bluuebunny/crossref_metadata_2025_split huggingface dataset:
Dissemination and pathogenesis of toxigenic Clostridium perfringens strains linked to neonatal intensive care units and Necrotising Enterocolitis
Authors: Kiu Raymond , Shaw Alex , Sim Kathleen , Bedwell Harley , Cornwell Emma , Pickard Derek , Belteki Gusztav , Malsom Jennifer , Philips Sarah , Young Gregory R , Schofield Zoe , Alcon-Giner Cristina , Berrington Janet E , Stewart Christopher , Dougan Gordon , Clarke Paul , Douce Gillian , Simon Kroll J , Hall Lindsay J 
Published: August 2021

ABSTRACTBackgroundClostridium perfringens is an anaerobic toxin-producing bacterium that has long been associated with intestinal diseases, particularly in neonatal humans and animals. More recently, infant gut microbiome studies have suggested an important link between C. perfringens and the devastating preterm-associated disease Necrotising Enterocolitis (NEC), but in-depth studies on this pathogen (genomics and mechanistic) are lacking.Methods/MaterialsWe isolated and whole-genome sequenced 274 infant-associated C. perfringens isolates from 5 hospitals across the UK between 2011-2016 (including longitudinal samples from 31 individuals). We performed in-depth genomic analyses, phenotypically characterised pathogenic traits of 10 strains (including 4 C. perfringens from NEC patients) and established a novel oral-challenge C57BL/6 mouse infection model for microbe-host studies.ResultsPore-forming toxin encoding genes pfoA and cpb2 were enriched within hypervirulent lineages that exclusively consisted of C. perfringens-associated NEC (CPA-NEC) strains, in addition to overabundance of colonisation factors. Importantly, we identified a circulating C. perfringens variant, eventually linked to a fatal CPA-NEC case. The variant was detected consistently within 6 individuals in two sister hospitals across a 40-day window, demonstrating for the first time the intra- and inter-hospital dissemination of C. perfringens. CPA-NEC isolates were determined phenotypically to be more virulent (linked with overabundance of gene pfoA) than isolates obtained from non-NEC preterm babies. In addition, two pfoA-positive CPA-NEC C. perfringens strains were confirmed to induce clinical inflammatory tissue lesions in vivo.ConclusionsHypervirulent lineages are linked to CPA-NEC, potentially due to the production of pore-forming toxins, coupled with higher metabolic, transmission, and pathogenic capacities. These studies indicate C. perfringens is an important bacterial pathogen in preterm infants and highlights the requirement for further investigation into development of intervention and therapeutic strategies.
Pharmacological and genetic activation of cAMP synthesis disrupts cholesterol utilization in Mycobacterium tuberculosis
Authors: Wilburn Kaley M. , Montague Christine R. , Qin Bo , Woods Ashley K. , Love Melissa S. , McNamara Case W. , Schultz Peter G. , Southard Teresa L. , Huang Lu , Petrassi H. Michael , VanderVen Brian C. 
Published: August 2021

