bluuebunny/crossref_metadata_2025_split huggingface dataset:
Extracellular electrophysiology on clonal human β-cell spheroids
Authors: Puginier Emilie , Fischer Karen Leal , Gaitan Julien , Lallouet Marie , Scotti Pier-Arnaldo , Raoux Matthieu , Lang Jochen 
Published: April 2024

AbstractPancreatic islets are important in nutrient homeostasis and improved cellular models of clonal origin may very useful especially in view of relatively scarce primary material. Close 3D contact and coupling between β-cells are a hallmark of physiological function improving signal/noise ratios. Extracellular electrophysiology using micro-electrode arrays (MEA) is technically far more accessible than single cell patch clamp, enables dynamic monitoring of electrical activity in 3D organoids and recorded multicellular slow potentials (SP) provide unbiased insight in cell-cell coupling. We have therefore asked whether 3D spheroids enhance clonal β-cell function such as electrical activity and hormone secretion using human EndoC-βH1, EndoC-βH5 and rodent INS-1 cells. EndoC-βH1 spheroids exhibited increased signals in terms of SP frequency and especially amplitude as compared to monolayers and even single cell action potentials (AP) were quantifiable. Enhanced electrical signature in spheroids was accompanied by an increase in the glucose stimulated insulin secretion index. EndoC-βH5 monolayers and spheroids gave electrophysiological profiles similar to EndoC-βH1, except for a higher electrical activity at 3 mM glucose, and exhibited moreover a biphasic profile. Again, physiological concentrations of GLP-1 increased AP frequency. Spheroids also exhibited a higher secretion index. INS-1 cells did not form stable spheroids, but overexpression of connexin 36, required for cell-cell coupling, increased glucose responsiveness, dampened basal activity and consequently augmented the stimulation index. In conclusion, spheroid formation enhances physiological function of the human clonal β-cell lines and these models may provide surrogates for primary islets in extracellular electrophysiology.
Evaluating the efficacy of protein quantification methods on membrane proteins
Authors: Löptien Jana , Vesting Sidney , Dobler Susanne , Mohammadi Shabnam 
Published: April 2024

AbstractProtein quantification is an important tool for a wide range of biological applications. The most common broadscale methods include the Lowry, bicinchoninic acid (BCA), and Coomassie Bradford assays. Despite their wide applicability, the mechanisms of action imply that these methods may not be ideal for large transmembrane proteins due to the proteins’ integration in the plasma membrane. Here, we investigate this problem by assessing the efficacy and applicability of these three common protein quantification methods on a candidate transmembrane protein – the Na,K-ATPase (NKA). We compared these methods to an ELISA, which we newly developed and describe here for the quantification of NKA. The use of a relative standard curve allows this ELISA to be easily adapted to other proteins and across the animal kingdom. Our results revealed that the three conventional methods significantly underestimate the concentration of NKA compared to the ELISA. Further, by applying the protein concentrations determined by the different methods to in vitro assays, we found that variation in the resulting data was consistently low when the assay reactions were prepared based on concentrations determined from the ELISA. Thus, when target protein concentrations vary across samples, the conventional quantification methods cannot produce reliable results in downstream applications. In contrast, the ELISA we describe here consistently provides robust results.
Antibody-dependent Intracellular neutralisation by TRIM21 is potentiated by HOIP and linear ubiquitin chains
Authors: Green Christopher , Winklhofer Konstanze F. , Fletcher Adam J. , James Leo C. , Skidmore John , Duce James , McEwan William A. 
Published: April 2024

