenic markers and form tubular networks. In conclusion, heparin-modified mineralized collagen scaffolds could be successfully functionalized with naturally occurring bioactive factor mixtures promoting cell migration and vascularization.
Overexpression Populus d-Type Cyclin Gene PsnCYCD1;1 Influences Cell Division and Produces Curved Leaf in Arabidopsis thaliana
Authors: Zheng Tangchun , Dai Lijuan , Liu Yi , Li Shuang , Zheng Mi , Zhao Zhongnan , Qu Guan-Zheng
Published: May 2021

d-type cyclins (CYCDs) are a special class of cyclins and play extremely important roles in plant growth and development. In the plant kingdom, most of the existing studies on CYCDs have been done on herbaceous plants, with few on perennial woody plants. Here, we identified a Populus d-type cyclin gene, PsnCYCD1;1, which is mainly transcribed in leaf buds and stems. The promoter of PsnCYCD1;1 activated GUS gene expression and transgenic Arabidopsis lines were strongly GUS stained in whole seedlings and mature anthers. Moreover, subcellular localization analysis showed the fluorescence signal of PsnCYCD1;1-GFP fusion protein is present in the nucleus. Furthermore, overexpression of the PsnCYCD1;1 gene in Arabidopsis can promote cell division and lead to small cell generation and cytokinin response, resulting in curved leaves and twisted inflorescence stems. Moreover, the transcriptional levels of endogenous genes, such as ASs, KNATs, EXP10, and PHB, were upregulated by PsnCYCD1;1. Together, our results indicated that PsnCYCD1;1 participates in cell division by cytokinin response, providing new information on controlling plant architecture in woody plants.
Antioxidant Potential of Aqueous Dispersions of Fullerenes C60, C70, and Gd@C82
Authors: Mikheev Ivan V. , Sozarukova Madina M. , Izmailov Dmitry Yu. , Kareev Ivan E. , Proskurnina Elena V. , Proskurnin Mikhail A.
Published: May 2021

The antioxidant potential (capacity and activity) of aqueous fullerene dispersions (AFD) of non-functionalized C60, C70, and Gd@C82 endofullerene (in micromolar concentration range) was estimated based on chemiluminescence measurements of the model of luminol and generation of organic radicals by 2,2′-azobis(2-amidinopropane) dihydrochloride (ABAP). The antioxidant capacity was estimated by the TRAP method, from the concentration of half-suppression, and from the suppression area in the initial period. All three approaches agree and show that the antioxidant capacity of AFDs increased in the order Gd@C82 < C70 < C60. Mathematical modeling of the long-term kinetics data was used for antioxidant activity estimation. The effect of C60 and C70 is found to be quenching of the excited product of luminol with ABAP-generated radical and not an actual antioxidant effect; quenching constants differ insignificantly. Apart from quenching with a similar constant, the AFD of Gd@C82 exhibits actual antioxidant action. The antioxidant activity in Gd@C82 is 300-fold higher than quenching constants.
DArTseq-Based High-Throughput SilicoDArT and SNP Markers Applied for Association Mapping of Genes Related to Maize Morphology
Authors: Tomkowiak Agnieszka , Bocianowski Jan , Spychała Julia , Grynia Joanna , Sobiech Aleksandra , Kowalczewski Przemysław Łukasz
Published: May 2021

