Actin polymerization state regulates osteogenic differentiation in human  adipose-derived stem cells
CONCLUSIONS: In summary, our data suggest that the actin polymerization state may promote the osteogenic differentiation of hASCs by regulating the protein expression of focal adhesion-associated proteins in a concentration-dependent manner. Our findings provide valuable information for exploring the mechanism of osteogenic differentiation in hASCs.PMID:33858321 | DOI:10.1186/s11658-021-00259-8 (Source: Mol Biol Cell)
Source: Mol Biol Cell - April 16, 2021 Category: Molecular Biology Authors: Bing Sun Rongmei Qu Tingyu Fan Yuchao Yang Xin Jiang Asmat Ullah Khan Zhitao Zhou Jingliao Zhang Kuanhai Wei Jun Ouyang Jingxing Dai Source Type: research

Uncovering SARS-CoV-2 kidney tropism
Nat Rev Mol Cell Biol. 2021 Apr 15. doi: 10.1038/s41580-021-00370-w. Online ahead of print.NO ABSTRACTPMID:33859371 | DOI:10.1038/s41580-021-00370-w (Source: Mol Biol Cell)
Source: Mol Biol Cell - April 16, 2021 Category: Molecular Biology Authors: Samira Musah Source Type: research

Establishment of Intestinal Epithelial Cell Monolayers and Their Use in Calcium Switch Assay for Assessment of Intestinal Tight Junction Assembly
Methods Mol Biol. 2021 Apr 17. doi: 10.1007/7651_2021_347. Online ahead of print.ABSTRACTIntestinal barrier function relies primarily on the assembly and integrity of tight junctions, which forms a size-selective barrier. This barrier restricts paracellular movement of solutes in various types of epithelia. Of note, extracellular Ca2+ concentration affects tight junction assembly. Therefore, the removal and re-addition of Ca2+ into cell culture medium of cultured intestinal epithelial cells causes destabilization and reassembly of tight junction to membrane periphery near apical surface, respectively. Based on this princip...
Source: Mol Biol Cell - April 16, 2021 Category: Molecular Biology Authors: Pawin Pongkorpsakol Wilasinee Satianrapapong Preedajit Wongkrasant Peter R Steinhagen Nuttha Tuangkijkul Nutthapoom Pathomthongtaweechai Chatchai Muanprasat Source Type: research

Clioquinol Attenuates Pulmonary Fibrosis Through Inactivation of Fibroblasts via Iron Chelation
In conclusion, our study demonstrated that CQ, acting as an iron chelator, attenuates experimental PF through inactivation of fibroblasts, providing support for targeting iron metabolism as a basis for PF treatment.PMID:33861690 | DOI:10.1165/rcmb.2020-0279OC (Source: Mol Biol Cell)
Source: Mol Biol Cell - April 16, 2021 Category: Molecular Biology Authors: Yumeng Zhu Jing Chang Ke Tan Steven K Huang Xin Liu Xiaofan Wang Mengshu Cao Hongmin Zhang Shuxin Li Xianglin Duan Yanzhong Chang Yumei Fan Pengxiu Cao Source Type: research

Territories or spaghetti? Chromosome organization exposed
Nat Rev Mol Cell Biol. 2021 Apr 14. doi: 10.1038/s41580-021-00372-8. Online ahead of print.NO ABSTRACTPMID:33854243 | DOI:10.1038/s41580-021-00372-8 (Source: Mol Biol Cell)
Source: Mol Biol Cell - April 15, 2021 Category: Molecular Biology Authors: Gabriel M C Longo Vassilis Roukos Source Type: research

Functional analysis of four Class III peroxidases from Chinese pear fruit: a critical role in lignin polymerization
Physiol Mol Biol Plants. 2021 Mar;27(3):515-522. doi: 10.1007/s12298-021-00949-9. Epub 2021 Feb 20.ABSTRACTPear fruit could be used as good medicine to relieve coughs, promote salivation, nourish lungs, and reduce the risk of many diseases for its phytochemical action. Lignin is a major secondary metabolite in Chinese pear fruit. Class III peroxidase (Class III PRX) is an important enzyme in the biosynthesis of lignin in plants. However, we poorly understand the role of PRXs in lignin biosynthesis in Chinese pear fruit. In our study, we cloned five PRXs from Chinese pear (Pyrus bretschneideri), namely PbPRX2, PbPRX22, PbPR...
Source: Mol Biol Cell - April 15, 2021 Category: Molecular Biology Authors: Xi Zhu Lan Jiang Yongping Cai Yunpeng Cao Source Type: research

Effect of thermospermine on expression profiling of different gene using massive analysis of cDNA ends (MACE) and vascular maintenance in Arabidopsis
Physiol Mol Biol Plants. 2021 Mar;27(3):577-586. doi: 10.1007/s12298-021-00967-7. Epub 2021 Mar 14.ABSTRACTArabidopsis thaliana polyamine oxidase 5 gene (AtPAO5) functions as a thermospermine (T-Spm) oxidase. Aerial growth of its knock-out mutant (Atpao5-2) was significantly repressed by low dose(s) of T-Spm but not by other polyamines. To figure out the underlying mechanism, massive analysis of 3'-cDNA ends was performed. Low dose of T-Spm treatment modulates more than two fold expression 1,398 genes in WT compared to 3186 genes in Atpao5-2. Cell wall, lipid and secondary metabolisms were dramatically affected in low dose...
Source: Mol Biol Cell - April 15, 2021 Category: Molecular Biology Authors: G H M Sagor Stefan Simm Dong Wook Kim Masaru Niitsu Tomonobu Kusano Thomas Berberich Source Type: research