AbstractAntibody-dependent intracellular neutralisation (ADIN) promotes the rapid proteasomal degradation of viruses and other large substrates in the cytosol. It is dependent on detection of intracellular virus-bound antibodies by the Fc receptor and E3 ligase TRIM21, followed by disassembly of the virus by the unfoldase VCP/p97. It is not known how VCP is recruited to TRIM21. We performed a limited siRNA knock-down screen of known VCP adaptors to determine their involvement in ADIN. Knock-down of HOIP, the only ubiquitin E3 ligase capable of generating linear ubiquitin chains, resulted in impaired virus neutralisation. HOIPIN-8, a HOIP inhibitor, showed concentration-dependent reduction in virus neutralisation. We found that the activity of HOIP in ADIN is dependent on its ubiquitin binding domains and its PUB domain which recruits VCP. Knock-down of OTULIN, the only deubiquitinating enzyme that exclusively cleaves linear ubiquitin chains, potentiated neutralisation of the virus. Our results expand the role of HOIP and linear ubiquitin chains in proteostasis.
Regulation of fatty acid delivery to metastases by tumor endothelium
Authors: Edwards Deanna N. , Wang Shan , Song Wenqiang , Kim Laura C. , Ngwa Verra M. , Hwang Yoonha , Ess Kevin C. , Boothby Mark R. , Chen Jin 
Published: April 2024

AbstractTumor metastasis, the main cause of death in cancer patients, requires outgrowth of tumor cells after their dissemination and residence in microscopic niches. Nutrient sufficiency is a determinant of such outgrowth1. Fatty acids (FA) can be metabolized by cancer cells for their energetic and anabolic needs but impair the cytotoxicity of T cells in the tumor microenvironment (TME)2, 3, thereby supporting metastatic progression. However, despite the important role of FA in metastatic outgrowth, the regulation of intratumoral FA is poorly understood. In this report, we show that tumor endothelium actively promotes tumor growth and restricts anti-tumor cytolysis by transferring FA into developing metastatic tumors. This process uses transendothelial fatty acid transport via endosome cargo trafficking in a mechanism that requires mTORC1 activity. Thus, tumor burden was significantly reduced upon endothelial-specific targeted deletion of Raptor, a unique component of the mTORC1 complex (RptorECKO). In vivo trafficking of a fluorescent palmitic acid analog to tumor cells and T cells was reduced in RptorECKOlung metastatic tumors, which correlated with improved markers of T cell cytotoxicity. Combination of anti-PD1 with RAD001/everolimus, at a low dose that selectively inhibits mTORC1 in endothelial cells4, impaired FA uptake in T cells and reduced metastatic disease, corresponding to improved anti-tumor immunity. These findings describe a novel mechanism of transendothelial fatty acid transfer into the TME during metastatic outgrowth and highlight a target for future development of therapeutic strategies.
Enhanced delivery of protein therapeutics with a diphtheria toxin-like platform that evades pre-existing neutralizing immunity
Authors: Gill Shivneet K. , Sugiman-Marangos Seiji N. , Beilhartz Greg L. , Mei Elizabeth , Taipale Mikko , Melnyk Roman A. 
Published: April 2024

ABSTRACTTargeted intracellular delivery of therapeutic peptides and proteins remains an important but unresolved goal in biotechnology. A promising approach is to engineer bacterial exotoxins that deliver their cytotoxic enzymes into cells and can be engineered to target cancer cells as is the case with immunotoxins. The well-studied diphtheria toxin translocation domain is ideally suited as a delivery platform as it has been shown to be capable of delivering a wide range of macromolecular cargo. Widespread deployment of DT-based therapeutics in humans, however, is complicated by the prevalence of pre-existing anti-DT antibodies from childhood vaccinations that reduce the exposure, efficacy and safety of this important class of protein drugs. Thus, there is a great need for delivery platforms with no pre-existing immunity in humans. Here, we describe the discovery and characterization of a distant diphtheria toxin homolog from the ancient reptile pathogenAustwickia chelonaethat we have named Chelona Toxin (CT). We show that CT is comparable to DT structure and function in all respects except that it is not recognized by pre-existing anti-DT antibodies present in human sera. Moreover, we demonstrate that the CT translocase is superior to the DT translocase at delivering therapeutic protein cargo into target cells. These findings highlight CT as a potentially class-enabling new chassis for developing safer and more efficacious immunotoxins and intracellular protein delivery platforms for cancer therapy.
Does bumblebee preference of continuous over interrupted strings in string-pulling tasks indicate means-end comprehension?
Authors: Wen Chao , Lu Yuyi , Solvi Cwyn , Dong Shunping , Wang Cai , Wen Xiujun , Xiao Haijun , Dong Shikui , Wen Junbao , Peng Fei , Chittka Lars 
Published: April 2024