Today, agricultural productivity is essential to meet the needs of a growing population, and is also a key tool in coping with climate change. Innovative plant breeding technologies such as molecular markers, phenotyping, genotyping, the CRISPR/Cas method and next-generation sequencing can help agriculture meet the challenges of the 21st century more effectively. Therefore, the aim of the research was to identify single-nucleotide polymorphisms (SNPs) and SilicoDArT markers related to select morphological features determining the yield in maize. The plant material consisted of ninety-four inbred lines of maize of various origins. These lines were phenotyped under field conditions. A total of 14 morphological features was analyzed. The DArTseq method was chosen for genotyping because this technique reduces the complexity of the genome by restriction enzyme digestion. Subsequently, short fragment sequencing was used. The choice of a combination of restrictases allowed the isolation of highly informative low copy fragments of the genome. Thanks to this method, 90% of the obtained DArTseq markers are complementary to the unique sequences of the genome. All the observed features were normally distributed. Analysis of variance indicated that the main effect of lines was statistically significant (p < 0.001) for all 14 traits of study. Thanks to the DArTseq analysis with the use of next-generation sequencing (NGS) in the studied plant material, it was possible to identify 49,911 polymorphisms, of which 33,452 are SilicoDArT markers and the remaining 16,459 are SNP markers. Among those mentioned, two markers associated with four analyzed traits deserved special attention: SNP (4578734) and SilicoDArT (4778900). SNP marker 4578734 was associated with the following features: anthocyanin coloration of cob glumes, number of days from sowing to anthesis, number of days from sowing to silk emergence and anthocyanin coloration of internodes. SilicoDArT marker 4778900 was associated with the following features: number of days from sowing to anthesis, number of days from sowing to silk emergence, tassel: angle between the axis and lateral branches and plant height. Sequences with a length of 71 bp were used for physical mapping. The BLAST and EnsemblPlants databases were searched against the maize genome to identify the positions of both markers. Marker 4578734 was localized on chromosome 7, the closest gene was Zm00001d022467, approximately 55 Kb apart, encoding anthocyanidin 3-O-glucosyltransferase. Marker 4778900 was located on chromosome 7, at a distance of 45 Kb from the gene Zm00001d045261 encoding starch synthase I. The latter observation indicated that these flanking SilicoDArT and SNP markers were not in a state of linkage disequilibrium.
Effects of Donor Cell Types on the Development of Bovine Embryos Using Cytoplasm Injection Cloning Technology
Authors: Xu Lianguang , Song Seok-Hwan , Idrees Muhammad , Mesalam Ayman , Joo Myeong-Don , Sidrat Tabinda , Wei Yiran , Lee Kyeong-Lim , Lu Wenfa , Kong Il-Keun
Published: May 2021

Cytoplasm injection cloning technology (CICT) is an efficient technique for evaluating the developmental potential of cloned embryos. In this study, we investigated the effects of donor cell type on the developmental potential and quality of cloned bovine embryos. Adult fibroblasts (AFs) and embryonic cells (ECs) were used as donor cells to clone bovine embryos using CICT. We initially used AF cells to develop cloned embryos and then cultured the cloned day-8 blastocysts for 10 days to obtain ECs as donor cells for second embryo cloning. We found that the bovine blastocysts cloned using AF cells had significantly reduced developmental rates, embryo quality, and ratios of inner cell mass (ICM) to the total number of cells compared to those using ECs as donor cells. Furthermore, there were significant differences in the DNA methyltransferase-, histone deacetylation-, apoptosis-, and development-related genes at the blastocyst stage in embryos cloned from AFs compared to those in embryos cloned from ECs. Our results suggest that using ECs as donor cells for nuclear transfer enhances the quantity and quality of cloned embryos. However, further investigation is required in terms of determining pregnancy rates and developing cloned embryos from different donor cell types.
Proteins Marking the Sequence of Genotoxic Signaling from Irradiated Mesenchymal Stromal Cells to CD34+ Cells
Authors: Kohl Vanessa , Drews Oliver , Costina Victor , Bierbaum Miriam , Jawhar Ahmed , Roehl Henning , Weiss Christel , Brendel Susanne , Kleiner Helga , Flach Johanna , Spiess Birgit , Seifarth Wolfgang , Nowak Daniel , Hofmann Wolf-Karsten , Fabarius Alice , Popp Henning D.
Published: May 2021

