bluuebunny/crossref_metadata_2025_split huggingface dataset:
Nano3P-seq: transcriptome-wide analysis of gene expression and tail dynamics using end-capture nanopore cDNA sequencing
Authors: Begik Oguzhan , Diensthuber Gregor , Liu Huanle , Delgado-Tejedor Anna , Kontur Cassandra , Niazi Adnan Muhammad , Valen Eivind , Giraldez Antonio J. , Beaudoin Jean-Denis , Mattick John S. , Novoa Eva Maria 
Published: September 2021

AbstractRNA polyadenylation plays a central role in RNA maturation, fate, and stability. In response to developmental cues, polyA tail lengths can vary, affecting the translation efficiency and stability of mRNAs. Here, we develop Nanopore 3’ end-capture sequencing (Nano3P-seq), a novel method that relies on nanopore cDNA sequencing to simultaneously quantify RNA abundance, tail composition and tail length dynamics at per-read resolution. By employing a template switching-based sequencing protocol, Nano3P-seq can sequence any given RNA molecule from its 3’ end, regardless of its polyadenylation status, without the need for PCR amplification or ligation of RNA adapters. We demonstrate that Nano3P-seq captures a wide diversity of RNA biotypes, providing quantitative estimates of RNA abundance and tail lengths in mRNA, lncRNA, sn/snoRNA, scaRNA, and rRNA molecules. We find that, in addition to mRNA and lncRNA, polyA tails can be identified in 16S mitochondrial rRNA in both mouse and zebrafish models. Moreover, we show that mRNA tail lengths are dynamically regulated during vertebrate embryogenesis at an isoform-specific level, correlating with mRNA decay. Finally, we identify non-A bases within polyA tails of various lengths and reveal their distribution during vertebrate embryogenesis. Overall, Nano3P-seq is a simple and robust method for accurately estimating transcript levels, tail lengths, and tail composition heterogeneity in individual reads, with minimal library preparation biases, both in the coding and non-coding transcriptome.
A curcumin direct protein (DiPro) biosensor for cell-free prototyping
Authors: Lesniewska Agata , Griffin Guy , Freemont Paul S , Polizzi Karen M , Moore Simon J 
Published: September 2021

AbstractIn synthetic biology, biosensors are routinely coupled to a gene expression cascade for detecting small molecules and physical signals. We posit that an alternative direct protein (DiPro) biosensor mechanism, could provide a new opportunity for rapid detection of specific chemicals. Herein, we reveal a fluorescent curcumin DiPro biosensor, based on the Escherichia coli double bond reductase (EcCurA) as a detection system. We characterise the EcCurA DiPro biosensor and propose enhanced curcumin fluorescence is generated through π-π stacking between protein and ligand. Using a cell-free synthetic biology approach, we use the EcCurA DiPro biosensor to fine tune 10 reaction parameters (cofactor, substrate, and enzyme levels) for cell-free biosynthesis, assisted through acoustic liquid handling robotics. Overall, we increase EcCurA-curcumin fluorescence by 80-fold. We speculate that a generic DiPro biosensor fluorescence mechanism can be further exploited for a wider range of chemicals that share intrinsic fluorescence and have a suitable binding protein.
Sequential rescue and repair of stalled and damaged ribosome by bacterial PrfH and RtcB
Authors: Tian Yannan , Zeng Fuxing , Raybarman Adrika , Carruthers Amy , Li Qingrong , Fatma Shirin , Huang Raven H. 
Published: September 2021

AbstractIn bacteria, rescue of stalled ribosomes due to 3’-truncated mRNAs is carried out by the ubiquitous trans-translation system as well as alternative ribosome-rescue factors such as ArfA and ArfB. It is unclear, however, how the stalled ribosomes caused by ribosomal damages are rescued. Here, we report that a bacterial system composed of PrfH and RtcB not only rescues a stalled ribosome resulting from a specific damage in the decoding center but also repairs the damage afterwards. Peptide release assays reveal that PrfH is only active with the damaged ribosome, but not with the intact one. A 2.55-angstrom cryo-EM structure of PrfH in complex with the damaged 70S ribosome provides molecular insight into specific recognition of the damage site by PrfH. RNA repair assays demonstrate that PrfH-coupled RtcB efficiently repairs the damaged 30S ribosomal subunit, but not the damaged tRNAs. Thus, our studies have uncovered a biological operation by a pair of bacterial enzymes, aiming to reverse the potentially lethal damage inflicted by an invading ribotoxin for cell survival.
Identification of touch neurons underlying dopaminergic pleasurable touch and sexual receptivity
Authors: Elias Leah J. , Schaffler Melanie , Succi Isabella , Foster William , Gradwell Mark , Bohic Manon , Ejoh Lindsay , Abraira Victoria , Abdus-Saboor Ishmail 
Published: September 2021

