bluuebunny/crossref_metadata_2025 huggingface dataset:
Detailed metabolic phenotyping of four tissue specific Cas9 transgenic mouse lines
Authors: Bond Simon T. , Zhuang Aowen , Yang Christine , Gould Eleanor A.M. , Sikora Tim , Liu Yingying , Fu Ying , Watt Kevin I. , Tan Yanie , Kiriazis Helen , Lancaster Graeme I. , Gregorevic Paul , Henstridge Darren C. , McMullen Julie R. , Meikle Peter J. , Calkin Anna C. , Drew Brian G.
Published: April 2021

CRISPR/Cas9 technology has revolutionized gene editing and fast tracked our capacity to manipulate genes of interest for the benefit of both research and therapeutic applications. Whilst many advances have, and continue to be made in this area, perhaps the most utilized technology to date has been the generation of knockout cells, tissues and animals by taking advantage of Cas9 function to promote indels in precise locations in the genome. Whilst the advantages of this technology are many fold, some questions still remain regarding the effects that long term expression of foreign proteins such as Cas9, have on mammalian cell function. Several studies have proposed that chronic overexpression of Cas9, with or without its accompanying guide RNAs, may have deleterious effects on cell function and health. This is of particular concern when applying this technology in vivo, where chronic expression of Cas9 in tissues of interest may promote disease-like phenotypes and thus confound the investigation of the effects of the gene of interest. Although these concerns remain valid, no study to our knowledge has yet to demonstrate this directly. Thus, in this study we used the lox-stop-lox (LSL) spCas9 ROSA26 transgenic (Tg) mouse line to generate four tissue-specific Cas9-Tg models with expression in the heart, liver, skeletal muscle and adipose tissue. We performed comprehensive phenotyping of these mice up to 20-weeks of age and subsequently performed molecular analysis of their organs. We demonstrated that Cas9 expression in these tissues had no detrimental effect on whole body health of the animals, nor did it induce any tissue-specific effects on energy metabolism, liver health, inflammation, fibrosis, heart function or muscle mass. Thus, our data suggests that these models are suitable for studying the tissue specific effects of gene deletion using the LSL-Cas9-Tg model, and that phenotypes observed utilizing these models can be confidently interpreted as being gene specific, and not confounded by the chronic overexpression of Cas9.
The nucleoporin Nup50 activates the Ran guanyl-nucleotide exchange factor RCC1 to promote mitotic NPC assembly
Authors: Holzer Guillaume , de Magistris Paola , Gramminger Cathrin , Sachdev Ruchika , Magalska Adriana , Schooley Allana , Anja Scheufen , Lennartz Birgitt , Tatarek-Nossol Marianna , Lue Hongqi , Linder Monika I. , Kutay Ulrike , Preisinger Christian , Moreno-Andres Daniel , Antonin Wolfram
Published: April 2021

During mitotic exit, thousands of nuclear pore complexes (NPCs) assemble concomitant with the nuclear envelope to build a transport-competent nucleus. We show here that Nup50 plays a crucial role in NPC assembly that is independent of its well-established function in nuclear transport. RNAi-mediated downregulation in cells or immunodepletion of the protein in Xenopus egg extracts interferes with NPC assembly. We define a conserved central region of 46 residues in Nup50 that is crucial for Nup153 and MEL28/ELYS binding, and NPC interaction. Surprisingly, neither NPC interaction nor binding of Nup50 to importin α, β, the GTPase Ran or chromatin is crucial for its function in the assembly process. Instead, we discovered that an N-terminal fragment of Nup50 can stimulate the Ran guanine exchange factor RCC1 and NPC assembly, indicating that Nup50 acts via the Ran system in mitotic NPC reformation. In support of this conclusion, Nup50 mutants defective in RCC1 binding and stimulation cannot replace the wild type protein in in vitro NPC assembly assays.
The Dendritic Ergic: Microtubule and Actin Cytoskeletons Mediate Stop-and-Go Movement of Mobile Carriers Between Stable Structures
Authors: de los Ángeles Juricic Urzúa María , Rojas Javiera Gallardo , Correa Andrés Couve , Cerda Mauricio , Gründler Steffen Härtel , González-Silva Carolina
Published: April 2021

The ER-to-Golgi intermediate compartment (ERGIC) is a membranous organelle that mediates protein transport between the endoplasmic reticulum (ER) and Golgi apparatus. In neurons, clusters of these vesiculotubular structures are situated in throughout the cell in proximity to the ER, passing cargo to the cis-Golgi cisternae located mainly in the perinuclear region. Although ERGIC markers have been identified in neurons, the distribution and dynamics of neuronal ERGIC structures have not been characterized.

Here, we argue that long-distance ERGIC transport occurs via an intermittent mechanism in neurons, with mobile elements moving between stationary structures. Using immunofluorescence microscopy, we detected discrete, irregular ERGIC structures in neural soma and dendrites. Slow live-cell imaging (2 frames/minute; 15 minutes) indicated that 8% of dendritic ERGIC structures were stable, remaining in place over long periods. On the other hand, fast live-cell imaging (2 frames/second; 180 seconds) captured mobile ERGIC structures advancing very short distances along dendrites. Importantly, these distances were consistent with the lengths between the stationary ERGIC structures. Kymography revealed ERGIC elements that moved intermittently, emerging from and fusing with stationary ERGIC structures. Surprisingly, this movement was apparently dependent not only on the integrity of the microtubule cytoskeleton, as has been previously reported, but on the actin cytoskeleton as well.