Non-targeted effects (NTE) of ionizing radiation may initiate myeloid neoplasms (MN). Here, protein mediators (I) in irradiated human mesenchymal stromal cells (MSC) as the NTE source, (II) in MSC conditioned supernatant and (III) in human bone marrow CD34+ cells undergoing genotoxic NTE were investigated. Healthy sublethal irradiated MSC showed significantly increased levels of reactive oxygen species. These cells responded by increasing intracellular abundance of proteins involved in proteasomal degradation, protein translation, cytoskeleton dynamics, nucleocytoplasmic shuttling, and those with antioxidant activity. Among the increased proteins were THY1 and GNA11/14, which are signaling proteins with hitherto unknown functions in the radiation response and NTE. In the corresponding MSC conditioned medium, the three chaperones GRP78, CALR, and PDIA3 were increased. Together with GPI, these were the only four altered proteins, which were associated with the observed genotoxic NTE. Healthy CD34+ cells cultured in MSC conditioned medium suffered from more than a six-fold increase in γH2AX focal staining, indicative for DNA double-strand breaks, as well as numerical and structural chromosomal aberrations within three days. At this stage, five proteins were altered, among them IQGAP1, HMGB1, and PA2G4, which are involved in malign development. In summary, our data provide novel insights into three sequential steps of genotoxic signaling from irradiated MSC to CD34+ cells, implicating that induced NTE might initiate the development of MN.
Arylamine Analogs of Methylene Blue: Substituent Effect on Aggregation Behavior and DNA Binding
Authors: Khadieva Alena , Mostovaya Olga , Padnya Pavel , Kalinin Valeriy , Grishaev Denis , Tumakov Dmitrii , Stoikov Ivan
Published: May 2021

The synthesis of new phenothiazine derivatives, analogs of Methylene Blue, is of particular interest in the design of new drugs, as well as in the development of a new generation of agents for photodynamic therapy. In this study, two new derivatives of phenothiazine, i.e., 3,7-bis(4-aminophenylamino)phenothiazin-5-ium chloride dihydrochloride (PTZ1) and 3,7-bis(4-sulfophenylamino)phenothiazin-5-ium chloride (PTZ2), are synthesized for the first time and characterized by NMR, IR spectroscopy, HRMS and elemental analysis. The interaction of the obtained compounds PTZ1 and PTZ2 with salmon sperm DNA is investigated. It is shown by UV-Vis spectroscopy and DFT calculations that substituents in arylamine fragments play a crucial role in dimer formation and interaction with DNA. In the case of PTZ1, two amine groups promote H-aggregate formation and DNA interactions through groove binding and intercalation. In the case of PTZ2, sulfanilic acid fragments prevent any dimer formation and DNA binding due to electrostatic repulsion. DNA interaction mechanisms are studied and confirmed by UV-vis and fluorescence spectroscopy in comparison with Methylene Blue. The obtained results open significant opportunities for the development of new drugs and photodynamic agents.
PERK/ATF4-Dependent ZFAS1 Upregulation Is Associated with Sorafenib Resistance in Hepatocellular Carcinoma Cells
Authors: Lin Jiunn-Chang , Yang Pei-Ming , Liu Tsang-Pai
Published: May 2021

Sorafenib, a multi-kinase inhibitor, is the first-line treatment for advanced hepatocellular carcinoma (HCC) patients. However, this drug only provides a short improvement of patients’ overall survival, and drug resistance is commonly developed. Thus, the identification of resistant factor(s) or biomarker(s) is needed to develop more efficient therapeutic strategies. Long, non-coding RNAs (lncRNAs) have recently been viewed as attractive cancer biomarkers and drive many important cancer phenotypes. A lncRNA, ZFAS1 (ZNFX1 antisense RNA 1) has been found to promote HCC metastasis. This study found that sorafenib induced ZFAS1 expression specifically in sorafenib-resistant HCC cells. Although ZFAS1 knockdown did not restore the sensitivity of HCC cells to sorafenib, its expression may act as a resistant biomarker for sorafenib therapy. Bioinformatics analysis predicted that sorafenib tended to induce pathways related to endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) in sorafenib-resistant HCC cells. In vitro experimental evidence suggested that sorafenib induced protein kinase RNA-like ER kinase (PERK)/activating transcription factor 4 (ATF4)-dependent ZFAS1 expression, and sorafenib resistance could be overcome by PERK/ATF inhibitors. Therefore, PERK/ATF4/ZFAS1 signaling axis might be an attractive therapeutic and prognostic biomarker for sorafenib therapy in HCC.
Roles of Plant Glycine-Rich RNA-Binding Proteins in Development and Stress Responses
Authors: Ma Liqun , Cheng Ke , Li Jinyan , Deng Zhiqi , Zhang Chunjiao , Zhu Hongliang
Published: May 2021