Efficient High-Density hiPSCs Expansion in Simple Dialysis Device
Methods Mol Biol. 2021 Apr 16. doi: 10.1007/7651_2021_391. Online ahead of print.ABSTRACTiPSCs are potential cell types that can be used for regenerative medicine. Suspension culture is the main approach to produce a sufficient amount of required cells to realize the application of hiPSCs for the transplantation of the large organ. The dialysis culture system holds the potential to reduce the cost by utilizing endogenous growth factors and recycle the remaining exogenous growth factors at the same time. However, the current large scale dialysis culture system was not optimized for expanding the high-density culture. Severa...
Source: Mol Biol Cell - April 15, 2021 Category: Molecular Biology Authors: Fuad Gandhi Torizal Hyunjin Choi Marie Shinohara Yasuyuki Sakai Source Type: research

Bronchopulmonary Dysplasia: Endothelial Cells in the Driver's Seat
Am J Respir Cell Mol Biol. 2021 Apr 15. doi: 10.1165/rcmb.2021-0145ED. Online ahead of print.NO ABSTRACTPMID:33856963 | DOI:10.1165/rcmb.2021-0145ED (Source: Mol Biol Cell)
Source: Mol Biol Cell - April 15, 2021 Category: Molecular Biology Authors: Christine U Vohwinkel Rubin M Tuder Source Type: research

Territories or spaghetti? Chromosome organization exposed
Nat Rev Mol Cell Biol. 2021 Apr 14. doi: 10.1038/s41580-021-00372-8. Online ahead of print.NO ABSTRACTPMID:33854243 | DOI:10.1038/s41580-021-00372-8 (Source: Mol Biol Cell)
Source: Mol Biol Cell - April 15, 2021 Category: Molecular Biology Authors: Gabriel M C Longo Vassilis Roukos Source Type: research

Functional analysis of four Class III peroxidases from Chinese pear fruit: a critical role in lignin polymerization
Physiol Mol Biol Plants. 2021 Mar;27(3):515-522. doi: 10.1007/s12298-021-00949-9. Epub 2021 Feb 20.ABSTRACTPear fruit could be used as good medicine to relieve coughs, promote salivation, nourish lungs, and reduce the risk of many diseases for its phytochemical action. Lignin is a major secondary metabolite in Chinese pear fruit. Class III peroxidase (Class III PRX) is an important enzyme in the biosynthesis of lignin in plants. However, we poorly understand the role of PRXs in lignin biosynthesis in Chinese pear fruit. In our study, we cloned five PRXs from Chinese pear (Pyrus bretschneideri), namely PbPRX2, PbPRX22, PbPR...
Source: Mol Biol Cell - April 15, 2021 Category: Molecular Biology Authors: Xi Zhu Lan Jiang Yongping Cai Yunpeng Cao Source Type: research

Effect of thermospermine on expression profiling of different gene using massive analysis of cDNA ends (MACE) and vascular maintenance in Arabidopsis
Physiol Mol Biol Plants. 2021 Mar;27(3):577-586. doi: 10.1007/s12298-021-00967-7. Epub 2021 Mar 14.ABSTRACTArabidopsis thaliana polyamine oxidase 5 gene (AtPAO5) functions as a thermospermine (T-Spm) oxidase. Aerial growth of its knock-out mutant (Atpao5-2) was significantly repressed by low dose(s) of T-Spm but not by other polyamines. To figure out the underlying mechanism, massive analysis of 3'-cDNA ends was performed. Low dose of T-Spm treatment modulates more than two fold expression 1,398 genes in WT compared to 3186 genes in Atpao5-2. Cell wall, lipid and secondary metabolisms were dramatically affected in low dose...
Source: Mol Biol Cell - April 15, 2021 Category: Molecular Biology Authors: G H M Sagor Stefan Simm Dong Wook Kim Masaru Niitsu Tomonobu Kusano Thomas Berberich Source Type: research

Association of cyclin-dependent kinase inhibitor 2B antisense RNA 1 gene expression and rs2383207 variant with breast cancer risk and survival
This study aimed to explore the association of lncRNA cyclin-dependent kinase inhibitor 2B antisense RNA 1 (CDKN2B-AS1) gene expression and the rs2383207A>G intronic variant with breast cancer (BC) risk and prognosis and to verify the molecular role and networks of this lncRNA in BC by bioinformatics gene analysis.METHODS: Serum CDKN2B-AS1 relative expression and rs2383207 genotypes were determined in 214 unrelated women (104 primary BC and 110 controls) using real-time PCR. Sixteen BC studies from The Cancer Genome Atlas (TCGA) including 8925 patients were also retrieved for validation of results.RESULTS: CDKN2B-AS1 se...
Source: Mol Biol Cell - April 14, 2021 Category: Molecular Biology Authors: Shahad W Kattan Yahya H Hobani Sameerah Shaheen Sara H Mokhtar Mohammad H Hussein Eman A Toraih Manal S Fawzy Hussein Abdelaziz Abdalla Source Type: research