Our results indicate that the dendritic ERGIC has a dual nature, with both stationary and mobile structures. The neural ERGIC network transports proteins via a stop-and-go movement that is mediated by the microtubule and actin cytoskeletons.
A Chromosome-level Assembly of a Wild Castor Genome Provides New Insights into the Adaptive Evolution in a Tropical Desert
Authors: Lu Jianjun , Pan Cheng , Fan Wei , Liu Wanfei , Zhao Huayan , Li Donghai , Wang Sen , Hu Lianlian , He Bing , Qian Kun , Qin Rui , Ruan Jue , Lin Qiang , Lü Shiyou , Cui Peng
Published: April 2021

Wild castor grows in the high-altitude tropical desert of the African Plateau, a region known for high ultraviolet radiations, strong lights and extremely dry conditions. To investigate the potential genetic basis of adaptation to both highland and tropical deserts, we generated a chromosome-level genome sequence of the wild castor accession WT05, with genome size of 316 Mb and scaffold and contig N50 sizes of 31.93 Mb and 8.96 Mb, respectively. Compared with cultivated castor and other Euphorbiaceae species, the wild castor exhibits positive selection and gene family expansion for genes involved in DNA repair, photosynthesis and abiotic stress responses. Genetic variations associated with positive selection were identified in several key genes, such asLIG1, DDB2, andRECG1, involved in nucleotide excision repair. Moreover, a study of genomic diversity among wild and cultivated accessions revealed genomic regions containing selection signatures associated with the adaptation to extreme environments. The identification of the genes and alleles with selection signatures provides insights into the genetic mechanisms underlying the adaptation of wild castor to the high-altitude tropical desert and would facilitate direct improvement of modern castor varieties.
Sequencing of methylase-accessible regions in integral circular extrachromosomal DNA reveals differences in chromatin structure
Authors: Chen Weitian , Weng Zhe , Xie Zhe , Xie Yeming , Zhang Chen , Chen Zhichao , Ruan Fengying , Wang Juan , Sun Yuxin , Fang Yitong , Guo Mei , Tong Yiqin , Li Yaning , Tang Chong
Published: April 2021

Although extrachromosomal DNA (ecDNA) has been intensively studied for several decades, the mechanisms underlying its tumorigenic effects have been revealed only recently. In the majority of conventional sequencing studies, the high-throughput short-read sequencing largely ignores the epigenetic status of most ecDNA regions except for the junctional areas. Here, we developed the sequencing of enzyme-accessible chromatin in circular DNA (CCDA-seq) method, which uses methylase to label open chromatin without fragmentation and exonuclease to enrich the ecDNA sequencing depth, followed by long-read nanopore sequencing. Using CCDA-seq, we observed significantly different patterns in nucleosome/regulator binding in ecDNA at a single-molecule resolution. These results deepen the understanding of ecDNA regulatory mechanisms.
Chromatin interaction aware gene regulatory modeling with graph attention networks
Authors: Karbalayghareh Alireza , Sahin Merve , Leslie Christina S.
Published: April 2021

Linking distal enhancers to genes and modeling their impact on target gene expression are longstanding unresolved problems in regulatory genomics and critical for interpreting non-coding genetic variation. Here we present a new deep learning approach called GraphReg that exploits 3D interactions from chromosome conformation capture assays in order to predict gene expression from 1D epigenomic data or genomic DNA sequence. By using graph attention networks to exploit the connectivity of distal elements up to 2Mb away in the genome, GraphReg more faithfully models gene regulation and more accurately predicts gene expression levels than state-of-the-art deep learning methods for this task. Feature attribution used with GraphReg accurately identifies functional enhancers of genes, as validated by CRISPRi-FlowFISH and TAP-seq assays, outperforming both CNNs and the recently proposed Activity-by-Contact model. Sequence-based GraphReg also accurately predicts direct transcription factor (TF) targets as validated by CRISPRi TF knockout experiments via in silico ablation of TF binding motifs. GraphReg therefore represents an important advance in modeling the regulatory impact of epigenomic and sequence elements.
Re-profiling of natural inhibitor via combinatorial drug screening: Brefeldin A variant design as an effective antagonist leading to EPAC2 structure modification and antibody design for identification
Authors: Uttarkar Akshay , Niranjan Vidya
Published: April 2021