In recent years, much progress has been made in elucidating the functional roles of plant glycine-rich RNA-binding proteins (GR-RBPs) during development and stress responses. Canonical GR-RBPs contain an RNA recognition motif (RRM) or a cold-shock domain (CSD) at the N-terminus and a glycine-rich domain at the C-terminus, which have been associated with several different RNA processes, such as alternative splicing, mRNA export and RNA editing. However, many aspects of GR-RBP function, the targeting of their RNAs, interacting proteins and the consequences of the RNA target process are not well understood. Here, we discuss recent findings in the field, newly defined roles for GR-RBPs and the actions of GR-RBPs on target RNA metabolism.
Catalytic Descriptors to Investigate Catalytic Power in the Reaction of Haloalkane Dehalogenase Enzyme with 1,2-Dichloroethane
Authors: Xin Xin , Li Chen , Gao Delu , Wang Dunyou
Published: May 2021

Enzymes play a fundamental role in many biological processes. We present a theoretical approach to investigate the catalytic power of the haloalkane dehalogenase reaction with 1,2-dichloroethane. By removing the three main active-site residues one by one from haloalkane dehalogenase, we found two reactive descriptors: one descriptor is the distance difference between the breaking bond and the forming bond, and the other is the charge difference between the transition state and the reactant complex. Both descriptors scale linearly with the reactive barriers, with the three-residue case having the smallest barrier and the zero-residue case having the largest. The results demonstrate that, as the number of residues increases, the catalytic power increases. The predicted free energy barriers using the two descriptors of this reaction in water are 23.1 and 24.2 kcal/mol, both larger than the ones with any residues, indicating that the water solvent hinders the reactivity. Both predicted barrier heights agree well with the calculated one at 25.2 kcal/mol using a quantum mechanics and molecular dynamics approach, and also agree well with the experimental result at 26.0 kcal/mol. This study shows that reactive descriptors can also be used to describe and predict the catalytic performance for enzyme catalysis.
Calreticulin Deficiency Disturbs Ribosome Biogenesis and Results in Retardation in Embryonic Kidney Development
Authors: Serin Nazli , Dihazi Gry H. , Tayyeb Asima , Lenz Christof , Müller Gerhard A. , Zeisberg Michael , Dihazi Hassan
Published: May 2021

Nephrogenesis is driven by complex signaling pathways that control cell growth and differentiation. The endoplasmic reticulum chaperone calreticulin (Calr) is well known for its function in calcium storage and in the folding of glycoproteins. Its role in kidney development is still not understood. We provide evidence for a pivotal role of Calr in nephrogenesis in this investigation. We show that Calr deficiency results in the disrupted formation of an intact nephrogenic zone and in retardation of nephrogenesis, as evidenced by the disturbance in the formation of comma-shaped and s-shaped bodies. Using proteomics and transcriptomics approaches, we demonstrated that in addition to an alteration in Wnt-signaling key proteins, embryonic kidneys from Calr−/− showed an overall impairment in expression of ribosomal proteins which reveals disturbances in protein synthesis and nephrogenesis. CRISPR/cas9 mediated knockout confirmed that Calr deficiency is associated with a deficiency of several ribosomal proteins and key proteins in ribosome biogenesis. Our data highlights a direct link between Calr expression and the ribosome biogenesis.
Regulation of ClC-2 Chloride Channel Proteostasis by Molecular Chaperones: Correction of Leukodystrophy-Associated Defect
Authors: Fu Ssu-Ju , Hu Meng-Chun , Hsiao Cheng-Tsung , Cheng An-Ting , Chen Tsung-Yu , Jeng Chung-Jiuan , Tang Chih-Yung
Published: May 2021