Structural insights into the cause of human < em > RSPH4A < /em > primary ciliary dyskinesia
Mol Biol Cell. 2021 Apr 14:mbcE20120806. doi: 10.1091/mbc.E20-12-0806. Online ahead of print.ABSTRACTCilia and flagella are evolutionarily conserved eukaryotic organelles involved in cell motility and signaling. In humans, mutations in Radial Spoke Head Protein 4 homolog A (RSPH4A) can lead to primary ciliary dyskinesia (PCD), a life-shortening disease characterized by chronic respiratory tract infections, abnormal organ positioning, and infertility. Despite its importance for human health, the location of RSPH4A in human cilia has not been resolved, and the structural basis of RSPH4A-/- PCD remains elusive. Here, we prese...
Source: Mol Biol Cell - April 14, 2021 Category: Molecular Biology Authors: Yanhe Zhao Justine Pinskey Jianfeng Lin Weining Yin Patrick R Sears Leigh A Daniels Maimoona A Zariwala Michael R Knowles Lawrence E Ostrowski Daniela Nicastro Source Type: research

The N-terminal Tail of < em > C. elegans < /em > CENP-A Interacts with KNL-2 and is Essential for Centromeric Chromatin Assembly
Mol Biol Cell. 2021 Apr 14:mbcE20120798. doi: 10.1091/mbc.E20-12-0798. Online ahead of print.ABSTRACTCentromeres are epigenetically defined by the centromere-specific histone H3 variant CENP-A. Specialized loading machinery, including the histone chaperone HJURP/Scm3, participates in CENP-A nucleosome assembly. However, Scm3/HJURP is missing from multiple lineages, including nematodes, with CENP-A-dependent centromeres. Here, we show that the extended N-terminal tail of C. elegans CENP-A contains a predicted structured region that is essential for centromeric chromatin assembly; removal of this region prevents CENP-A loadi...
Source: Mol Biol Cell - April 14, 2021 Category: Molecular Biology Authors: Christian de Groot Jack Houston Bethany Davis Adina Gerson-Gurwitz Joost Monen Pablo Lara-Gonzalez Karen Oegema Andrew K Shiau Arshad Desai Source Type: research

Structures of chaperone-associated assembly intermediates reveal coordinated mechanisms of proteasome biogenesis
Nat Struct Mol Biol. 2021 Apr 12. doi: 10.1038/s41594-021-00583-9. Online ahead of print.ABSTRACTThe proteasome mediates most selective protein degradation. Proteolysis occurs within the 20S core particle (CP), a barrel-shaped chamber with an α7β7β7α7 configuration. CP biogenesis proceeds through an ordered multistep pathway requiring five chaperones, Pba1-4 and Ump1. Using Saccharomyces cerevisiae, we report high-resolution structures of CP assembly intermediates by cryogenic-electron microscopy. The first structure corresponds to the 13S particle, which consists of a complete α-ring, partial &...
Source: Mol Biol Cell - April 13, 2021 Category: Molecular Biology Authors: Helena M Schnell Richard M Walsh Shaun Rawson Mandeep Kaur Meera K Bhanu Geng Tian Miguel A Prado Angel Guerra-Moreno Joao A Paulo Steven P Gygi Jeroen Roelofs Daniel Finley John Hanna Source Type: research

Correction to: Current status and impending progress for cassava structural genomics
Plant Mol Biol. 2021 Apr 13. doi: 10.1007/s11103-021-01139-7. Online ahead of print.NO ABSTRACTPMID:33847871 | DOI:10.1007/s11103-021-01139-7 (Source: Mol Biol Cell)
Source: Mol Biol Cell - April 13, 2021 Category: Molecular Biology Authors: Jessica B Lyons Jessen V Bredeson Ben N Mansfeld Guillaume Jean Bauchet Jeffrey Berry Adam Boyher Lukas A Mueller Daniel S Rokhsar Rebecca S Bart Source Type: research

Sheep Erythrocyte Preparation for Hemolytic Tests Exploring Complement Functional Activities
Methods Mol Biol. 2021;2227:61-67. doi: 10.1007/978-1-0716-1016-9_6.ABSTRACTSheep erythrocytes (SE) are commonly used in complement functional tests. Non sensitized SE are useful to study the FH activity of cell protection. Indeed, as the cell surface of sheep erythrocytes is rich in sialic acids, Factor H (FH) is able to bind on it and therefore they represent a model of nonactivating surface. Because of their high capacity of complement regulation SE need to be modified to explore other functionality of the complement pathways, like the Complement hemolytic 50 (CH50) or the AP C3 convertase decay assays. For these tests,...
Source: Mol Biol Cell - April 13, 2021 Category: Molecular Biology Authors: Melchior Chabannes Pauline Bordereau Paula Vieira Martins Marie-Agn ès Dragon-Durey Source Type: research