Drug discovery can be impactful with re-purposing and combinatorial strategies leading to potential pharmaceutical outputs. Epac2 has been a target of interest for various physiological conditions where suppression is inevitable to achieve desired therapeutic effect. Epac isoforms inhibition is crucial in vascular functions to prevent chronic inflammation leading to hypertension and myocardial infarction. An attempt utilizing brefeldin A, a natural inhibitor was subjected to substitution of 3 side chain groups with 43 fragments via combinatorial strategy. This resulted in generating a library of 79507 brefeldin A variants. High throughput virtual screening yielded 68,043 variants followed by precision docking providing 117 lead like brefeldin A variants. The best docked variant (3-((1R,2E,6R,10E,11aS,13S,14A)-6-(methylsulfonamido)-13-(3-methylureido)-4-oxo-4,6,7,8,9,11a,12,13,14,14a-decahydro-1H-cyclopenta[f][1]oxacyclotridecin-1-yl)-2,3-dihydro-1Himidazol-1-ium) has an increased binding efficiency of −10.841 kcal/mol. Simulation studies up to 200ns of complex lead to re-orientation of target tertiary structure resulted in RMSD change of 30.221 Å, suggesting the epac2 structure modification leading to unavailability of RAS-GEF domain and its interaction with Rap1b. A single domain antibody was designed to bind specifically to re-structured epac2 for potential identification over the native target structure. The resulting Brefeldin variant can be potentially labelled as a most effective antagonist against epac2 which induces theoretically irreversible structural re-conformation. This study also provides a robustin-silicoworkflow for searching of chemical space, generating and screening of combination libraries and the efficient utilization of known inhibitor.
An improved molecular crowding sensor CRONOS for detection of crowding changes in membrane-less organelles under pathological conditions
Authors: Miyagi Tamami , Yamanaka Yoshiaki , Harada Yuichiro , Narumi Satoshi , Hayamizu Yuhei , Kuroda Masahiko , Kanekura Kohsuke
Published: April 2021

Membrane-less organelles (MLOs) formed by liquid-liquid phase separation (LLPS) play pivotal roles in biological processes. During LLPS, proteins and nucleotides are extremely condensed, resulting in changes of their conformation and biological functions. Disturbed LLPS homeostasis in MLOs cause fatal diseases such as amyotrophic lateral sclerosis. Therefore, it is important to detect changes of the degree of crowding in MLOs. However, it has not been investigated well due to lack of an appropriate method. To address this, we developed a genetically-encoded molecular crowding sensor CRONOS that senses the degree of macromolecular crowding in MLOs using fluorescence resonance energy transfer (FRET) system. CRONOS is a very bright biosensor with wider dynamic range and detect changes in the macromolecular volume fraction better than the previously reported mCer-mCit sensor in solution. By fusing to scaffold protein of each MLO, we successfully delivered CRONOS to MLO of interest and detected previously undescribed difference of the degree of crowding in each MLO. If not tagged, CRONOS localized to interstitial space of MLOs, giving us the crowding information of inspace. CRONOS also detected changes of degree of macromolecular crowding in nucleolus induced by environmental stress or inhibition of transcription. These findings suggest that CRONOS can be a useful tool for determination of molecular crowding and detection of pathological changes in MLOs in live cells.
ReFeaFi: Genome-wide prediction of regulatory elements driving transcription initiation
Authors: Umarov Ramzan , Li Yu , Arakawa Takahiro , Takizawa Satoshi , Gao Xin , Arner Erik
Published: April 2021

Regulatory elements control gene expression through transcription initiation (promoters) and by enhancing transcription at distant regions (enhancers). Accurate identification of regulatory elements is fundamental for annotating genomes and understanding gene expression patterns. While there are many attempts to develop computational promoter and enhancer identification methods, reliable tools to analyze long genomic sequences are still lacking. Prediction methods often perform poorly on the genome-wide scale because the number of negatives is much higher than that in the training sets. To address this issue, we propose a dynamic negative set updating scheme with a two-model approach, using one model for scanning the genome and the other one for testing candidate positions. The developed method achieves good genome-level performance and maintains robust performance when applied to other species, without re-training. Moreover, the unannotated predicted regulatory regions made on the human genome are enriched for disease-associated variants, suggesting them to be potentially true regulatory elements rather than false positives. We validated high scoring “false positive” predictions using reporter assay and all tested candidates were successfully validated, demonstrating the ability of our method to discover novel human regulatory regions.
A new contact killing toxin permeabilizes cells and belongs to a large protein family
Authors: Crisan Cristian V. , Chandrashekar Harshini , Everly Catherine , Steinbach Gabi , Hill Shannon E. , Yunker Peter J. , Lieberman Raquel R. , Hammer Brian K.
Published: April 2021

Vibrio choleraeis an aquatic Gram-negative bacterium that causes severe diarrheal cholera disease when ingested by humans. To eliminate competitor cells in both the external environment and inside hosts,V. choleraeuses the Type VI Secretion System (T6SS). The T6SS is a macromolecular weapon employed by many Gram-negative bacteria to deliver cytotoxic proteins into adjacent cells. In addition to canonical T6SS gene clusters encoded by all sequencedV. choleraeisolates, strain BGT49 encodes an additional locus, which we named auxiliary cluster 4 (Aux 4). The Aux 4 cluster is located on a mobile genetic element and can be used by killer cells to eliminate bothV. choleraeandEscherichia colicells in a T6SS-dependent manner. A putative toxin encoded in the cluster, which we name TpeV (Type VIPermeabilizingEffectorVibrio), shares no homology to known proteins and does not contain motifs or domains indicative of function. Ectopic expression of TpeV in the periplasm ofE. colipermeabilizes cells and disrupts the membrane potential. Using confocal microscopy, we confirm that susceptible target cells become permeabilized when competed with killer cells harboring the Aux 4 cluster. We also determine thattpiV, the gene located immediately downstream oftpeV, encodes an immunity protein that neutralizes the toxicity of TpeV. Finally, we show that TpeV homologs are broadly distributed across important animal and plant pathogens and are localized in proximity to other T6SS genes. Our results suggest that TpeV is a toxin that belongs to a large family of T6SS proteins.