AbstractThere is a growing appreciation for the idea that bacterial utilization of host-derived lipids, including cholesterol, supports Mycobacterium tuberculosis (Mtb) pathogenesis. This has generated interest in identifying novel antibiotics that can disrupt cholesterol utilization by Mtb in vivo. Here we identify a novel small molecule agonist (V-59) of the Mtb adenylyl cyclase Rv1625c, which stimulates 3’, 5’-cyclic adenosine monophosphate (cAMP) synthesis and inhibits cholesterol utilization by Mtb. Similarly, using a complementary genetic approach that induces bacterial cAMP synthesis independent of Rv1625c, we demonstrate that inducing cAMP synthesis is sufficient to inhibit cholesterol utilization in Mtb. Although the physiological roles of individual adenylyl cyclase enzymes in Mtb are largely unknown, here we demonstrate that the transmembrane region of Rv1625c is required for cholesterol metabolism. Finally, in this work the pharmacokinetic properties of Rv1625c agonists are optimized, producing an orally-available Rv1625c agonist that impairs Mtb pathogenesis in infected mice. Collectively, this work demonstrates a novel role for Rv1625c and cAMP signaling in controlling cholesterol metabolism in Mtb and establishes that cAMP signaling can be pharmacologically manipulated for the development of new antibiotic strategies.Author SummaryThe recalcitrance of Mycobacterium tuberculosis (Mtb) to conventional antibiotics has created a need to identify novel pharmacological mechanisms to inhibit Mtb pathogenesis. There is a growing understanding of the metabolic adaptations Mtb adopts during infection to support its survival and pathogenesis. This has generated interest in identifying small molecule compounds that effectively inhibit these in vivo metabolic adaptations, while overcoming challenges like poor pharmacokinetic properties or redundancy in target pathways. The Mtb cholesterol utilization pathway has repeatedly been speculated to be a desirable antibiotic target, but compounds that successfully inhibit this complex pathway and are suitable for use in vivo are lacking. Here, we establish that stimulating cAMP synthesis in Mtb is a mechanism that is sufficient to block cholesterol utilization by the bacterium, preventing the release of key metabolic intermediates that are derived from breakdown of the cholesterol molecule. For the first time, we identify small molecule agonists of the Mtb adenylyl cyclase Rv1625c that have promising pharmacological properties and are suitable for use during in vivo studies. These Rv1625c agonists increase cAMP synthesis, inhibit cholesterol utilization by Mtb, and disrupt Mtb pathogenesis in mouse models of chronic infection.
High-efficiency CRISPR gene editing in C. elegans using Cas9 integrated into the genome
Authors: Schwartz Matthew L. , Davis M. Wayne , Rich Matthew S. , Jorgensen Erik M. 
Published: August 2021

AbstractGene editing in C. elegans using plasmid-based CRISPR reagents requires microinjection of many animals to produce a single edit. Germline silencing of plasmid-borne Cas9 is a major cause of inefficient editing. Here, we present a set of C. elegans strains that constitutively express Cas9 in the germline from an integrated transgene. These strains markedly improve the success rate for plasmid-based CRISPR edits. For simple GFP insertions, 60 – 100% of injected animals typically produce edited progeny, depending on the target locus. Template-guided editing from an extrachromosomal array is maintained over multiple generations. We have built strains with the Cas9 transgene on multiple chromosomes. Additionally, each Cas9 locus also contains a heatshock-driven Cre recombinase for selectable marker removal and a bright fluorescence marker for easy outcrossing. These integrated Cas9 strains greatly reduce the workload for producing individual genome edits.Author SummaryGermlines have evolved specialized mechanisms to protect themselves from invasions by transposons and viruses, which create barriers to genome editing techniques. For example, transgenes are silenced in the germline of the nematode C. elegans, thereby creating a barrier to CRISPR editing by Cas9. To facilitate gene editing, we built a collection of C. elegans strains in which Cas9 is never silenced. CRISPR is significantly more efficient in these animals, decreasing the effort researchers need to expend to get edited animals. The strains are available in multiple genetic backgrounds, and contain accessory transgenes to simplify downstream genetics. Together, these strains enable efficient, low-cost genome editing in C. elegans.
Genetic screens identify connections between ribosome recycling and nonsense mediated decay
Authors: D’Orazio Karole N. , Lessen Laura N. , Veltri Anthony J. , Neiman Zachary , Pacheco Miguel , Loll-Krippleber Raphael , Brown Grant W. , Green Rachel 
Published: August 2021