Hemolytic Tests Exploring Factor H Functional Activities
Methods Mol Biol. 2021;2227:69-81. doi: 10.1007/978-1-0716-1016-9_7.ABSTRACTImpairment of the complement regulatory protein Factor H (FH) is implicated in the physiopathological mechanisms of different diseases like atypical hemolytic and uremic syndrome and C3 glomerulopathies. It may be due to genetic abnormalities or acquired with the development of autoantibodies. FH has several ligands; therefore, the exploration of its functions requires to perform different tests. Among them, two hemolytic tests are very useful because they give specific and complementary information about FH functions. The first one is dedicated to...
Source: Mol Biol Cell - April 13, 2021 Category: Molecular Biology Authors: Melchior Chabannes Shambhuprasad K Togarsimalemath Marie-Agn ès Dragon-Durey Source Type: research

Complement C3 Deposition on Endothelial Cells Revealed by Flow Cytometry
Methods Mol Biol. 2021;2227:97-105. doi: 10.1007/978-1-0716-1016-9_9.ABSTRACTThe three pathways of the complement system converge toward the cleavage of the central complement component C3 into its activated fragments, with C3b being able to bind covalently to the activating surface. The endothelial cells are among the major targets for complement attack in pathological conditions, as the atypical hemolytic uremic syndrome. Therefore, study of complement C3 deposition on endothelial cells by flow cytometry is a sensitive test to measure complement activation. This test can be used as a research or clinical tool to test com...
Source: Mol Biol Cell - April 13, 2021 Category: Molecular Biology Authors: Idris Boudhabhay Anne Grunenwald Lubka T Roumenina Source Type: research

Detection of C3 Nephritic Factor by Hemolytic Assay
Methods Mol Biol. 2021;2227:147-158. doi: 10.1007/978-1-0716-1016-9_15.ABSTRACTC3 nephritic Factor (C3NeF) is autoantibody that binds neoepitopes of the C3 convertase C3bBb, resulting in a stabilization of the enzyme. First functional characterizations of C3NeF were performed by hemolytic assays using preactivated sheep erythrocytes (bearing C3b). Sheep erythrocytes are beforehand sensitized with an anti-sheep red blood cell stroma antibody produced in rabbit (hemolysin). Sensitized sheep erythrocytes will initiate cascade complement activation via the classic pathway, followed by alternative pathway amplification loop, re...
Source: Mol Biol Cell - April 13, 2021 Category: Molecular Biology Authors: Melchior Chabannes V éronique Frémeaux-Bacchi Sophie Chauvet Source Type: research

Complement Detection in Mouse Kidneys by Immunofluorescence
Methods Mol Biol. 2021;2227:179-189. doi: 10.1007/978-1-0716-1016-9_17.ABSTRACTImmunofluorescence staining of tissues has become a reliable and informative technique used in a diverse set of applications, ranging from simple detection of an antigen of interest in a specific location to the semiquantitative analysis of spatial relationships between multiple antigens and/or cell types. During complement activation, circulating complement proteins are covalently fixed to target tissues, providing a durable marker of complement activation in the tissue, and many of these proteins can be readily detected by immunofluorescence m...
Source: Mol Biol Cell - April 13, 2021 Category: Molecular Biology Authors: Jennifer Laskowski Joshua M Thurman Source Type: research

Complement Detection in Human Tumors by Immunohistochemistry and Immunofluorescence
Methods Mol Biol. 2021;2227:191-203. doi: 10.1007/978-1-0716-1016-9_18.ABSTRACTTumors contain a complement rich microenvironment in which all cell types (e.g., tumor cells and stromal cells) are able to produce different proteins. We developed immunohistochemistry (IHC) assays allowing to identify on paraffin embedded tumor sections, not only the complement producing cells but also the complement activation fragments which result from activation of complement cascade within the tumor. The local production of complement can be detected by cytoplasmic staining, whereas the activation fragments are localized at the surface of...
Source: Mol Biol Cell - April 13, 2021 Category: Molecular Biology Authors: Marie V Daugan Margot Revel Laetitia Lacroix Catherine Saut ès-Fridman Wolf H Fridman Lubka T Roumenina Source Type: research

Single-Stranded DNA-Binding Proteins in the Archaea
Methods Mol Biol. 2021;2281:23-47. doi: 10.1007/978-1-0716-1290-3_2.ABSTRACTSingle-stranded (ss) DNA-binding proteins are found in all three domains of life where they play vital roles in nearly all aspects of DNA metabolism by binding to and stabilizing exposed ssDNA and acting as platforms onto which DNA-processing activities can assemble. The ssDNA-binding factors SSB and RPA are extremely well conserved across bacteria and eukaryotes, respectively, and comprise one or more OB-fold ssDNA-binding domains. In the third domain of life, the archaea, multiple types of ssDNA-binding protein are found with a variety of domain ...
Source: Mol Biol Cell - April 13, 2021 Category: Molecular Biology Authors: Najwa Taib Simonetta Gribaldo Stuart A MacNeill Source Type: research

In Vivo Binding of Single-Stranded DNA-Binding Protein to Stalled Replication Fork Helicases
Methods Mol Biol. 2021;2281:81-91. doi: 10.1007/978-1-0716-1290-3_5.ABSTRACTUnderstanding protein-protein interactions is key to unraveling protein function in vivo. Here we describe a dual/triple-plasmid system that enables co-expression of two, or three, recombinant proteins harboring different affinity tags in the same Escherichia coli cell. This novel protein expression system provides a platform to understand protein-protein interactions and enables researchers to study protein complex formation and in vivo localization.PMID:33847953 | DOI:10.1007/978-1-0716-1290-3_5 (Source: Mol Biol Cell)
Source: Mol Biol Cell - April 13, 2021 Category: Molecular Biology Authors: Cong Yu Piero R Bianco Source Type: research

