bluuebunny/crossref_metadata_2025_split huggingface dataset:
Spatacsin regulates directionality of lysosome trafficking
Authors: Pierga Alexandre , Matusiak Raphaël , Cauhapé Margaux , Branchu Julien , Boutry Maxime , Darios Frédéric 
Published: June 2022

AbstractThe endoplasmic reticulum (ER) forms contacts with the lysosomal compartment, regulating lysosome positioning and motility. The movement of lysosomes are controlled by the attachment of molecular motors to their surface. However, the molecular mechanisms by which ER controls lysosome dynamics are still elusive. Here, we demonstrate that spatacsin is an ER-resident protein that regulates ER-lysosomes contacts to promote lysosome motility, shown by the presence of tubular lysosomes. Tubular lysosomes, which are highly dynamic, are entangled in a network of tubular ER. Screening for spatacsin partners required for tubular lysosome formation showed spatacsin to act by regulating protein degradation. We demonstrate that spatacsin promotes the degradation of its partner AP5Z1, which regulates the relative amount of spastizin and AP5Z1 at lysosomes. Spastizin and AP5Z1 contribute to lysosome trafficking by interacting with anterograde and retrograde motor proteins, kinesin KIF13A and dynein/dynactin subunit p150Glued, respectively. Ultimately, investigations in polarized neurons demonstrated that spatacsin-regulated degradation of AP5Z1 controls the directionality of lysosomes trafficking. Collectively, our results identify spatacsin as a protein regulating the directionality of lysosome trafficking.
Co-immunization with pre-erythrocytic antigens alongside circumsporozoite protein can enhance sterile protection againstPlasmodiumsporozoite infection
Authors: Vigdorovich Vladimir , Patel Hardik , Watson Alexander , Raappana Andrew , Reynolds Laura , Selman William , Beeman Suzannah , Edlefsen Paul T. , Kappe Stefan H.I. , Sather D. Noah 
Published: June 2022

AbstractMalaria-causingPlasmodiumparasites have a complex life cycle and present numerous antigen targets that may contribute to protective immune responses. The currently recommended vaccine—RTS,S—functions by targeting theP. falciparumcircumsporozoite protein (CSP), which is the most abundant surface protein of the sporozoite form responsible for initiating infection of the human host. Despite showing only moderate efficacy, RTS,S has established a strong foundation for the development of next-generation subunit vaccines. Our previous work characterizing the sporozoite surface proteome identified additional non-CSP antigens that may be useful as immunogens individually or in combination with CSP. In this study, we examined eight such antigens using the rodent malaria parasiteP. yoeliias a model system. We demonstrate that despite conferring weak protection individually, co-immunizing each of several of these antigens alongside CSP, could significantly enhance the sterile protection achieved by CSP immunization alone. Thus, our work provides compelling evidence that a multi-antigen pre-erythrocytic vaccine approach may enhance protection compared to CSP-only vaccines. This lays the groundwork for further studies aimed at testing the identified antigen combinations in human vaccination trials that assess efficacy with controlled human malaria infection.ImportanceThe currently approved malaria vaccine targets a single parasite protein (CSP) and only results in partial protection. We tested several additional vaccine targets in combination with CSP to identify those that could enhance protection from infection upon challenge in the mouse malaria model. In identifying several such enhancing vaccine targets, our work indicates that a multi-protein immunization approach may be a promising avenue to achieving higher levels of protection from infection. Our work identified several candidate leads for follow-up in the models relevant for human malaria, and provides an experimental framework for efficiently carrying out such screens for other combinations of vaccine targets.
Selection for translational efficiency in genes associated with alphaproteobacterial gene transfer agents
Authors: Kogay Roman , Zhaxybayeva Olga 
Published: June 2022