AbstractThe decay of messenger RNA with a premature termination codon (PTC) by nonsense mediated decay (NMD) is an important regulatory pathway for eukaryotes and an essential pathway in mammals. NMD is typically triggered by the ribosome terminating at a stop codon that is aberrantly distant from the poly-A tail. Here, we use a fluorescence screen to identify factors involved in NMD inS. cerevisiae. In addition to the known NMD factors, including the entire UPF family (UPF1, UPF2 and UPF3), as well asNMD4andEBS1, we identify factors known to function in post-termination recycling and characterize their contribution to NMD. We then use a series of modified reporter constructs that block both elongating and scanning ribosomes downstream of stop codons and demonstrate that a deficiency in recycling of 80S ribosomes or 40S subunits stabilizes NMD substrates. These observations inS. cerevisiaeexpand on recently reported data in mammals indicating that the 60S recycling factor ABCE1 is important for NMD (1, 2) by showing that increased activities of both elongating and scanning ribosomes (80S or 40S) in the 3’UTR correlate with a loss of NMD.Author SummaryIn this work, we aim to understand the mechanism of targeting mRNAs for decay via the long-studied nonsense mediated decay (NMD) pathway. We demonstrate that efficient large and small subunit ribosome recycling are necessary components of NMD. We go on to provide evidence that either scanning or actively translating ribosomes in the 3’UTR disrupt the decay of NMD targets. Our work highlights the importance of the composition of the 3’UTR in NMD signaling and emphasizes the need for this region to indeed be untranslated for NMD to occur. Exon junction complexes (EJCs) in the 3’UTR are known to induce NMD, however, in the budding yeast system used here, the NMD targets are EJC-free. Therefore, our data support a model in which factors other than EJCs may accumulate in the 3’UTR and provide a signal for NMD.
Selection for non-specific adhesion is a driver of FimH evolution increasing Escherichia coli biofilm capacity
Authors: Yoshida Mari , Thiriet-Rupert Stanislas , Mayer Leonie , Beloin Christophe , Ghigo Jean-Marc 
Published: August 2021

ABSTRACTBacterial interactions with surfaces rely on the coordinated expression and interplay of surface exposed adhesion factors. However, how bacteria dynamically modulate their vast repertoire of adhesins to achieve surface colonization is not yet well-understood. We used experimental evolution and positive selection for improved adhesion to investigate how an initially poorly adherent Escherichia coli strain increased its adhesion capacities to abiotic surfaces. We showed that all identified evolved clones acquired mutations located almost exclusively in the lectin domain of fimH, the gene coding for the α-D-mannose-specific tip adhesin of type 1 fimbriae. While most of these fimH mutants showed reduced mannose- binding ability, they all displayed enhanced binding to abiotic surfaces, indicating a trade-off between FimH-mediated specific and non-specific adhesion properties. Several of the identified mutations were already reported in FimH lectin domain of pathogenic and environmental E. coli, suggesting that, beyond patho-adaptation, FimH microevolution favoring non-specific surface adhesion could constitute a selective advantage for natural E. coli isolates. Consistently, although E. coli deleted for the fim operon still evolves an increased adhesion capacity, mutants selected in the Δfim background are outcompeted by fimH mutants revealing clonal interference for adhesion. Our study therefore provides insights into the plasticity of E. coli adhesion potential and shows that evolution of type 1 fimbriae is a major driver of the adaptation of natural E. coli to colonization.
The sRNA MicC downregulates hilD translation to control the SPI1 T3SS in Salmonella enterica serovar Typhimurium
Authors: Cakar Fatih , Golubeva Yekaterina A. , Vanderpool Carin K. , Slauch James M. 
Published: August 2021

ABSTRACTSalmonella enterica serovar Typhimurium invades the intestinal epithelium and induces inflammatory diarrhea using the Salmonella pathogenicity island 1 (SPI1) type III secretion system (T3SS). Expression of the SPI1 T3SS is controlled by three AraC-like regulators, HilD, HilC and RtsA, which form a feed-forward regulatory loop that leads to activation of hilA, encoding the main transcriptional regulator of the T3SS structural genes. This complex system is affected by numerous regulatory proteins and environmental signals, many of which act at the level of hilD mRNA translation or HilD protein function. Here, we show that the sRNA MicC blocks translation of the hilD mRNA by base pairing near the ribosome binding site. This binding blocks translation but does not induce degradation of the hilD message. Our data indicate that micC is transcriptionally activated by SlyA, and SlyA feeds into the SPI1 regulatory network solely through MicC. Transcription of micC is negatively regulated by the OmpR/EnvZ two-component system, but this regulation is dependent on SlyA. OmpR/EnvZ control SPI1 expression partially through MicC, but also affect expression through other mechanisms. MicC-mediated regulation plays a role during infection, as evidenced by an increase in Salmonella fitness in the intestine in the micC deletion mutant that is dependent on the SPI1 T3SS. These results further elucidate the complex regulatory network controlling SPI1 expression and add to the list of sRNAs that control this primary virulence factor.IMPORTANCEThe Salmonella SPI1 T3SS is the primary virulence factor required for causing intestinal disease and initiating systemic infection. The system is regulated in response to a large variety of environmental and physiological factors such that the T3SS is expressed at only the appropriate time and place in the host during infection. Here we show how the sRNA MicC affects expression of the system. This work adds to our detailed mechanistic studies aimed at a complete understanding of the regulatory circuit.
Reconstruction of phospholipid synthesis by combing in vitro fatty acid synthesis and cell-free gene expression
Authors: Eto Sumie , Matsumura Rumie , Fujimi Mai , Shimane Yasuhiro , Berhanu Samuel , Kasama Takeshi , Kuruma Yutetsu 
Published: August 2021