Following Trypanosoma cruzi RPA-DNA Interaction Using Fluorescent In Situ Hybridization Coupled with Immunofluorescence (FISH/IF)
Methods Mol Biol. 2021;2281:209-215. doi: 10.1007/978-1-0716-1290-3_12.ABSTRACTFluorescent in situ hybridization coupled with immunofluorescence (FISH/IF) is an assay that has been widely used to study DNA-protein interactions. The technique is based on the use of a fluorescent nucleic acid probe and fluorescent antibodies to reveal the localization of a DNA sequence and a specific protein in the cell. The interaction can be inferred by the quantification of the co-localization between the protein and the DNA. Here, we describe a detailed FISH/IF methodology that our group used to study RPA-telomere interaction in the path...
Source: Mol Biol Cell - April 13, 2021 Category: Molecular Biology Authors: Raphael S Pavani Maria Carolina Elias Source Type: research

Quantifying the Affinity of Trypanosoma cruzi RPA-1 to the Single-Stranded DNA Overhang of the Telomere Using Surface Plasmon Resonance
Methods Mol Biol. 2021;2281:217-228. doi: 10.1007/978-1-0716-1290-3_13.ABSTRACTSurface plasmon resonance (SPR) biosensors provide real-time binding affinity measurements between a pair of biomolecules, characterizing its interaction dynamics. An example of Trypanosoma cruzi's RPA-1 and a single-stranded DNA telomere sequence is presented with detailed guidelines and fundamentals for SPR technology.PMID:33847961 | DOI:10.1007/978-1-0716-1290-3_13 (Source: Mol Biol Cell)
Source: Mol Biol Cell - April 13, 2021 Category: Molecular Biology Authors: Marcela de Oliveira Vitarelli Maria Carolina Elias Source Type: research

Optical Tweezers to Investigate the Structure and Energetics of Single-Stranded DNA-Binding Protein-DNA Complexes
Methods Mol Biol. 2021;2281:273-288. doi: 10.1007/978-1-0716-1290-3_17.ABSTRACTOptical tweezers enable the isolation and mechanical manipulation of individual nucleoprotein complexes. Here, we describe how to use this technique to interrogate the mechanical properties of individual protein-DNA complexes and extract information about their overall structural organization.PMID:33847965 | DOI:10.1007/978-1-0716-1290-3_17 (Source: Mol Biol Cell)
Source: Mol Biol Cell - April 13, 2021 Category: Molecular Biology Authors: Jos é A Morin Fernando Cerr ón Francisco J Cao-Garc ía Borja Ibarra Source Type: research

Targeted Integration Through Transformation of Hydroxyurea-Arrested Cells
Methods Mol Biol. 2021;2307:139-145. doi: 10.1007/978-1-0716-1414-3_9.ABSTRACTHomologous recombination is required to specifically target DNA to a desired genomic locus. Non-homologous end joining is the predominant form of recombination in Yarrowia lipolytica. Transformation of this organism with linear DNA therefore results in random integration of the introduced DNA into the genome. In this protocol, hydroxyurea-mediated cell cycle arrest is applied to significantly increase the rate of homologous recombination during transformation and enhance targeted integration.PMID:33847987 | DOI:10.1007/978-1-0716-1414-3_9 (Source: Mol Biol Cell)
Source: Mol Biol Cell - April 13, 2021 Category: Molecular Biology Authors: Vasiliki Tsakraklides Source Type: research

Engineering Yarrowia lipolytica for Production of Fatty Alcohols with YaliBrick Vectors
Methods Mol Biol. 2021;2307:159-173. doi: 10.1007/978-1-0716-1414-3_11.ABSTRACTBiosynthesis of fatty alcohol holds great promise as substitute to replace petroleum-derived fatty alcohols to mitigate environmental concerns and reduce earth's carbon footprint. In this protocol, we detail the procedures of how to use the YaliBrick gene assembly platform to achieve modular assembly of fatty alcohol pathway in Y. lipolytica. To limit fatty alcohol oxidation, we will also describe the hydroxyurea-based protocols for the efficient disruption of POX1 gene, encoding the fatty acyl coenzyme A in Y. lipolytica, with the homologous ar...
Source: Mol Biol Cell - April 13, 2021 Category: Molecular Biology Authors: Wanqi Sun Zhiliang Yang Peng Xu Source Type: research

Droplet-Based Microfluidic High-Throughput Screening of Enzyme Mutant Libraries Secreted by Yarrowia lipolytica
Methods Mol Biol. 2021;2307:205-219. doi: 10.1007/978-1-0716-1414-3_14.ABSTRACTYarrowia lipolytica has emerged as an attractive solution for screening enzyme activities thanks to the numerous tools available for heterologous protein production and its strong secretory ability. Nowadays, activity screening for improved enzymes mostly relies on the evaluation of independent clones in microtiter plates. However, even with highly robotized screening facilities, the relatively low throughput and high cost of the technology do not enable the screening of large diversities, which significantly reduce the probability of isolating ...
Source: Mol Biol Cell - April 13, 2021 Category: Molecular Biology Authors: Thomas Beneyton Tristan Rossignol Source Type: research