AbstractGene transfer agents (GTAs) are virus-like elements that are encoded by some bacterial and archaeal genomes. The production of GTAs can be induced by the carbon depletion and results in host lysis and release of virus-like particles that contain mostly random fragments of the host DNA. The remaining members of a GTA-producing population act as GTA recipients by producing proteins needed for the GTA-mediated DNA acquisition. Here, we detect a codon usage bias towards codons with more readily available tRNAs in the RcGTA-like GTA genes of alphaproteobacterial genomes. Such bias likely improves the translational efficacy during GTA gene expression. While the strength of codon usage bias fluctuates substantially among individual GTA genes and across taxonomic groups, it is especially pronounced in Sphingomonadales, whose members are known to inhabit nutrient-depleted environments. By screening genomes for gene families with similar trends in codon usage biases to those in GTA genes, we found a gene that likely encodes head completion protein in some GTAs were it appeared missing, and 13 genes previously not implicated in GTA lifecycle. The latter genes are involved in various molecular processes, including the homologous recombination and transport of scarce organic matter. Our findings provide insights into the role of selection for translational efficiency in evolution of GTA genes, and outline genes that are potentially involved in the previously hypothesized integration of GTA-delivered DNA into the host genome.ImportanceHorizontal gene transfer (HGT) is a fundamental process that drives evolution of microorganisms. HGT can result a rapid dissemination of beneficial genes within and among microbial communities, and can be achieved via multiple mechanisms. One peculiar HGT mechanism involves viruses “domesticated” by some bacteria and archaea (their hosts). These so-called gene transfer agents (GTAs) are encoded in hosts’ genomes, produced under starvation conditions, and cannot propagate themselves as viruses. We show that GTA genes are under selection to improve efficiency of their translation when the host activates GTA production. The selection is especially pronounced in bacteria that occupy nutrient-depleted environments. Intriguingly, several genes involved in DNA incorporation into a genome are under similar selection pressure, suggesting that they may facilitate integration of GTA-delivered DNA into the host genome. Our findings underscore the potential importance of GTAs as a mechanism of HGT under nutrient-limited conditions, which are widespread in microbial habitats.
In vivoseamless genetic engineering via CRISPR-triggered single-strand annealing
Authors: Aguilar Gustavo , Bauer Milena , Vigano M. Alessandra , Schnider Sophie T. , Brügger Lukas , Jiménez-Jiménez Carlos , Guerrero Isabel , Affolter Markus 
Published: June 2022

AbstractPrecise genome engineering is essential for both basic and applied research. CRISPR/Cas accelerated the speed and ease by which defined exogenous sequences are integrated into specific loci. Nevertheless, knock-in generation in multicellular animals remains challenging, partially due to the complexity of insertion screening. Even when achieved, the analysis of protein localization can still be unfeasible in highly packed tissues, where spatial and temporal control of gene labeling would be ideal. Here, we describe SEED/Harvest, an efficient knock-in method based on homology-directed (HDR) and single-strand annealing (SSA) repair pathways. HDR mediates the integration of a switchable cassette. Upon a subsequent CRISPR-triggered repair event, resolved by SSA, the cassette is seamlessly removed. Germline excision of SEED cassettes allows for fast and robust knock-in generation with both fluorescent proteins and short protein tags in tandem. Tissue-specific expression of Cas9 results in somatic cassette excision, conferring spatio-temporal control of protein labelling and the conditional rescue of mutants. Finally, to achieve conditional protein labeling and manipulation of short tag knock-ins, we have developed a toolbox based on rational engineering and functionalization of the ALFA nanobody.
Structural basis for RNA-mediated assembly of type V CRISPR-associated transposons
Authors: Schmitz Michael , Querques Irma , Oberli Seraina , Chanez Christelle , Jinek Martin 
Published: June 2022

SummaryCRISPR systems have been co-opted by Tn7-like elements to direct RNA-guided transposition. Type V-K CRISPR-associated transposons rely on the concerted activities of the pseudonuclease Cas12k, the AAA+ ATPase TnsC, the Zn-finger protein TniQ, and the transposase TnsB. Here we present a cryo-electron microscopic structure of a target DNA-bound Cas12k-transposon recruitment complex comprising RNA-guided Cas12k, TniQ, TnsC and, unexpectedly, the ribosomal protein S15. Complex assembly on target DNA results in complete R-loop formation mediated by critical interactions between TniQ and the trans-activating crRNA, and is coupled with TniQ-dependent nucleation of a TnsC filament. In vivo transposition assays corroborate our structural findings, and biochemical and functional analyses of S15 supports its role as a bona fide component of the type V crRNA-guided transposition machinery. Altogether, our work uncovers key aspects of the mechanisms underpinning RNA-mediated assembly of CRISPR-associated transposons that will guide their development as programmable site-specific gene insertion tools.
Transient and DNA-free in vivo CRISPR/Cas9 genome edition for flexible modelling of endometrial carcinogenesis
Authors: Navaridas Raúl , Vidal-Sabanés Maria , Ruiz-Mitjana Anna , Perramon-Güell Aida , Megino-Luque Cristina , Llobet-Navas David , Matias-Guiu Xavier , Egea Joaquim , Encinas Mario , Bardia Lídia , Colombelli Julien , Dolcet Xavier 
Published: June 2022