AbstractBumblebees (Bombus terrestris) have been shown to engage in string-pulling behavior to access rewards. The objective of this study was to elucidate whether bumblebees display means-end comprehension in a string-pulling task. We presented bumblebees with two options: one where a string is connected to an artificial flower containing a reward and the other presenting an interrupted string. Bumblebees displayed a consistent preference for pulling connected strings over interrupted ones after training with a stepwise pulling technique. When exposed to novel string colors, bees continued to exhibit a bias towards pulling the connected string. This suggests that bumblebees engage in featural generalization of the visual display of the string connected to the flower in this task. If the view of the string connected to the flower was restricted during the training phase, the proportion of bumblebees choosing the connected strings significantly decreased. Similarly, when the bumblebees were confronted with coiled connected strings during the testing phase, they failed to identify and reject the interrupted strings. This finding underscores the significance of visual consistency in enabling the bumblebees to perform the task successfully. Our results suggest that bumblebees’ ability to distinguish between continuous strings and interrupted strings relies on a combination of image matching and associative learning, rather than means-end understanding. These insights contribute to a deeper understanding of the cognitive processes employed by bumblebees when tackling complex spatial tasks.
Cryo-EM Structure of AAV2 Rep68 bound to integration site AAVS1: Insights into the mechanism of DNA melting
Authors: Jaiswal R. , Santosh V. , Braud B , Washington A. , Escalante Carlos R. 
Published: April 2024

AbstractThe Rep68 protein from Adeno-Associated Virus (AAV) is a multifunctional SF3 helicase that performs most of the DNA transactions required for the viral life cycle. During AAV DNA replication, Rep68 assembles at the origin and catalyzes the DNA melting and nicking reactions during the hairpin rolling replication process to complete the second-strand synthesis of the AAV genome. Here, we report the Cryo-EM structures of Rep68 bound to double-stranded DNA (dsDNA) containing the sequence of the AAVS1 integration site in different nucleotide-bound states. In the apo state, Rep68 forms a heptameric complex around DNA, with three Origin Binding Domains (OBDs) bound to the Rep Binding Site (RBS) sequence and three other OBDs forming transient dimers with them. The AAA+domains form an open ring with no interactions between subunits and with DNA. We hypothesize the heptameric quaternary structure is necessary to load onto dsDNA. In the ATPγS-bound state, a subset of three subunits binds the nucleotide, undergoing a large conformational change, inducing the formation of intersubunit interactions interaction and interaction with three consecutive DNA phosphate groups. Moreover, the induced conformational change positions three phenylalanine residues to come in close contact with the DNA backbone, producing a distortion in the DNA. We propose that the phenylalanine residues can potentially act as a hydrophobic wedge in the DNA melting process.
Electric charge controls plasmodesma conductivity
Authors: Howell Alexander H. , Christensen Anneline H. , James Vincent , Vasina Viktoriya V. , Jensen Kaare H. , Foley James , Evans James E. , Stone Howard A. , Peters Winfried S. , Knoblauch Michael 
Published: April 2024

AbstractWhile plant cells are enclosed by rigid cell walls that counteract intracellular hydrostatic pressure1, their plasma membrane, cytosol, and endoplasmic reticulum (ER) remain connected through plasmodesmata, nanoscopic cell wall pores2. Plasmodesmal cell-to-cell transport occurs in the cytosolic sleeve between the plasma membrane and the ER membrane3–5, and is generally thought to be limited by the size of the moving particle alone6. Given that biological membranes carry negative electric surface charges7–9, this steric notion conflicts with physical theory of ion diffusion in nanometer-sized pores with charged walls10. Quantifying the movements of differently sized and charged fluorescent dyes inTradescantiastamen hairs, we found that anionic fluorophores of up to 1 kDa traversed plasmodesmata whereas much smaller cationic ones did not. While this agrees with theoretical expectations of different size exclusion limits for cations and anions, it questions current dogma concerning plasmodesma function and also structure, as it implies positively rather than negatively charged surfaces within plasmodesmal pores. Our findings call for re-evaluations of current models of symplasmic transport, especially of charged molecules like the phytohormone auxin (indole-acetic acid) and certain amino acids.
Genome-wide identification of MAPK gene family and comparative transcriptional profiling among different organ and stress response in two jute species
Authors: Ahmed Borhan , Tabassum Anika , Bashar Kazi Khayrul , Ullah MW , Aktar Nasima , Roni MS , Hasan Fakhrul , Alam Mobashwer 
Published: April 2024