Western Blotting of Membrane-Bound Proteins in Yarrowia lipolytica
Methods Mol Biol. 2021;2307:233-247. doi: 10.1007/978-1-0716-1414-3_16.ABSTRACTWith the discovery of Western blotting as first described by Towbin et al. in 1979, the transfer and visualization of electrophoretically separated proteins on membranes has become the de facto method for the qualitative and quantitative detection of proteins of interest. In this method, proteins are resolved by electrophoresis on a polyacrylamide gel, followed by a transfer of the separated proteins onto a nitrocellulose or polyvinyl difluoride (PVDF) membrane. Once immobilized on these membranes, the protein of interest can be detected and vis...
Source: Mol Biol Cell - April 13, 2021 Category: Molecular Biology Authors: Adithya Ramesh Ian Wheeldon Source Type: research

Science: priceless, but costly
Mol Biol Cell. 2021 Apr 15;32(8):635-637. doi: 10.1091/mbc.E20-12-0768.ABSTRACTWorking as a researcher is very satisfying. However, it comes with a price. This is a story about growing up as a scientist in the field of molecular biology. Starting as a young, rather naive researcher, I learned, step by step, not only the facts about my favorite RNA molecules but also the demands and downsides of academia. Going through my recent "scientific awakening," I fully acknowledged the rules of the game: to write, to publish, to patent, to apply for grants and awards, and finally, to engage in all forms of coscientific end...
Source: Mol Biol Cell - April 13, 2021 Category: Molecular Biology Authors: Marta Gabryelska Source Type: research

TREM2 Dictates Antibacterial Defense and Viability of Bone Marrow-derived Macrophages During Bacterial Infection
This study was designed to investigate the organelle source and biological activity of ROS in the context of TREM2-mediated immune defense during E.coli infection. Bone marrow-derived macrophages (BMDMs) were transfected with TREM2-overexpressing adenoviruses or control viruses, and challenged with E.coli. The BMDMs were administered to mouse models with local E.coli infection. Additionally, monocytic TREM2 expression, NOX2 levels and pyroptosis were detected in patients with bacterial sepsis. General ROS production was found to be comparable between TREM2-overexpressing and control BMDMs upon E.coli challenge. The deficie...
Source: Mol Biol Cell - April 13, 2021 Category: Molecular Biology Authors: ShiYue Yang Yang Yang FeiFei Wang QinYu Luo Yan Zhang Fei Zheng Qiang Shu QiXing Chen XiangMing Fang Source Type: research

Influenza Antiviral Subversion: Now the Host is in on the Act
Am J Respir Cell Mol Biol. 2021 Apr 13. doi: 10.1165/rcmb.2021-0094ED. Online ahead of print.NO ABSTRACTPMID:33848230 | DOI:10.1165/rcmb.2021-0094ED (Source: Mol Biol Cell)
Source: Mol Biol Cell - April 13, 2021 Category: Molecular Biology Authors: Erin N Yepsen Kevin S Harrod Source Type: research

Inflammasome Genetic Variants, Macrophage Function, and Clinical Outcomes in Cystic Fibrosis
Am J Respir Cell Mol Biol. 2021 Apr 13. doi: 10.1165/rcmb.2020-0257OC. Online ahead of print.ABSTRACTCystic fibrosis (CF) is characterized by chronic airway infection, inflammation, and tissue damage that lead to progressive respiratory failure. NLRP3 and NLRC4 are cytoplasmic pattern recognition receptors that activate the inflammasome, initiating a caspase-1 mediated response. We hypothesized that gain of function inflammasome responses are associated with worse outcomes in children with CF. We genotyped nonsynonymous variants in NLRP3 and the NLRC4 pathway from individuals in the Early Pseudomonas Infection Control (EPI...
Source: Mol Biol Cell - April 13, 2021 Category: Molecular Biology Authors: Andrew D Graustein William R Berrington Kati J Buckingham Felicia K Nguyen Lara L Joudeh Margaret Rosenfeld Michael J Bamshad Ronald L Gibson Thomas R Hawn Mary J Emond Source Type: research

Airway Basal Cells in COPD: A Continuum or a Dead End?
Am J Respir Cell Mol Biol. 2021 Apr 13. doi: 10.1165/rcmb.2021-0150ED. Online ahead of print.NO ABSTRACTPMID:33848453 | DOI:10.1165/rcmb.2021-0150ED (Source: Mol Biol Cell)
Source: Mol Biol Cell - April 13, 2021 Category: Molecular Biology Authors: Renat Shaykhiev Source Type: research

Gut-specific arylphorin mediates midgut regenerative response against cry-induced damage in Achaea janata
Comp Biochem Physiol B Biochem Mol Biol. 2021 Apr 10:110600. doi: 10.1016/j.cbpb.2021.110600. Online ahead of print.ABSTRACTDevelopment of insect resistance to biopesticides is a current and pertinent global issue. Earlier, it was established that lepidopteran larvae can recover from Bt intoxication via a midgut regenerative response and subsequently generate resistance. Molecular aspects of restoration of the midgut integrity following toxin exposure are emerging recently. In the present study, we bring out the pivotal role of gut arylphorin in mediating the midgut regenerative response following sublethal Bt exposure in ...
Source: Mol Biol Cell - April 13, 2021 Category: Molecular Biology Authors: Narender K Dhania Vinod K Chauhan Dasari Abhilash Vivek Thakur R K Chaitanya Shourya Dutta-Gupta Aparna Dutta-Gupta Source Type: research