ABSTRACTThe CRISPR/Cas9 technology has emerged as a powerful tool to generate mouse models of disease. Endometrial cancer is the most common malignancy of the female genital tract. In the present study, we have developed a pipeline for the generation of somatically engineered mouse models of endometrial cancer by in vivo electroporation-mediated delivery of Cas9 ribonucleoprotein into the uterine cavity. By using mT/mG dual-fluorescent reporter mice, we show that this system allows an efficient genomic edition specifically in epithelial endometrial cells. As a proof of its applicability for endometrial cancer modeling, we designed Cas9 ribonucleoprotein targeting Pten, the most frequently tumor suppressor gene mutated in this type of cancer. Pten-targeting ribonucleoprotein delivery into the uterine cavity caused loss of expression of PTEN protein in epithelial endometrial cells that resulted in the development of endometrial lesions. We also validated this technique for gene edition in other important endometrial driver genes such as p53 or Fbxw7. By co-targeting LoxP sites of mT/mG reporter mice and Pten, we demonstrate the generation of differentially edited cell populations that may be a useful tool to model tumoral heterogeneity. Moreover, the combination of CRISPR/Cas9 technology in mT/mG dual-reporter mice and light-sheet microscopy represents an interesting approach for in vivo cancer cell tracing. This methodology opens a new door for future rapid, flexible, customizable and multiplexable in vivo modeling of endometrial cancer.
Use of a MAIT activating ligand, 5-OP-RU, as a mucosal adjuvant in a murine model of Vibrio cholerae O1 vaccination
Authors: Jensen Owen , Trivedi Shubhanshi , Li Kelin , Aubé Jeffrey , Hale J. Scott , Ryan Edward T. , Leung Daniel T. 
Published: June 2022

AbstractBackgroundMucosal-associated invariant T (MAIT) cells are innate-like T cells enriched in the mucosa with capacity for B cell help. We hypothesize that targeting MAIT cells, using a MAIT activating ligand as an adjuvant, could improve mucosal vaccine responses to bacterial pathogens.MethodsWe utilized murine models of Vibrio cholerae vaccination to test the adjuvant potential of the MAIT activating ligand, 5-(2-oxopropylideneamino)-6-D-ribitylaminouracil (5-OP-RU). We measured V. cholerae-specific antibody and antibody-secreting cell responses, and used flow cytometry to examine MAIT cell and B cell phenotype, in blood, bronchoalveolar lavage fluid (BALF), and mucosal tissues, following intranasal vaccination with live V. cholerae O1 or a V. cholerae O1 polysaccharide conjugate vaccine.ResultsWe report significant expansion of MAIT cells in the lungs of 5-OP-RU treated mice, and increases in BALF V. cholerae O-specific-polysaccharide IgG responses in our conjugate vaccine model adjuvanted with low-dose 5-OP-RU. No significant differences in humoral responses were found in our live V. cholerae vaccination model.ConclusionsUsing a murine model, we demonstrate the potential, as well as the limitations, of targeting MAIT cells to improve antibody responses to a mucosal cholera vaccine. Our study highlights the need for future research optimizing MAIT cell targeting for improving mucosal vaccines.One Sentence SummaryTargeting mucosal-associated invariant T (MAIT) cells with a mucosal adjuvant in an intranasal cholera vaccine model resulted in significant expansion of lung MAIT cells, but limited improvements in cholera-specific antibody responses.
Exploration, representation and rationalization of the conformational phase-space of N-glycans
Authors: Grothaus Isabell Louise , Bussi Giovanni , Ciacchi Lucio Colombi 
Published: June 2022