The ClC-2 channel plays a critical role in maintaining ion homeostasis in the brain and the testis. Loss-of-function mutations in the ClC-2-encoding human CLCN2 gene are linked to the white matter disease leukodystrophy. Clcn2-deficient mice display neuronal myelin vacuolation and testicular degeneration. Leukodystrophy-causing ClC-2 mutant channels are associated with anomalous proteostasis manifesting enhanced endoplasmic reticulum (ER)-associated degradation. The molecular nature of the ER quality control system for ClC-2 protein remains elusive. In mouse testicular tissues and Leydig cells, we demonstrated that endogenous ClC-2 co-existed in the same protein complex with the molecular chaperones heat shock protein 90β (Hsp90β) and heat shock cognate protein (Hsc70), as well as the associated co-chaperones Hsp70/Hsp90 organizing protein (HOP), activator of Hsp90 ATPase homolog 1 (Aha1), and FK506-binding protein 8 (FKBP8). Further biochemical analyses revealed that the Hsp90β-Hsc70 chaperone/co-chaperone system promoted mouse and human ClC-2 protein biogenesis. FKBP8 additionally facilitated membrane trafficking of ClC-2 channels. Interestingly, treatment with the Hsp90-targeting small molecule 17-allylamino-17-demethoxygeldanamycin (17-AAG) substantially boosted ClC-2 protein expression. Also, 17-AAG effectively increased both total and cell surface protein levels of leukodystrophy-causing loss-of-function ClC-2 mutant channels. Our findings highlight the therapeutic potential of 17-AAG in correcting anomalous ClC-2 proteostasis associated with leukodystrophy.
Somatostatin, a Presynaptic Modulator of Glutamatergic Signal in the Central Nervous System
Authors: Pittaluga Anna , Roggeri Alessandra , Vallarino Giulia , Olivero Guendalina
Published: May 2021

Somatostatin is widely diffused in the central nervous system, where it participates to control the efficiency of synaptic transmission. This peptide mainly colocalizes with GABA, in inhibitory, GABA-containing interneurons from which it is actively released in a Ca2+ dependent manner upon application of depolarizing stimuli. Once released in the synaptic cleft, somatostatin acts locally, or it diffuses in the extracellular space through “volume diffusion”, a mechanism(s) of distribution which mainly operates in the cerebrospinal fluid and that assures the progression of neuronal signalling from signal-secreting sender structures towards receptor-expressing targeted neurons located extrasynaptically, in a non-synaptic, inter-neuronal form of communication. Somatostatin controls the efficiency of central glutamate transmission by either modulating presynaptically the glutamate exocytosis or by metamodulating the activity of glutamate receptors colocalized and functionally coupled with somatostatin receptors in selected subpopulations of nerve terminals. Deciphering the role of somatostatin in the mechanisms of “volume diffusion” and in the “receptor-receptor interaction” unveils new perspectives in the central role of this fine tuner of synaptic strength, paving the road to new therapeutic approaches for the cure of central disorders.
Apex Resection in Zebrafish (Danio rerio) as a Model of Heart Regeneration: A Video-Assisted Guide
Authors: Ellman Ditte Gry , Slaiman Ibrahim Mohamad , Mathiesen Sabrina Bech , Andersen Kristian Skriver , Hofmeister Wolfgang , Ober Elke Annette , Andersen Ditte Caroline
Published: May 2021

Ischemic heart disease is one of the leading causes of deaths worldwide. A major hindrance to resolving this challenge lies in the mammalian hearts inability to regenerate after injury. In contrast, zebrafish retain a regenerative capacity of the heart throughout their lifetimes. Apex resection (AR) is a popular zebrafish model for studying heart regeneration, and entails resecting 10–20% of the heart in the apex region, whereafter the regeneration process is monitored until the heart is fully regenerated within 60 days. Despite this popularity, video tutorials describing this technique in detail are lacking. In this paper we visualize and describe the entire AR procedure including anaesthesia, surgery, and recovery. In addition, we show that the concentration and duration of anaesthesia are important parameters to consider, to balance sufficient levels of sedation and minimizing mortality. Moreover, we provide examples of how zebrafish heart regeneration can be assessed both in 2D (immunohistochemistry of heart sections) and 3D (analyses of whole, tissue cleared hearts using multiphoton imaging). In summary, this paper aims to aid beginners in establishing and conducting the AR model in their laboratory, but also to spur further interest in improving the model and its evaluation.
PDS5A and PDS5B in Cohesin Function and Human Disease
Authors: Zhang Nenggang , Coutinho Luiza E. , Pati Debananda
Published: May 2021