Structures of chaperone-associated assembly intermediates reveal coordinated mechanisms of proteasome biogenesis
Nat Struct Mol Biol. 2021 Apr 12. doi: 10.1038/s41594-021-00583-9. Online ahead of print.ABSTRACTThe proteasome mediates most selective protein degradation. Proteolysis occurs within the 20S core particle (CP), a barrel-shaped chamber with an α7β7β7α7 configuration. CP biogenesis proceeds through an ordered multistep pathway requiring five chaperones, Pba1-4 and Ump1. Using Saccharomyces cerevisiae, we report high-resolution structures of CP assembly intermediates by cryogenic-electron microscopy. The first structure corresponds to the 13S particle, which consists of a complete α-ring, partial &...
Source: Mol Biol Cell - April 13, 2021 Category: Molecular Biology Authors: Helena M Schnell Richard M Walsh Shaun Rawson Mandeep Kaur Meera K Bhanu Geng Tian Miguel A Prado Angel Guerra-Moreno Joao A Paulo Steven P Gygi Jeroen Roelofs Daniel Finley John Hanna Source Type: research

Correction to: Current status and impending progress for cassava structural genomics
Plant Mol Biol. 2021 Apr 13. doi: 10.1007/s11103-021-01139-7. Online ahead of print.NO ABSTRACTPMID:33847871 | DOI:10.1007/s11103-021-01139-7 (Source: Mol Biol Cell)
Source: Mol Biol Cell - April 13, 2021 Category: Molecular Biology Authors: Jessica B Lyons Jessen V Bredeson Ben N Mansfeld Guillaume Jean Bauchet Jeffrey Berry Adam Boyher Lukas A Mueller Daniel S Rokhsar Rebecca S Bart Source Type: research

Sheep Erythrocyte Preparation for Hemolytic Tests Exploring Complement Functional Activities
Methods Mol Biol. 2021;2227:61-67. doi: 10.1007/978-1-0716-1016-9_6.ABSTRACTSheep erythrocytes (SE) are commonly used in complement functional tests. Non sensitized SE are useful to study the FH activity of cell protection. Indeed, as the cell surface of sheep erythrocytes is rich in sialic acids, Factor H (FH) is able to bind on it and therefore they represent a model of nonactivating surface. Because of their high capacity of complement regulation SE need to be modified to explore other functionality of the complement pathways, like the Complement hemolytic 50 (CH50) or the AP C3 convertase decay assays. For these tests,...
Source: Mol Biol Cell - April 13, 2021 Category: Molecular Biology Authors: Melchior Chabannes Pauline Bordereau Paula Vieira Martins Marie-Agn ès Dragon-Durey Source Type: research

Hemolytic Tests Exploring Factor H Functional Activities
Methods Mol Biol. 2021;2227:69-81. doi: 10.1007/978-1-0716-1016-9_7.ABSTRACTImpairment of the complement regulatory protein Factor H (FH) is implicated in the physiopathological mechanisms of different diseases like atypical hemolytic and uremic syndrome and C3 glomerulopathies. It may be due to genetic abnormalities or acquired with the development of autoantibodies. FH has several ligands; therefore, the exploration of its functions requires to perform different tests. Among them, two hemolytic tests are very useful because they give specific and complementary information about FH functions. The first one is dedicated to...
Source: Mol Biol Cell - April 13, 2021 Category: Molecular Biology Authors: Melchior Chabannes Shambhuprasad K Togarsimalemath Marie-Agn ès Dragon-Durey Source Type: research

Complement C3 Deposition on Endothelial Cells Revealed by Flow Cytometry
Methods Mol Biol. 2021;2227:97-105. doi: 10.1007/978-1-0716-1016-9_9.ABSTRACTThe three pathways of the complement system converge toward the cleavage of the central complement component C3 into its activated fragments, with C3b being able to bind covalently to the activating surface. The endothelial cells are among the major targets for complement attack in pathological conditions, as the atypical hemolytic uremic syndrome. Therefore, study of complement C3 deposition on endothelial cells by flow cytometry is a sensitive test to measure complement activation. This test can be used as a research or clinical tool to test com...
Source: Mol Biol Cell - April 13, 2021 Category: Molecular Biology Authors: Idris Boudhabhay Anne Grunenwald Lubka T Roumenina Source Type: research

Detection of C3 Nephritic Factor by Hemolytic Assay
Methods Mol Biol. 2021;2227:147-158. doi: 10.1007/978-1-0716-1016-9_15.ABSTRACTC3 nephritic Factor (C3NeF) is autoantibody that binds neoepitopes of the C3 convertase C3bBb, resulting in a stabilization of the enzyme. First functional characterizations of C3NeF were performed by hemolytic assays using preactivated sheep erythrocytes (bearing C3b). Sheep erythrocytes are beforehand sensitized with an anti-sheep red blood cell stroma antibody produced in rabbit (hemolysin). Sensitized sheep erythrocytes will initiate cascade complement activation via the classic pathway, followed by alternative pathway amplification loop, re...
Source: Mol Biol Cell - April 13, 2021 Category: Molecular Biology Authors: Melchior Chabannes V éronique Frémeaux-Bacchi Sophie Chauvet Source Type: research