IMPORTANCE

Bacteria live in polymicrobial communities where competition for resources and space is essential for survival. Proteobacteria use the T6SS to eliminate neighboring cells and cause disease. However, the mechanisms by which many T6SS toxins kill or inhibit susceptible target cells are poorly understood. The sequence of the TpeV toxin we describe here is unlike any previously described protein. We demonstrate that it has antimicrobial activity by permeabilizing cells, eliminating membrane potentials and causing severe cytotoxicity. TpeV homologs are found near known T6SS genes in human, animal and plant bacterial pathogens, indicating that the toxin is a representative member of a broadly distributed protein family. We propose that TpeV-like toxins contribute to the fitness and pathogenicity of many bacteria. Finally, since antibiotic resistance is a critical global health threat, the discovery of new antimicrobial mechanisms could lead to the development of new treatments against resistant strains.
Light-dependent inhibition of clathrin-mediated endocytosis in yeast
Authors: Prischich Davia , del Dedo Javier Encinar , Cambra Maria , Prat Judit , Camarero Nuria , Nevola Laura , Martín-Quirós Andrés , Rebollo Elena , Giralt Ernest , Isabel Geli María , Gorostiza Pau
Published: April 2021

Clathrin-mediated endocytosis (CME) is an essential cellular process, which is evolutionarily conserved among eukaryotes. Yeast constitutes a powerful genetic model to dissect the complex endocytic machinery, yet there is a lack of pharmacological agents that could complement genetics in selectively and reversibly interfere with CME in these organisms. TL2 is a light-regulated peptide inhibitor that targets the AP2/β-arrestin interaction and that can photocontrol CME with high spatiotemporal precision in mammalian cells. Here, we study endocytic protein dynamics by live-cell imaging of the fluorescently tagged coat-associated protein Sla1-GFP and demonstrate that TL2 retains its inhibitory activity inS. cerevisiaespheroplasts, thus providing a unique tool for acute and reversible CME modulation in yeast.
Species and strain cultivation of skin, oral, and gut microbiota
Authors: Fleming Elizabeth , Pabst Victor , Hoyt Amelia , Zhou Wei , Hardy Rachel , Peterson Anna , Beach Ryan , Ondouah-Nzutchi Yvette , Dong Jinhong , Oh Julia
Published: April 2021

Genomics-driven discovery of microbial species have provided extraordinary insights into the biodiversity of human microbiota. High resolution genomics to investigate species- and strain-level diversity and mechanistic studies, however, rely on the availability of individual microbes from a complex microbial consortia. Here, we describe and validate a streamlined workflow for cultivating microbes from the skin, oral, and gut microbiota, informed by metagenomic sequencing, mass spectrometry, and strain profiling.
Callosal fiber length scales with brain size according to functional lateralization, evolution, and development
Authors: Yang Liyuan , Zhao Chenxi , Xiong Yirong , Zhong Suyu , Wu Di , Peng Shaoling , Thiebaut de Schotten Michel , Gong Gaolang
Published: April 2021

Brain size significantly impacts the organization of white matter fibers. Fiber length scaling – the degree to which fiber length varies according to brain size – was overlooked. We investigated how fiber lengths within the corpus callosum, the most prominent white matter tract, vary according to brain size. The results showed substantial variation in length scaling among callosal fibers, replicated in two large healthy cohorts (∼2000 individuals). The underscaled callosal fibers mainly connected the precentral gyrus and parietal cortices, whereas the overscaled callosal fibers mainly connected the prefrontal cortices. The variation in such length scaling was biologically meaningful: larger scaling corresponded to larger neurite density index but smaller fractional anisotropy values; cortical regions connected by the callosal fibers with larger scaling were more lateralized functionally as well as phylogenetically and ontogenetically more recent than their counterparts. These findings highlight an interaction between interhemispheric communication and organizational and adaptive principles underlying brain development and evolution.

Significance Statement

Brain size varies across evolution, development, and individuals. Relative to small brains, the neural fiber length in large brains is inevitably increased, but the degree of such increase may differ between fiber tracts. Such a difference, if it exists, is valuable for understanding adaptive neural principles in large versus small brains during evolution and development. The present study showed a substantial difference in the length increase between the callosal fibers that connect the two hemispheres, replicated in two large healthy cohorts. Altogether, our study demonstrates that reorganization of interhemispheric fibers length according to brain size is intrinsically related to fiber composition, functional lateralization, cortical myelin content, evolutionary and developmental expansion.
The prostate cancer therapy enzalutamide compared with abiraterone acetate/prednisone impacts motivation for exploration, spatial learning and alters dopaminergic transmission in aged castrated mice
Authors: Nicola Celeste , Dubois Martine , Campart Cynthia , Sagheer Tareq Al , Desrues Laurence , Schapman Damien , Galas Ludovic , Lange Marie , Joly Florence , Castel Hélène
Published: April 2021