Precocious dissociation of sisters 5 (PDS5) is an associate protein of cohesin that is conserved from yeast to humans. It acts as a regulator of the cohesin complex and plays important roles in various cellular processes, such as sister chromatid cohesion, DNA damage repair, gene transcription, and DNA replication. Vertebrates have two paralogs of PDS5, PDS5A and PDS5B, which have redundant and unique roles in regulating cohesin functions. Herein, we discuss the molecular characteristics and functions of PDS5, as well as the effects of its mutations in the development of diseases and their relevance for novel therapeutic strategies.
Genetic Dominant Variants in STUB1, Segregating in Families with SCA48, Display In Vitro Functional Impairments Indistinctive from Recessive Variants Associated with SCAR16
Authors: Pakdaman Yasaman , Berland Siren , Bustad Helene J. , Erdal Sigrid , Thompson Bryony A. , James Paul A. , Power Kjersti N. , Ellingsen Ståle , Krooni Martin , Berge Line I. , Sexton Adrienne , Bindoff Laurence A. , Knappskog Per M. , Johansson Stefan , Aukrust Ingvild
Published: May 2021

Variants in STUB1 cause both autosomal recessive (SCAR16) and dominant (SCA48) spinocerebellar ataxia. Reports from 18 STUB1 variants causing SCA48 show that the clinical picture includes later-onset ataxia with a cerebellar cognitive affective syndrome and varying clinical overlap with SCAR16. However, little is known about the molecular properties of dominant STUB1 variants. Here, we describe three SCA48 families with novel, dominantly inherited STUB1 variants (p.Arg51_Ile53delinsProAla, p.Lys143_Trp147del, and p.Gly249Val). All the patients developed symptoms from 30 years of age or later, all had cerebellar atrophy, and 4 had cognitive/psychiatric phenotypes. Investigation of the structural and functional consequences of the recombinant C-terminus of HSC70-interacting protein (CHIP) variants was performed in vitro using ubiquitin ligase activity assay, circular dichroism assay and native polyacrylamide gel electrophoresis. These studies revealed that dominantly and recessively inherited STUB1 variants showed similar biochemical defects, including impaired ubiquitin ligase activity and altered oligomerization properties of the CHIP. Our findings expand the molecular understanding of SCA48 but also mean that assumptions concerning unaffected carriers of recessive STUB1 variants in SCAR16 families must be re-evaluated. More investigations are needed to verify the disease status of SCAR16 heterozygotes and elucidate the molecular relationship between SCA48 and SCAR16 diseases.
The miR-106b/NR2F2-AS1/PLEKHO2 Axis Regulates Migration and Invasion of Colorectal Cancer through the MAPK Pathway
Authors: Liu Shuzhen , An Guoyan , Cao Qing , Li Tong , Jia Xinyu , Lei Lei
Published: May 2021

Increasing numbers of miRNAs have been observed as oncogenes or tumor suppressors in colorectal cancer (CRC). It was recently reported that hsa-miR-106b-5p (miR-106b) promoted CRC cell migration and invasion. However, there were also studies showing contradictory results. Therefore, in the present study, we further explore the role of miR-106b and its downstream networks in the carcinogenesis of CRC. We observed that the expression of miR-106b is significantly increased in Pan-Cancer and CRC tissues compared with normal tissues from The Cancer Genome Atlas (TCGA) database. Furthermore, we used Transwell, Cell Counting Kit-8, and colony formation assays to clarify that miR-106b promotes the migratory, invasive, and proliferative abilities of CRC cells. For the first time, we systematically screened the target mRNAs and lncRNAs of miR-106b using TCGA database and the bioinformatics algorithms. Dual-luciferase reporter assay confirmed that NR2F2-AS1 and PLEKHO2 are the direct targets of miR-106b. Furthermore, NR2F2-AS1 acts as a competing endogenous RNA (ceRNA) to regulate PLEKHO2 expression by sponging miR-106b. The results of Gene set enrichment analysis (GSEA) and Western blot indicated that they play important roles in CRC progression by regulating MAPK pathway. Thus, miR-106b/NR2F2-AS1/PLEKHO2/MAPK signaling axis may suggest the potential usage in CRC treatment.
PCSK9 Biology and Its Role in Atherothrombosis
Authors: Barale Cristina , Melchionda Elena , Morotti Alessandro , Russo Isabella
Published: May 2021