AbstractDespite their fundamental biological relevance, structure-property relationships inN-glycans are fundamentally lacking, and their highly multidimensional compositional and conformational phase-spaces remain largely unexplored. The torsional flexibility of the glycosidic linkages and the ring dynamics result in wide, rugged free-energy landscapes that are difficult to sample in molecular dynamics simulations. We show that a novel enhanced-sampling scheme combining replica-exchange with solute and collective-variable tempering, enabling transitions over all relevant energy barriers, delivers converged distributions of solvatedN-glycan conformers. Several dimensionality-reduction algorithms are compared and employed to generate conformational free-energy maps in two-dimensions. Together with an originally developed conformation-based nomenclature scheme that uniquely identify glycan conformers, our modelling procedure is applied to reveal the effect of chemical substitutions on the conformational ensemble of selected high-mannose-type and complex glycans. Moreover, the structure-prediction capabilities of two commonly used glycan force fields are assessed via the theoretical prediction of experimentally available NMR J-coupling constants. The results confirm the key role of especiallyωand ψ torsion angles in discriminating between different conformational states, and suggest an intriguing correlation between the torsional and ring-puckering degrees of freedom that may be biologically relevant.Abstract Figure
eDNA-stimulated cell dispersion fromCaulobacter crescentusbiofilms upon oxygen limitation is dependent on a toxin-antitoxin system
Authors: Berne Cecile , Zappa Sébastien , Brun Yves V. 
Published: June 2022

ABSTRACTIn their natural environment, most bacteria preferentially live as complex surface-attached multicellular colonies called biofilms. Biofilms begin with a few cells adhering to a surface, where they multiply to form a mature colony. When conditions deteriorate, cells can leave the biofilm. This dispersion is thought to be an important process that modifies the overall biofilm architecture and that promotes colonization of new environments. InCaulobacter crescentusbiofilms, extracellular DNA (eDNA) is released upon cell death and prevents newborn cells from joining the established biofilm. Thus, eDNA promotes the dispersal of newborn cells and the subsequent colonization of new environments. These observations suggest that eDNA is a cue for sensing detrimental environmental conditions in the biofilm. Here we show that the toxin-antitoxin ParDE4stimulates cell death in areas of a biofilm with decreased O2availability. In conditions where O2availability is low, eDNA concentration is correlated with cell death. Cell dispersal away from biofilms is decreased whenparDE4is deleted, probably due to the lower local eDNA concentration. Expression ofparDE4is positively regulated by O2and the expression of this operon is decreased in biofilms where O2availability is low. Thus, PCD by an O2-regulated toxin-antitoxin system stimulates dispersal away from areas of a biofilm with decreased O2availability and favors colonization of a new, more hospitable environment.
Making a pathogen? Evaluating the impact of protist predation on the evolution of virulence inSerratia marcescens
Authors: Hopkins Heather A. , Lopezguerra Christian , Lau Meng-Jia , Raymann Kasie 
Published: June 2022

AbstractOpportunistic pathogens are environmental microbes that are generally harmless and only occasionally cause disease. Unlike obligate pathogens, the growth and survival of opportunistic pathogens does not rely on host infection or transmission. Their versatile lifestyles make it challenging to decipher how and why virulence has evolved in opportunistic pathogens. The Coincidental Evolution Hypothesis (CEH) postulates that virulence results from exaptation or pleiotropy, i.e., traits evolved for adaptation to living in one environment that have a different function in another. In particular, adaptation to avoid or survive protist predation has been suggested to contribute to the evolution of bacterial virulence (the training grounds hypothesis). Here we used experimental evolution to determine how the selective pressure imposed by a protist predator impacts the virulence and fitness of a ubiquitous environmental opportunistic bacterial pathogen that has acquired multi-drug resistance:Serratia marcescens. To this aim, we evolvedS. marcescensin the presence or absence of generalist protist predator,Tetrahymena thermophila. After 60 days of evolution, we evaluated genotypic and phenotypic changes by comparing evolvedS. marcescensto the ancestral strain. Whole genome shotgun (WGS) sequencing of the entire evolved populations and individual isolates revealed numerous cases of parallel evolution, many more than statistically expected by chance, in genes associated with virulence. Our phenotypic assays suggested that evolution in the presence of a predator maintained virulence, whereas evolution in the absence of a predator resulted in attenuated virulence. We also found a significant correlation between virulence, biofilm formation, and grazing resistance. Overall, our results provide evidence that bacterial virulence and virulence related traits are maintained by selective pressures imposed by protist predation.
Evaluation of standard treatments for managing adult Japanese patients with inadequately controlled moderate‐to‐severe atopic dermatitis: Two‐year data from the ADDRESS‐J disease registry
Authors: Katoh Norito , Saeki Hidehisa , Kataoka Yoko , Etoh Takafumi , Teramukai Satoshi , Takagi Hiroki , Fujita Hiroyuki , Ardeleanu Marius , Rizova Elena , Arima Kazuhiko ,   the ADDRESS‐J Investigators
Published: June 2022