Cognitive side effects after cancer treatment, threatening quality of life (QoL) and adherence to treatments, now constitute a major challenge in oncology. Abiraterone acetate plus prednisone (AAP) and enzalutamide (ENZ) are next generation therapy (NGT) administered with androgen deprivation therapy to metastatic castration-resistant prostate cancer (mCRPC) patients. NGT significantly improved mCRPC overall survival but neurological side effects such as fatigue and cognitive impairment have been recently reported. We developed a behavioral 17 months-aged and castrated mouse model receivingper osAAP or ENZ during 5 days per week for six consecutive weeks. After behavioral tests, brain and plasma were collected for immunohistochemical studies. The objective was to elucidate the impact of NGT on spontaneous activity, cognitive functions and emotional reactivity, as well as neurobiological functions. ENZ exposure reduced spontaneous activity and exploratory behavior associated with a decreased tyrosine hydroxylase (TH)-dopaminergic activity in the substantia nigra pars compacta and the ventral tegmental area. A decrease in TH+-DA afferent fibers and Phospho-DARPP32-related dopaminergic neuronal activities in the striatum and the ventral hippocampus, highlighted ENZ-induced dopaminergic regulation whithin the nigrostriatal and mesolimbocortical pathways. ENZ and AAP treatments did not substantially modify spatial learning and memory or behavioral flexibility performances, but ENZ led to a thygmotaxis behavior impacting the cognitive score, and reduced c-fos-related activity of NeuN+-neurons in the dorsal hippocampus. These results establish the consequences of the mCRPC treatment ENZ in aged castrated mouse motivation to exploration and cognition, of particular importance for future management of patients elderly postrate cancer patients and their QoL.
NRPS-gene disruption in the apple-pathogen,Neonectria ditissima
Authors: Vélëz Heriberto , Bourras Salim , Garkava-Gustavsson Larisa , Dalman Kerstin
Published: April 2021

Apple production in Sweden and elsewhere is being threaten by the fungus,Neonectria ditissima, which causes a disease known as Fruit Tree Canker. The disease can cause extensive damages and the removal of diseased-wood and heavily infected trees can be laborious and expensive. Currently, there is no way to eradicate the fungus from infected trees and our knowledge of the infection process is limited. Thus, in order to target and modify genes efficiently, the genetic transformation technique developed forN. ditissimaback in 2003 was modified. We report on the upgraded protocol and show that protoplasts were viable, able to uptake foreign DNA, and able to regenerate back into a mycelial colony, either as targeted gene-disruption mutants or as ectopic mutants expressing GFP.
The monitoring system is attuned to others’ actions during dyadic motor
interactions
Authors: Moreau Quentin , Tieri Gaetano , Era Vanessa , Aglioti Salvatore Maria , Candidi Matteo
Published: April 2021

Successful interpersonal motor interactions necessitate the simultaneous
monitoring of our own and our partner’s actions. To characterize the dynamics of
the action monitoring system for tracking self and other behaviors during dyadic
synchronous interactions, we combined EEG recordings and immersive Virtual
Reality in two tasks where participants were asked to coordinate their actions
with those of a Virtual Partner (VP). The two tasks differed in the features to
be monitored: the Goal task required participants to
predict and monitor the VP’s reaching goal; the
Spatial task required participants to predict and
monitor the VP’s reaching trajectory. In both tasks, the VP performed unexpected
movement corrections to which the participant needed to adapt. By comparing the
neural activity locked to the detection of unexpected changes in the VP action
(other-monitoring) or to the participants’ action-replanning (self-monitoring),
we show that during interpersonal interactions the monitoring system is more
attuned to others’ than to one’s own actions. Additionally, distinctive neural
responses to VP’s unexpected goals and trajectory corrections were found: goal
corrections were reflected both in early fronto-central and later posterior
neural responses while trajectory deviations from the expected movement were
reflected only in later and posterior responses. Since these responses were
locked to the partner’s behavior and not to one’s own, our results indicate that
during interpersonal interactions the action monitoring system is dedicated to
evaluating the partner’s movements. Hence, our results reveal an eminently
social role of the monitoring system during motor interactions.

Significance Statement

Non-verbal synchronous interpersonal interactions require the
monitoring of both our actions and those of our partners. Understanding
the neural underpinnings of this ability with a focus on the dynamics
between self- and other-monitoring is fundamental to the comprehension
of social coordination. By combining EEG and immersive Virtual Reality
we demonstrate that the monitoring system is more attuned to others’
actions than to our own. In two tasks, we show that the neural activity
associated with unexpected corrections in the goal or the trajectory of
an action are locked to the partner’s actions rather than to the
participants’ subsequent adaptation. This pattern of results highlights
a social mode adopted by the monitoring system to handle motor
interactions.
Evaluation of efficacy-versusaffinity-driven agonism with biased GLP-1R ligands P5 and exendin-F1
Authors: Marzook Amaara , Chen Shiqian , Pickford Phil , Lucey Maria , Wang Yifan , Corrêa Ivan R , Broichhagen Johannes , Hodson David J , Salem Victoria , Rutter Guy A , Tan Tricia M , Bloom Stephen R , Tomas Alejandra , Jones Ben
Published: April 2021