It is now about 20 years since the first case of a gain-of-function mutation involving the as-yet-unknown actor in cholesterol homeostasis, proprotein convertase subtilisin/kexin type 9 (PCSK9), was described. It was soon clear that this protein would have been of huge scientific and clinical value as a therapeutic strategy for dyslipidemia and atherosclerosis-associated cardiovascular disease (CVD) management. Indeed, PCSK9 is a serine protease belonging to the proprotein convertase family, mainly produced by the liver, and essential for metabolism of LDL particles by inhibiting LDL receptor (LDLR) recirculation to the cell surface with the consequent upregulation of LDLR-dependent LDL-C levels. Beyond its effects on LDL metabolism, several studies revealed the existence of additional roles of PCSK9 in different stages of atherosclerosis, also for its ability to target other members of the LDLR family. PCSK9 from plasma and vascular cells can contribute to the development of atherosclerotic plaque and thrombosis by promoting platelet activation, leukocyte recruitment and clot formation, also through mechanisms not related to systemic lipid changes. These results further supported the value for the potential cardiovascular benefits of therapies based on PCSK9 inhibition. Actually, the passive immunization with anti-PCSK9 antibodies, evolocumab and alirocumab, is shown to be effective in dramatically reducing the LDL-C levels and attenuating CVD. While monoclonal antibodies sequester circulating PCSK9, inclisiran, a small interfering RNA, is a new drug that inhibits PCSK9 synthesis with the important advantage, compared with PCSK9 mAbs, to preserve its pharmacodynamic effects when administrated every 6 months. Here, we will focus on the major understandings related to PCSK9, from its discovery to its role in lipoprotein metabolism, involvement in atherothrombosis and a brief excursus on approved current therapies used to inhibit its action.
Gastric Serotonin Biosynthesis and Its Functional Role in L-Arginine-Induced Gastric Proton Secretion
Authors: Holik Ann-Katrin , Schweiger Kerstin , Stoeger Verena , Lieder Barbara , Reiner Angelika , Zopun Muhammet , Hoi Julia K. , Kretschy Nicole , Somoza Mark M. , Kriwanek Stephan , Pignitter Marc , Somoza Veronika
Published: May 2021

Among mammals, serotonin is predominantly found in the gastrointestinal tract, where it has been shown to participate in pathway-regulating satiation. For the stomach, vascular serotonin release induced by gastric distension is thought to chiefly contribute to satiation after food intake. However, little information is available on the capability of gastric cells to synthesize, release and respond to serotonin by functional changes of mechanisms regulating gastric acid secretion. We investigated whether human gastric cells are capable of serotonin synthesis and release. First, HGT-1 cells, derived from a human adenocarcinoma of the stomach, and human stomach specimens were immunostained positive for serotonin. In HGT-1 cells, incubation with the tryptophan hydroxylase inhibitor p-chlorophenylalanine reduced the mean serotonin-induced fluorescence signal intensity by 27%. Serotonin release of 147 ± 18%, compared to control HGT-1 cells (set to 100%) was demonstrated after treatment with 30 mM of the satiating amino acid L-Arg. Granisetron, a 5-HT3 receptor antagonist, reduced this L-Arg-induced serotonin release, as well as L-Arg-induced proton secretion. Similarly to the in vitro experiment, human antrum samples released serotonin upon incubation with 10 mM L-Arg. Overall, our data suggest that human parietal cells in culture, as well as from the gastric antrum, synthesize serotonin and release it after treatment with L-Arg via an HTR3-related mechanism. Moreover, we suggest not only gastric distension but also gastric acid secretion to result in peripheral serotonin release.
Neuroinflammation and Neuronal Loss in the Hippocampus Are Associated with Immediate Posttraumatic Seizures and Corticosterone Elevation in Rats
Authors: Komoltsev Ilia G. , Frankevich Stepan O. , Shirobokova Natalia I. , Volkova Aleksandra A. , Onufriev Mikhail V. , Moiseeva Julia V. , Novikova Margarita R. , Gulyaeva Natalia V.
Published: May 2021