AbstractAtopic dermatitis (AD), a chronic relapsing inflammatory skin disease with a high disease burden, is one of the most common dermatological conditions in Japan. Herein, we report the disease profiles and current AD treatment during 2‐year management of Japanese adults with moderate‐to‐severe AD. ADDRESS‐J was a prospective, longitudinal, observational study that evaluated real‐world effectiveness and safety of current AD treatments of adult patients with moderate‐to‐severe AD (Investigator's Global Assessment score 3 or 4) in Japan. The maximum follow‐up period was 2 years. Among 300 patients enrolled, 288 had ≥1 post‐baseline evaluation and were analyzed (mean age, 35.5 years; 60.1% male). Almost all patients (99.7%) received topical therapy; the most commonly used therapy was topical corticosteroids of the second‐highest potency (86.5%) (e.g., 0.1% mometasone furoate) followed by medium‐potency topical corticosteroids (50.3%) (e.g., 0.05% clobetasol butyrate). At month 12 of the study, 10.4% of patients had Investigator's Global Assessment 0/1, similarly at month 24 (10.8%). A total of 132 patients (45.8%) had ≥1 AD flare‐up during the observation period, with the majority of first flares occurring within the first year of the study. Various physician‐ and patient‐reported outcomes improved considerably during the first 3 months of the study, with only minor changes after this time. In this cohort, 16.7% of patients had skin infections requiring treatment; 7.3% had adverse events (AE) potentially related to treatment; 1.7% had serious AE; and 1.0% had treatment discontinuations due to AE. Limitations include missing data at later timepoints and the inclusion criteria limiting generalizability. In summary, this analysis of the ADDRESS‐J study showed that some patients with moderate or severe AD respond to conventional therapies, while others do not. For those with inadequately controlled moderate‐to‐severe AD, the newly emerged systemic agents, such as biologics, may provide a potential strategy for long‐term disease management.
Small farmlands can serve as open habitat for birds in subtropical cities of southern China
Authors: Lee Myung‐Bok 
Published: June 2022

AbstractGreenspace or green cover is one of the major factors affecting urban avian diversity. In China, small farmlands producing vegetables are relatively common in cities. Although these farmlands could be a part of greenspaces, their habitat value for birds is rarely known. I investigated how these farmlands influenced winter bird community in two cities of southern China, using the multivariate latent variable model. I considered three green cover types: crop (mostly vegetables), weed (pioneer spontaneous vegetation), and wood (tree/shrub). I calculated the percentage of each green cover at local (50 m radius circular area) and landscape (500 m radius circular area) scales. Of 30 bird species, the abundance of 13 species decreased as crop cover decreased with increasing woody cover at the local scale, whereas the abundance of only two bird species increased with decreasing crop cover. Habitat preference of species mediated these responses, leading to a strong association between the open habit trait and the negative responses. Open habitat bird species richness was also negatively affected by decreasing local crop cover. Tree/shrub bird species richness showed a positive response to decreasing crop cover but the explanatory power of the local scale model was low. Variables at the landscape scale were rarely associated with species abundance or richness. These results suggest that small vegetable‐dominant farmlands can serve as open habitat for birds in urban areas where greenspace largely consists of tree/shrub vegetation. The results also indicate that urban bird diversity is more influenced by local habitat features than landscape‐scale features.