The glucagon-like peptide-1 receptor (GLP-1R) is an important regulator of glucose homeostasis and has been successfully targeted for the treatment of type 2 diabetes. Recently described biased GLP-1R agonists with selective reductions in β-arrestinversusG protein coupling show improved metabolic actionsin vivo. However, two prototypical G protein-favouring GLP-1R agonists, P5 and exendin-F1, are reported to show divergent effects on insulin secretion. In this study we aimed to resolve this discrepancy by performing a side-by-side characterisation of these two ligands across a variety ofin vitroandin vivoassays. Exendin-F1 showed reduced acute efficacyversusP5 for several readouts, including recruitment of mini-G proteins, G protein-coupled receptor kinases (GRKs) and β-arrestin-2. Maximal responses were also lower for both GLP-1R internalisation and the presence of active GLP-1R-mini-Gscomplexes in early endosomes with exendin-F1 treatment. In contrast, prolonged insulin secretionin vitroand sustained anti-hyperglycaemic efficacy in mice were both greater with exendin-F1 than with P5. We conclude that the particularly low acute efficacy of exendin-F1 and associated reductions in GLP-1R downregulation appear to be more important than preservation of endosomal signalling to allow sustained insulin secretion responses. This has implications for the ongoing development of affinity-versusefficacy-driven biased GLP-1R agonists as treatments for metabolic disease.
Necrosis-induced apoptosis promotes regeneration inDrosophilawing imaginal discs
Authors: Klemm Jacob , Stinchfield Michael J. , Harris Robin E.
Published: April 2021

Regeneration is a complex process that requires a coordinated genetic response to tissue loss. Signals from dying cells are crucial to this process and are best understood in the context of regeneration following programmed cell death, like apoptosis. Conversely, regeneration following unregulated forms of death such as necrosis have yet to be fully explored. Here we have developed a method to investigate regeneration following necrosis using theDrosophilawing imaginal disc. We show that necrosis stimulates regeneration at an equivalent level to that of apoptosis-mediated cell death and activates a similar response at the wound edge involving localized JNK signaling. Unexpectedly however, necrosis also results in significant apoptosis far from the site of ablation, which we have termed necrosis-induced apoptosis (NiA). This apoptosis occurs independent of changes at the wound edge and importantly does not rely on JNK signaling. Furthermore, we find that blocking NiA limits proliferation and subsequently inhibits regeneration, suggesting that tissues damaged by necrosis can activate programmed cell death at a distance from the injury to promote regeneration.
Establishment of a Patient-Derived, Magnetic Levitation-Based, 3D Spheroid Granuloma Model for Human Tuberculosis
Authors: Kotze Leigh A. , Beltran Caroline G.G. , Lang Dirk , Loxton Andre G. , Cooper Susan , Meiring Maynard , Glanzmann Brigitte , Kinnear Craig , Walzl Gerhard , du Plessis Nelita
Published: April 2021

Tuberculous granulomas that develop in response toMycobacterium tuberculosis(M.tb) infection are highly dynamic entities shaped by the host immune response and disease kinetics. Within this microenvironment, immune cell recruitment, polarization and activation is driven not only by co-existing cell types and multi-cellular interactions, but also byM.tb-mediated changes involving metabolic heterogeneity, epigenetic reprogramming and rewiring of the transcriptional landscape of host cells. There is an increased appreciation of thein vivocomplexity, versatility and heterogeneity of the cellular compartment that constitutes the tuberculosis (TB) granuloma, and the difficulty in translating findings from animal models to human disease. Here we describe a novel biomimeticin vitro3-dimentional (3D) human lung granuloma model, resembling early “innate” and “adaptive” stages of the TB granuloma spectrum, and present results of histological architecture, host transcriptional characterization, mycobacteriological features, cytokine profiles and spatial distribution of key immune cells. A range of manipulations of immune cell populations in these granulomas will allow the study of host/pathogen pathways involved in the outcome of infection, as well as pharmacological interventions.

IMPORTANCE

Tuberculosis is a highly infectious disease, with granulomas as its hallmark. Granulomas play an important role in the control of M.tb infection and as such are crucial indicators for our understanding of host resistance to TB. Correlates of risk and protection to M.tb are still elusive, and the granuloma provides the perfect environment in which to study the immune response to infection and broaden our understanding thereof; however, human granulomas are difficult to obtain, and animal models are costly and do not always faithfully mimic human immunity. In fact, most TB research is conducted in vitro on immortalized or primary immune cells and cultured in 2D on flat, rigid plastic, which does not reflect in vivo characteristics. We have therefore conceived a 3D, human in vitro granuloma model which allows researchers to study features of granuloma-forming diseases, in an 3D structural environment resembling in vivo granuloma architecture and cellular orientation.
γδT Cells Are Required for CD8+T Cell Response to Vaccinia Viral Infection
Authors: Dai Rui , Huang Xiaopei , Yang Yiping
Published: April 2021

Vaccinia virus (VV) is the most studied member of the poxvirus family, is responsible for the successful elimination of smallpox worldwide, and has been developed as a vaccine vehicle for infectious diseases and cancer immunotherapy. We have previously shown that the unique potency of VV in the activation of CD8+T cell response is dependent on efficient activation of the innate immune system through Toll-like receptor (TLR)-dependent and -independent pathways. However, it remains incompletely defined what regulate CD8+T cell response to VV infection. In this study, we showed that γδT cells play an important role in promoting CD8+T cell response to VV infection. We found that γδT cells can directly present viral antigens in the context MHC-I for CD8+T cell activation to VVin vivo, and we further demonstrated that cell-intrinsic MyD88 signaling in γδT cells is required for activation of γδT cells and CD8+T cells. These results illustrate a critical role for γδT cells in the regulation of adaptive T cell response to viral infection and may shed light on the design of more effective vaccine strategies based on manipulation of γδT cells.