AbstractPhospholipid synthesis is a fundamental process that promotes cell propagation and, presently, is the most challenging issue in artificial cell research aimed at reconstituting living cells from biomolecules. Here, we constructed a cell-free phospholipid synthesis system that combines in vitro fatty acid synthesis and a cell-free gene expression system that synthesizes acyltransferases for phospholipid synthesis. Fatty acids were synthesized from acetyl-CoA and malonyl-CoA, then continuously converted into phosphatidic acids by the cell-free synthesized acyltransferases. Because the system can avoid the accumulation of synthetic intermediates that suppress the reaction, the yield of phospholipid has significantly improved from previous schemes (up to 400 µM). Additionally, by adding enzymes for recycling CoA, we synthesized phosphatidic acids from acetic acid and bicarbonate as carbon sources. The constructed system is available to express the genes from pathogenic bacteria and to analyze the synthesized phospholipids. By encapsulating our system inside giant vesicles, it would be possible to construct the artificial cells in which the membrane grows and divides sustainably.
Rapid encoding of task regularities in the human hippocampus guides sensorimotor timing
Authors: Polti Ignacio , Nau Matthias , Kaplan Raphael , van Wassenhove Virginie , Doeller Christian F. 
Published: August 2021

AbstractThe brain encodes the statistical regularities of the environment in a task-specific yet flexible and generalizable format. Here, we seek to understand this process by converging two parallel lines of research, one centered on sensorimotor timing, and the other on cognitive mapping in the hippocampal system. By combining functional magnetic resonance imaging (fMRI) with a fastpaced time-to-contact (TTC) estimation task, we found that the hippocampus signaled behavioral feedback received in each trial as well as performance improvements across trials along with reward-processing regions. Critically, it signaled performance improvements independent from the tested intervals, and its activity accounted for the trial-wise regression-to-the-mean biases in TTC estimation. This suggests that the hippocampus supports the rapid encoding of temporal context even on short time scales in a behavior-dependent manner. Our results emphasize the central role of the hippocampus in statistical learning and position it at the core of a brain-wide network updating sensorimotor representations in real time for flexible behavior.
Abundances of transfer RNA modifications and transcriptional levels for tRNA-modifying enzymes are sex-specific in mosquitoes
Authors: Kelley Melissa , Uhran Melissa R. , Herbert Cassandra , Yoshida George , Watts Emmarie , Limbach Patrick A. , Benoit Joshua B. 
Published: August 2021

AbstractAs carriers of multiple human diseases, understanding the mechanisms behind mosquito reproduction may have implications for remediation strategies. Transfer RNA (tRNA) acts as the adapter molecule of amino acids and are key components in protein synthesis and a critical factor in the function of tRNAs is chemical modifications. Here, we provide an assessment of tRNA modifications between sexes for three mosquito species and examine correlated transcript levels underlying key proteins involved in tRNA modification. Thirty-three tRNA modifications were detected among mosquito species and most of these modifications are higher in females compared to males. Analysis of previous male and female RNAseq datasets indicated a similar increase in tRNA modifying enzymes in females, supporting our observed female enrichment of tRNA modifications. Tissues-specific expressional studies revealed high transcript levels for tRNA modifying enzymes in the ovaries for Aedes aegypti, but not male reproductive tissues. These studies suggest that tRNA modifications may be critical to reproduction in mosquitoes, representing a potential novel target for control.
A clinically validated human saliva metatranscriptomic test for global systems biology studies
Authors: Toma Ryan , Cai Ying , Ogundijo Oyetunji , Gline Stephanie , Demusaj Diana , Duval Nathan , Torres Pedro , Camacho Francine , Banavar Guruduth , Vuyisich Momchilo 
Published: August 2021