AbstractMitogen activated protein kinase (MAPK) cascade is evolutionary conserved universal signal transduction module that plays central role in the growth and development of plants as well as in biotic and abiotic stress response. Although, MAPKs have been investigated in several model plants, no systematic analysis has been conducted in jute species (Corchorus olitoriusandC. capsularis) even though, their genome sequencing has been completed. In the present study we identified 11 and 12 putative MAPKs inC. olitoriusandC. capsularisusing their genomic database, respectively. Here we provide a comprehensive bioinformatics analysis of the MAPK family from bothCorchorusspecies including identification and nomenclature, chromosomal localization, sequence alignment, domain and Motif, gene structure, phylogenetic, functional analysis and investigation of expression analysis in response to abiotic stress and fiber cell development. The phylogenetic analysis of predicted MAPKs were clustered into four different clades and assigned with specific name based on their orthology based evolutionary relationship withArabidopsis. Structural analysis of the MAPK genes revealed that there was a large variation among the exon number in bothCorchorusspecies ranged from 2 to 11 but genes with the same clade had similar exon-intron structure. The sequence alignment analysis concede the presence of several conserved domain and motif including crucial signature phosphorylation motif TDY or TEY where first one is harbor in group D sequence and rest of the sequence contain TEY motif in their activation loop. Transcriptome analysis against salinity, drought along with fiber cell formation showed that MAPK4-1 genes in both jute genome highly expressed and may play a potential role in jute on adverse condition as well as jute fiber formation. These findings yielded new insights into the transcriptional control of MAPK gene expression, provide an improved understanding of abiotic stress responses and signaling transduction in jute, that lead to potential applications in the genetic improvement of jute cultivars.
Investigating the Structural Impact and Conformational Dynamics of a Sequence Variant (c.242G>A) inTMIEGene Provoking Usher Syndrome
Authors: Ishaq Saqib , Usmani Shabir Ahmad , Habib Obaid , Tahir Raheel , Aziz Abdul , Rahman Siddiq Ur , Huiying Liang 
Published: April 2024

AbstractUsher syndrome (USH) is a retinal autosomal recessive genetic disorder, characterized by congenital severe-to-profound sensorineural hearing loss, retinitis pigmentosa (RP), and rarely vestibular dysfunction. A transmembrane inner ear gene TMIE causing autosomal recessive usher syndrome hearing loss, which may open up interesting perspectives into the function of this protein in inner ear. This disease is linked with mutations in TMIE gene. In this study delineates the pathogenic association, miss-fold aggregation, and conformational paradigm of a missense variant (c.242G>A) resulting into (p.Arg81His) in TMIE gene segregating usher syndrome through a molecular dynamics simulations approach. The transmembrane inner ear expressed protein assumes a critical role as its helices actively engage in binding with specific target DNA base pairs. The alteration observed in the mutant protein, characterized by an outward repositioning of the proximal helical portion, which is attributed to the absence of preceding beta-hairpins in the C-terminal region. This structural modification results in the loss of hydrogen bonds, exposure of hydrophobic residues to the solvent, and a consequential transformation of helices into loops, ultimately leading to functional impairment in the TMIE protein. These notable modifications in the stability and conformation of the mutant protein were verified through essential dynamics analysis, revealing that a point mutation induces distinct overall motions and correlations between proteins, ultimately resulting in usher syndrome. The current study provides insilico evidences of Usher syndrome hearing loss disease as protein folding disorder. The energy calculation also revealed that there is a difference of −251.211Kj/mol which also indicates that the SNP has significantly decreased the stability of protein consequently folding into Usher syndrome. This study contributes molecular insights into the structural correlation between the TMIE protein and usher syndrome. The docking analysis highlight various interaction between wild and mutant structure emphasizing key residues involved in hydrogen and hydrophobic interaction.
APOBEC shapes tumor evolution and age at onset of lung cancer in smokers
Authors: Zhang Tongwu , Sang Jian , Hoang Phuc H. , Zhao Wei , Rosenbaum Jennifer , Johnson Kofi Ennu , Klimczak Leszek J. , McElderry John , Klein Alyssa , Wirth Christopher , Bergstrom Erik N. , Díaz-Gay Marcos , Vangara Raviteja , Colon-Matos Frank , Hutchinson Amy , Lawrence Scott M. , Cole Nathan , Zhu Bin , Przytycka Teresa M. , Shi Jianxin , Caporaso Neil E. , Homer Robert , Pesatori Angela C. , Consonni Dario , Imielinski Marcin , Chanock Stephen J. , Wedge David C. , Gordenin Dmitry A. , Alexandrov Ludmil B. , Harris Reuben S. , Landi Maria Teresa 
Published: April 2024