Importance

Targeting the immune systems has powerful potentials to treat many disorders, such as some cancers and viral infections. By understanding how the immune system responds to model infections, we can better determine strategies to manipulate our immune systems. Vaccinia virus is responsible for the worldwide elimination of smallpox and produces one of the longest immune responses known in humans. We know from previous findings that NK cells are required for initial immune response and CD8+T cells are required for the elimination of the virus. How CD8+T cells are activated in response to Vaccinia virus is not fully understood. This manuscript found that γδT cells activate CD8+T cells in response to Vaccinia virus infection through MyD88 pathway
IGF2BP2 Promotes Cancer Progression by Degrading the RNA Transcript Encoding a v-ATPase Subunit
Authors: Latifkar A. , Wang F. , Mullmann J.J. , Fernandez I.R. , Ling L. , Fischbach C. , Weiss R.S. , Lin H. , Cerione R.A. , Antonyak M.A.
Published: April 2021

Insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2) binds to various RNA transcripts and promotes cancer progression, although little is known regarding its regulation. Here we show IGF2BP2 is a substrate of the deacetylase and tumor suppressor sirtuin 1 (SIRT1) and regulates the expression of the vacuolar ATPase subunit ATP6V1A. SIRT1 down-regulation in aggressive cancers leads to increased acetylation of IGF2BP2 which recruits the XRN2 nuclease to degrade the ATP6V1A transcript, decreasing its expression. This impairs lysosomal function and results in the production of a secretome that enhances cancer cell proliferation and metastasis. These findings describe a previously unrecognized role for IGF2BP2 in the degradation of an mRNA transcript essential for lysosomal function and highlight how its sirtuin-regulated acetylation state can have significant biological and disease consequences.

One Sentence Summary

Acetylation of the RNA binding protein IGF2BP2, upon down-regulation of SIRT1, leads to degradation of the transcript encoding ATP6V1A and impaired lysosomal function in aggressive cancer cells.
Deficiency of the paternally inherited geneMagel2alters the development of separation-induced vocalization and maternal behavior in mice
Authors: Bosque Ortiz Gabriela M. , Santana Gustavo M. , Dietrich Marcelo O.
Published: April 2021

The behavior of offspring results from the combined expression of maternal and paternal genes. Genomic imprinting silences some genes in a parent-of-origin specific manner, a process that, among all animals, occurs only in mammals. How genomic imprinting affects the behavior of mammalian offspring, however, remains poorly understood. Here, we studied how the loss of the paternally inherited geneMagel2in mouse pups affects the emission of separation-induced ultrasonic vocalizations (USV). Using quantitative analysis of more than one hundred thousand USVs, we characterized the rate of vocalizations as well as their spectral features from postnatal days 6 to 12 (P6-P12), a critical phase of mouse development during which pups fully depend on the mother for survival. Our analyses show thatMagel2deficient offspring emit separation-induced vocalizations at lower rates and with altered spectral features. We also show that dams display altered behavior towards their ownMagel2deficient offspring. In a test to compare the retrieval of two pups, dams retrieve wildtype control pups first and faster thanMagel2deficient offspring. These results suggest that the loss ofMagel2impairs the expression of separation-induced vocalization in pups as well as maternal behavior, both of which support the pups’ growth and development.
Naturalistic audio-movies reveal common spatial organization across “visual” cortices of different blind individuals
Authors: Musz Elizabeth , Loiotile Rita , Chen Janice , Bedny Marina
Published: April 2021

Occipital cortices of different sighted people contain analogous maps of visual information (e.g., foveal vs. peripheral space). In congenital blindness, “visual” cortices enhance responses to nonvisual stimuli. Do deafferented visual cortices of different blind people represent common informational maps? We leverage a naturalistic stimulus paradigm and inter-subject pattern similarity analysis to address this question. Blindfolded sighted (S, n=22) and congenitally blind (CB, n=22) participants listened to three auditory excerpts from movies; a naturalistic spoken narrative; and matched degraded auditory stimuli (i.e., shuffled sentences and backwards speech) while undergoing fMRI scanning. In a parcel-based whole brain analysis, we measured the spatial activity patterns evoked by each unique, ten-second segment of each auditory clip. We then compared each subject’s spatial pattern to that of all other subjects in the same group (CB or S) within and across segments. In both blind and sighted groups, segments of meaningful auditory stimuli produced distinctive patterns of activity that were shared across individuals. Crucially, only in the CB group, this segment-specific, cross-subject pattern similarity effect emerged in visual cortex, but only for meaningful naturalistic stimuli and not backwards speech. These results suggest that spatial activity patterns within deafferented visual cortices encode meaningful, segment-level information contained in naturalistic auditory stimuli, and that these representations are spatially organized in a similar fashion across blind individuals.