AbstractWe report here the development of a high throughput, automated, inexpensive, and clinically validated saliva metatranscriptome test that requires less than 100 microliters of saliva. The RNA in the samples is preserved at the time of collection. Samples can then be stored and transported at ambient temperatures for up to 28 days without significantly affecting the test results. Another important feature of the sample preservative is that it inactivates all classes of pathogens, enabling safe transportation globally. Given the unique set of convenience, low cost, safety, and technical performance, this saliva metatranscriptomic test can be integrated into longitudinal, global scale, systems biology studies that will lead to an accelerated development of precision medicine diagnostic and therapeutic tools.Method SummaryThis report describes an important improvement to saliva transcriptome analysis. While current methods are complicated and expensive, the method reported here includes at-home sample collection, global shipping at ambient temperatures and pathogen inactivation at the point of collection; it uses fully automated, clinically validated and licensed laboratory and bioinformatic analyses.
High expression ofVRT2increases the number of rudimentary basal spikelets in wheat
Authors: Backhaus Anna E. , Lister Ashleigh , Tomkins Melissa , Adamski Nikolai M. , Simmonds James , Macaulay Iain , Morris Richard J. , Haerty Wilfried , Uauy Cristobal 
Published: August 2021

AbstractSpikelets are the fundamental building blocks ofPoaceaeinflorescences and their development and branching patterns determine the various inflorescence architectures and grain yield of grasses. In wheat, the central spikelets produce the most and largest grains, while spikelet size gradually decreases acro- and basipetally, giving rise to the characteristic lanceolate shape of wheat spikes. The acropetal gradient correlates with the developmental age of spikelets, however the basal spikelets are developed first and the cause of their small size and rudimentary development is unclear. Here, we adapted G&T-seq, a low-input transcriptomics approach, to characterise gene expression profiles within spatial sections of individual spikes before and after the establishment of the lanceolate shape. We observed larger differences in gene expression profiles between the apical, central and basal sections of a single spike than between any section belonging to consecutive developmental timepoints. We found thatSVPMADS-box transcription factors, includingVRT-A2, are expressed highest in the basal section of the wheat spike and display the opposite expression gradient to flowering E-classSEP1genes. Based on multi-year field trials and transgenic lines, we show that higher expression ofVRT-A2in the basal sections of the spike is associated with increased numbers of rudimentary basal spikelets. Our results, supported by computational modelling, suggest that the delayed transition of basal spikelets from vegetative to floral developmental programmes results in the lanceolate shape of wheat spikes. This study highlights the value of spatially resolved transcriptomics to gain new insights into developmental genetics pathways of grass inflorescences.One sentence summaryLarge transcriptional gradients existwithina wheat spike and are associated with rudimentary basal spikelet development, resulting in the characteristic lanceolate shape of wheat spikes.
Uptake of Fe-fraxetin complexes, an IRT1 independent strategy for iron acquisition in Arabidopsis thaliana
Authors: Robe Kevin , Stassen Max , Chamieh Joseph , Gonzalez Philippe , Hem Sonia , Santoni Véronique , Dubos Christian , Izquierdo Esther 
Published: August 2021

SUMMARYIron (Fe) is a micronutrient essential for plant growth and development. Iron uptake in alkaline soil is a challenge for most plants. In this study, we investigated the role of the catechol coumarins fraxetin and esculetin in plant Fe acquisition and their Fe chelating properties.Mass spectrometry and capillary electrophoresis were used to characterize Fe-coumarin complexes. To understand the role of these complexes, genetic, molecular and biochemical approaches were deployed.We demonstrated that catechol coumarins are taken up by Arabidopsis thaliana root via an ATP dependent mechanism and that plants defective in IRT1 activity (the main high affinity Fe importer) or bHLH121 (a key regulator of Fe deficiency responses) can be complemented by exogenous supply of fraxetin and to a lesser extent of esculetin. We also showed that Fe and fraxetin can form stable complexes at neutral to alkaline pH that can be taken up by the plant.Overall, these results indicate that at high pH, fraxetin can improve Fe nutrition by directly transporting Fe(III) into the root, circumventing the FRO2/IRT1 system, in a similar way as phytosiderophores do in grasses. This strategy may explain how non-grass species can thrive in alkaline soils.