ABSTRACTAPOBEC enzymes are part of the innate immunity and are responsible for restricting viruses and retroelements by deaminating cytosine residues1,2. Most solid tumors harbor different levels of somatic mutations attributed to the off-target activities of APOBEC3A (A3A) and/or APOBEC3B (A3B)3–6. However, how APOBEC3A/B enzymes shape the tumor evolution in the presence of exogenous mutagenic processes is largely unknown. Here, by combining deep whole-genome sequencing with multi-omics profiling of 309 lung cancers from smokers with detailed tobacco smoking information, we identify two subtypes defined by low (LAS) and high (HAS) APOBEC mutagenesis. LAS are enriched for A3B-like mutagenesis andKRASmutations, whereas HAS for A3A-like mutagenesis andTP53mutations. UnlikeAPOBEC3A,APOBEC3Bexpression is strongly associated with an upregulation of the base excision repair pathway. Hypermutation by unrepaired A3A and tobacco smoking mutagenesis combined withTP53-induced genomic instability can trigger senescence7, apoptosis8, and cell regeneration9, as indicated by high expression of pulmonary healing signaling pathway, stemness markers and distal cell-of-origin in HAS. The expected association of tobacco smoking variables (e.g., time to first cigarette) with genomic/epigenomic changes are not observed in HAS, a plausible consequence of frequent cell senescence or apoptosis. HAS have more neoantigens, slower clonal expansion, and older age at onset compared to LAS, particularly in heavy smokers, consistent with high proportions of newly generated, unmutated cells and frequent immuno-editing. These findings show how heterogeneity in mutational burden across co-occurring mutational processes and cell types contributes to tumor development, with important clinical implications.
Functional septate junctions between cyst cells are required for survival of transit amplifying male germ cells expressing Bag of marbles
Authors: Berry Cameron W. , Fuller Margaret T. 
Published: April 2024

AbstractIn adult stem cell lineages, the cellular microenvironment plays essential roles to ensure the proper balance of self-renewal, differentiation and regulated elimination of differentiating cells. Although regulated death of progenitor cells undergoing proliferation or early differentiation is a feature of many tissues, mechanisms that initiate this pruning remain unexplored, particularly in the male germline, where up to 30% of the germline is eliminated before the meiotic divisions. We conducted a targeted screen to identify functional regulators required in somatic support cells for survival or differentiation at early steps in the male germ line stem cell lineage. Cell type-specific knockdown in cyst cells uncovered novel roles of genes in germline stem cell differentiation, including a previously unappreciated role of the Septate Junction (SJ) in preventing cell death of differentiating germline progenitors. Loss of the SJ in the somatic cyst cells resulted in elimination of transit-amplifying spermatogonia by the 8-cell stage. Germ cell death was spared in males mutant for the differentiation factorbamindicating that intact barriers surrounding transit amplifying progenitors are required to ensure germline survival once differentiation has initiated.