Complement Detection in Mouse Kidneys by Immunofluorescence
Methods Mol Biol. 2021;2227:179-189. doi: 10.1007/978-1-0716-1016-9_17.ABSTRACTImmunofluorescence staining of tissues has become a reliable and informative technique used in a diverse set of applications, ranging from simple detection of an antigen of interest in a specific location to the semiquantitative analysis of spatial relationships between multiple antigens and/or cell types. During complement activation, circulating complement proteins are covalently fixed to target tissues, providing a durable marker of complement activation in the tissue, and many of these proteins can be readily detected by immunofluorescence m...
Source: Mol Biol Cell - April 13, 2021 Category: Molecular Biology Authors: Jennifer Laskowski Joshua M Thurman Source Type: research

Complement Detection in Human Tumors by Immunohistochemistry and Immunofluorescence
Methods Mol Biol. 2021;2227:191-203. doi: 10.1007/978-1-0716-1016-9_18.ABSTRACTTumors contain a complement rich microenvironment in which all cell types (e.g., tumor cells and stromal cells) are able to produce different proteins. We developed immunohistochemistry (IHC) assays allowing to identify on paraffin embedded tumor sections, not only the complement producing cells but also the complement activation fragments which result from activation of complement cascade within the tumor. The local production of complement can be detected by cytoplasmic staining, whereas the activation fragments are localized at the surface of...
Source: Mol Biol Cell - April 13, 2021 Category: Molecular Biology Authors: Marie V Daugan Margot Revel Laetitia Lacroix Catherine Saut ès-Fridman Wolf H Fridman Lubka T Roumenina Source Type: research

Single-Stranded DNA-Binding Proteins in the Archaea
Methods Mol Biol. 2021;2281:23-47. doi: 10.1007/978-1-0716-1290-3_2.ABSTRACTSingle-stranded (ss) DNA-binding proteins are found in all three domains of life where they play vital roles in nearly all aspects of DNA metabolism by binding to and stabilizing exposed ssDNA and acting as platforms onto which DNA-processing activities can assemble. The ssDNA-binding factors SSB and RPA are extremely well conserved across bacteria and eukaryotes, respectively, and comprise one or more OB-fold ssDNA-binding domains. In the third domain of life, the archaea, multiple types of ssDNA-binding protein are found with a variety of domain ...
Source: Mol Biol Cell - April 13, 2021 Category: Molecular Biology Authors: Najwa Taib Simonetta Gribaldo Stuart A MacNeill Source Type: research

In Vivo Binding of Single-Stranded DNA-Binding Protein to Stalled Replication Fork Helicases
Methods Mol Biol. 2021;2281:81-91. doi: 10.1007/978-1-0716-1290-3_5.ABSTRACTUnderstanding protein-protein interactions is key to unraveling protein function in vivo. Here we describe a dual/triple-plasmid system that enables co-expression of two, or three, recombinant proteins harboring different affinity tags in the same Escherichia coli cell. This novel protein expression system provides a platform to understand protein-protein interactions and enables researchers to study protein complex formation and in vivo localization.PMID:33847953 | DOI:10.1007/978-1-0716-1290-3_5 (Source: Mol Biol Cell)
Source: Mol Biol Cell - April 13, 2021 Category: Molecular Biology Authors: Cong Yu Piero R Bianco Source Type: research

Following Trypanosoma cruzi RPA-DNA Interaction Using Fluorescent In Situ Hybridization Coupled with Immunofluorescence (FISH/IF)
Methods Mol Biol. 2021;2281:209-215. doi: 10.1007/978-1-0716-1290-3_12.ABSTRACTFluorescent in situ hybridization coupled with immunofluorescence (FISH/IF) is an assay that has been widely used to study DNA-protein interactions. The technique is based on the use of a fluorescent nucleic acid probe and fluorescent antibodies to reveal the localization of a DNA sequence and a specific protein in the cell. The interaction can be inferred by the quantification of the co-localization between the protein and the DNA. Here, we describe a detailed FISH/IF methodology that our group used to study RPA-telomere interaction in the path...
Source: Mol Biol Cell - April 13, 2021 Category: Molecular Biology Authors: Raphael S Pavani Maria Carolina Elias Source Type: research

Quantifying the Affinity of Trypanosoma cruzi RPA-1 to the Single-Stranded DNA Overhang of the Telomere Using Surface Plasmon Resonance
Methods Mol Biol. 2021;2281:217-228. doi: 10.1007/978-1-0716-1290-3_13.ABSTRACTSurface plasmon resonance (SPR) biosensors provide real-time binding affinity measurements between a pair of biomolecules, characterizing its interaction dynamics. An example of Trypanosoma cruzi's RPA-1 and a single-stranded DNA telomere sequence is presented with detailed guidelines and fundamentals for SPR technology.PMID:33847961 | DOI:10.1007/978-1-0716-1290-3_13 (Source: Mol Biol Cell)
Source: Mol Biol Cell - April 13, 2021 Category: Molecular Biology Authors: Marcela de Oliveira Vitarelli Maria Carolina Elias Source Type: research