Significance Statement

Recent neuroimaging studies show that the so-called “visual” cortices activate during non-visual tasks in people who are born blind. Do the visual cortices of people who are born blind develop similar representational maps? While congenitally blind individuals listened to naturalistic auditory stimuli (i.e., sound clips from movies), distinct timepoints within each stimulus elicited unique spatial activity patterns in visual cortex, and these patterns were shared across different people. These findings suggest that in blindness, the visual cortices encode meaningful information embedded in naturalistic auditory signals in a spatially distributed manner, and that a common representational map can emerge in visual cortex independent of visual experience.
Genome-wide association study reveals candidate genes for flowering time in cowpea (Vigna unguiculata[L.] Walp)
Authors: Paudel Dev , Dareus Rocheteau , Rosenwald Julia , Muñoz-Amatriaín María , Rios Esteban F.
Published: April 2021

Cowpea (Vigna unguiculata[L.] Walp., diploid, 2n= 22) is a major crop used as a protein source for human consumption as well as a quality feed for livestock. It is drought and heat tolerant and has been bred to develop varieties that are resilient to changing climates. Plant adaptation to new climates and their yield are strongly affected by flowering time. Therefore, understanding the genetic basis of flowering time is critical to advance cowpea breeding. The aim of this study was to perform genome-wide association studies (GWAS) to identify marker trait associations for flowering time in cowpea using single nucleotide polymorphism (SNP) markers. A total of 367 accessions from a cowpea minicore collection were evaluated in Ft. Collins, CO in 2019 and 2020, and 292 accessions were evaluated in Citra, FL in 2018. These accessions were genotyped using the Cowpea iSelect Consortium Array that contained 51,128 SNPs. GWAS revealed seven reliable SNPs for flowering time that explained 8-12% of the phenotypic variance. Candidate genes includingFT, GI, CRY2, LSH3, UGT87A2, LIF2, andHTA9that are associated with flowering time were identified for the significant SNP markers. Further efforts to validate these loci will help to understand their role in flowering time in cowpea, and it could facilitate the transfer of some of this knowledge to other closely related legume species.
Diverse ATPase proteins in mobilomes constitute a large potential sink for prokaryotic host ATP
Authors: Shim Hyunjin , Shivram Haridha , Lei Shufei , Doudna Jennifer A. , Banfield Jillian F.
Published: April 2021

Prokaryote mobilome genomes rely on host machineries for survival and replication. Given that mobile genetic elements (MGEs) derive their energy from host cells, we investigated the diversity of ATP-utilizing proteins in MGE genomes to determine whether they might be associated with proteins that could suppress related host proteins that consume host energy. A comprehensive search of 353 huge phage genomes revealed that up to 9% of the proteins have ATPase domains. For example, ATPase proteins constitute ~3% of the genomes of Lak phages with ~550 kbp genomes that occur in the microbiomes of humans and other animals. Statistical analysis shows the number of ATPase proteins increases linearly with genome length, consistent with a large sink for host ATP during replication of megaphages. Using metagenomic data from diverse environments, we found 505 mobilome proteins with ATPase domains fused to diverse functional domains. Among these composite ATPase proteins, 61.6% have known functional domains that could contribute to host energy diversion during the mobilome life cycle. As many have domains that are known to interact with nucleic acids and proteins, we infer that numerous ATPase proteins are used during replication and for protection from host immune systems. We found a set of uncharacterized ATPase proteins with nuclease and protease activities, displaying unique domain architectures that are energy intensive based on the presence of multiple ATPase domains. In many cases, these composite ATPase proteins genomically co-localize with small proteins in genomic contexts that are reminiscent of toxin-antitoxin systems and phage helicase-antibacterial helicase systems. Small proteins that function as inhibitors may be a common strategy for control of cellular processes, thus could inspire the development of new nucleic acid and protein manipulation tools, with diverse biotechnological applications.
High-throughput analysis of B3GLCT regulation predicts phenotype of Peters’ Plus Syndrome in line with the miRNA Proxy Hypothesis
Authors: Thu Chu T. , Chung Jonathan Y. , Dhawan Deepika , Vaiana Christopher A. , Mahal Lara K.
Published: April 2021

MicroRNAs (miRNAs, miRs) finely tune protein expression and target networks of 100s-1000s of genes that control specific biological processes. They are critical regulators of glycosylation, one of the most diverse and abundant posttranslational modifications. In recent work, miRs have been shown to predict the biological functions of glycosylation enzymes, leading to the “miRNA proxy hypothesis” which states, “if a miR drives a specific biological phenotype…, the targets of that miR will drive the same biological phenotype.” Testing of this powerful hypothesis is hampered by our lack of knowledge about miR targets. Target prediction suffers from low accuracy and a high false prediction rate. Herein, we develop a high-throughput experimental platform to analyze miR:target interactions, miRFluR. We utilize this system to analyze the interactions of the entire human miRome with beta-3-glucosyltransferase (B3GLCT), a glycosylation enzyme whose loss underpins the congenital disorder Peters’ Plus Syndrome. Although this enzyme is predicted by multiple algorithms to be highly targeted by miRs, we identify only 27 miRs that downregulate B3GLCT, a >96% false positive rate for prediction. Functional enrichment analysis of these validated miRs predict phenotypes associated with Peters’ Plus Syndrome, although B3GLCT is not in their known target network. Thus, biological phenotypes driven by B3GLCT may be driven by the target networks of miRs that regulate this enzyme, providing additional evidence for the miRNA Proxy Hypothesis.