Hippocampal damage after traumatic brain injury (TBI) is associated with late posttraumatic conditions, such as depression, cognitive decline and epilepsy. Mechanisms of selective hippocampal damage after TBI are not well understood. In this study, using rat TBI model (lateral fluid percussion cortical injury), we assessed potential association of immediate posttraumatic seizures and changes in corticosterone (CS) levels with neuroinflammation and neuronal cell loss in the hippocampus. Indices of distant hippocampal damage (neurodegeneration and neuroinflammation) were assessed using histological analysis (Nissl staining, Iba-1 immunohistochemical staining) and ELISA (IL-1β and CS) 1, 3, 7 and 14 days after TBI or sham operation in male Wistar rats (n = 146). IL-1β was elevated only in the ipsilateral hippocampus on day 1 after trauma. CS peak was detected on day 3 in blood, the ipsilateral and contralateral hippocampus. Neuronal cell loss in the hippocampus was demonstrated bilaterally; in the ipsilateral hippocampus it started earlier than in the contralateral. Microglial activation was evident in the hippocampus bilaterally on day 7 after TBI. The duration of immediate seizures correlated with CS elevation, levels of IL-1β and neuronal loss in the hippocampus. The data suggest potential association of immediate post-traumatic seizures with CS-dependent neuroinflammation-mediated distant hippocampal damage.
Dexamethasone Downregulates Autophagy through Accelerated Turn-Over of the Ulk-1 Complex in a Trabecular Meshwork Cells Strain: Insights on Steroid-Induced Glaucoma Pathogenesis
Authors: Sbardella Diego , Tundo Grazia Raffaella , Coletta Massimo , Manni Gianluca , Oddone Francesco
Published: May 2021

Steroid-induced glaucoma is a severe pathological condition, sustained by a rapidly progressive increase in intraocular pressure (IOP), which is diagnosed in a subset of subjects who adhere to a glucocorticoid (GC)-based therapy. Molecular and clinical studies suggest that either natural or synthetic GCs induce a severe metabolic dysregulation of Trabecular Meshwork Cells (TMCs), an endothelial-derived histotype with phagocytic and secretive functions which lay at the iridocorneal angle in the anterior segment of the eye. Since TMCs physiologically regulate the composition and architecture of trabecular meshwork (TM), which is the main outflow pathway of aqueous humor, a fluid which shapes the eye globe and nourishes the lining cell types, GCs are supposed to trigger a pathological remodeling of the TM, inducing an IOP increase and retina mechanical compression. The metabolic dysregulation of TMCs induced by GCs exposure has never been characterized at the molecular detail. Herein, we report that, upon dexamethasone exposure, a TMCs strain develops a marked inhibition of the autophagosome biogenesis pathway through an enhanced turnover of two members of the Ulk-1 complex, the main platform for autophagy induction, through the Ubiquitin Proteasome System (UPS).
New Insights into Alterations in PL Proteins Affecting Their Binding to DNA after Exposure of Mytilus galloprovincialis to Mercury—A Possible Risk to Sperm Chromatin Structure?
Authors: Lettieri Gennaro , Notariale Rosaria , Carusone Nadia , Giarra Antonella , Trifuoggi Marco , Manna Caterina , Piscopo Marina
Published: May 2021

Mercury (Hg) is a highly toxic and widespread pollutant. We previously reported that the exposure of Mytilus galloprovincialis for 24 h to doses of HgCl2 similar to those found in seawater (range 1–100 pM) produced alterations in the properties of protamine-like (PL) proteins that rendered them unable to bind and protect DNA from oxidative damage. In the present work, to deepen our studies, we analyzed PL proteins by turbidimetry and fluorescence spectroscopy and performed salt-induced release analyses of these proteins from sperm nuclei after the exposure of mussels to HgCl2 at the same doses. Turbidity assays indicated that mercury, at these doses, induced PL protein aggregates, whereas fluorescence spectroscopy measurements showed mercury-induced conformational changes. Indeed, the mobility of the PLII band changed in sodium dodecyl sulphate-polyacrylamide gel electrophoresis, particularly after exposure to 10-pM HgCl2, confirming the mercury-induced structural rearrangement. Finally, exposure to HgCl2 at all doses produced alterations in PL-DNA binding, detectable by DNA absorption spectra after the PL protein addition and by a decreased release of PLII and PLIII from the sperm nuclei. In conclusion, in this paper, we reported Hg-induced PL protein alterations that could adversely affect mussel reproductive activity, providing an insight into the molecular mechanism of Hg-related infertility.