AbstractPleasurable touch during social behavior is the key to building familial bonds and meaningful connections. One form of social touch occurs during sexual encounters. Although sexual behavior is initiated in part by touch, and touch is ongoing throughout copulation, the identity and role of sensory neurons that transduce sexual touch remain unknown. A population of sensory neurons labeled by the G-protein coupled receptor Mrgprb4 detect stroking touch in mice, however, these neurons have never been implicated in any natural social behaviors. Here, we study the social relevance of Mrgprb4-lineage neurons by genetically engineering mice to allow activation or ablation of this population and reveal that these neurons are required for sexual receptivity and sufficient to induce dopamine release in the brain. Even in social isolation, optogenetic stimulation of Mrgprb4-lineage neurons through the back skin is sufficient to induce a conditioned place preference and a striking dorsiflexion resembling the lordotic copulatory posture in females. In the absence of Mrgprb4-lineage neurons, female mice no longer find male mounts rewarding: sexual receptivity is supplanted by aggression and a coincident decline in dopaminergic release in the mesolimbic reward pathway. In addition to sexual behavior, Mrgprb4-lineage neurons are also required for social postures induced by female-to-female back touch. Together, these findings establish that Mrgprb4-lineage neurons are the first neurons of a skin-to-brain circuit encoding the rewarding quality of social touch.
The 22q11.2 region regulates presynaptic gene-products linked to schizophrenia
Authors: Nehme Ralda , Pietiläinen Olli , Artomov Mykyta , Tegtmeyer Matthew , Bell Christina , Ganna Andrea , Singh Tarjinder , Trehan Aditi , Valakh Vera , Sherwood John , Manning Danielle , Peirent Emily , Malik Rhea , Guss Ellen J. , Hawes Derek , Beccard Amanda , Bara Anne M. , Hazelbaker Dane Z. , Zuccaro Emanuela , Genovese Giulio , Loboda Alexander A , Neumann Anna , Lilliehook Christina , Kuismin Outi , Hamalainen Eija , Kurki Mitja , Hultman Christina M. , Kähler Anna K. , Paulo Joao A. , Madison Jon , Cohen Bruce , McPhie Donna , Adolfsson Rolf , Perlis Roy , Dolmetsch Ricardo , Farhi Samouil , McCarroll Steven , Hyman Steven , Neale Ben , Barrett Lindy E. , Harper Wade , Palotie Aarno , Daly Mark , Eggan Kevin 
Published: September 2021

AbstractTo study how the 22q11.2 deletion predisposes to psychiatric disease, we generated induced pluripotent stem cells from deletion carriers and controls, as well as utilized CRISPR/Cas9 to introduce the heterozygous deletion into a control cell line. Upon differentiation into neural progenitor cells, we found the deletion acted in trans to alter the abundance of transcripts associated with risk for neurodevelopmental disorders including Autism Spectrum Disorder. In more differentiated excitatory neurons, altered transcripts encoded presynaptic factors and were associated with genetic risk for schizophrenia, including common (per-SNP heritability p (τc)= 4.2 x 10-6) and rare, loss of function variants (p = 1.29×10-12). These findings suggest a potential relationship between cellular states, developmental windows and susceptibility to psychiatric conditions with different ages of onset. To understand how the deletion contributed to these observed changes in gene expression, we developed and applied PPItools, which identifies the minimal protein-protein interaction network that best explains an observed set of gene expression alterations. We found that many of the genes in the 22q11.2 interval interact in presynaptic, proteasome, and JUN/FOS transcriptional pathways that underlie the broader alterations in psychiatric risk gene expression we identified. Our findings suggest that the 22q11.2 deletion impacts genes and pathways that may converge with risk loci implicated by psychiatric genetic studies to influence disease manifestation in each deletion carrier.
Combinatorial G x G x E CRISPR screening and functional analysis highlights SLC25A39 in mitochondrial GSH transport
Authors: Shi Xiaojian , Reinstadler Bryn , Shah Hardik , To Tsz-Leung , Byrne Katie , Summer Luanna , Calvo Sarah E. , Goldberger Olga , Doench John G. , Mootha Vamsi K. , Shen Hongying 
Published: September 2021

AbstractThe SLC25 carrier family consists of 53 transporters that shuttle nutrients and co-factors across mitochondrial membranes1-3. The family is highly redundant and their transport activities coupled to metabolic state. Here, we introduce a pooled, dual CRISPR screening strategy that knocks out pairs of transporters in four metabolic states — glucose, galactose, OXPHOS inhibition, and absence of pyruvate — designed to unmask the inter-dependence of these genes. In total, we screened 63 genes in four metabolic states, corresponding to 2016 single and pair-wise genetic perturbations. We recovered 19 gene-by-environment (GxE) interactions and 9 gene-by-gene (GxG) interactions. One GxE interaction hit illustrated that the fitness defect in the mitochondrial folate carrier (SLC25A32) KO cells was genetically buffered in galactose due to a lack of substrate in de novo purine biosynthesis. Another GxE interaction hit revealed non-equivalence of the paralogous ATP/ADP exchangers (ANTs) with ANT2 specifically required during OXPHOS inhibition. GxG analysis highlighted a buffering interaction between the iron transporter SLC25A37 and the poorly characterized SLC25A39. Mitochondrial metabolite profiling, organelle transport assays, and structure-guided mutagenesis suggests SLC25A39 is critical for mitochondrial glutathione (GSH) transport. Our work underscores the importance of systemetically investigating family-wide genetic interactions between mitochondrial transporters across many metabolic environments.
Imaging Flow Cytometry reveals a dual role for exopolysaccharides in biofilms: To promote self-adhesion while repelling non-self-community members
Authors: Maan Harsh , Povolotsky Tatyana L. , Porat Ziv , Kolodkin-Gal Ilana 
Published: September 2021

AbstractIn nature, bacteria are establishing differentiated communities referred to as biofilms. These multicellular communities are held together by self-produced polymers that allow the community members to adhere to the surface as well as to neighbor bacteria. Here, we report that exopolysaccharides prevent Bacillus subtilis from co-aggregating with a distantly related bacterium Bacillus mycoides, while maintaining their role in promoting self-adhesion and co-adhesion with phylogenetically related bacterium, Bacillus atrophaeus. The defensive role of the exopolysaccharides is due to the specific regulation of bacillaene. Single cell analysis of biofilm and free-living bacterial cells using imaging flow cytometry confirmed a specific role for the exopolysaccharides in microbial competition repelling B. mycoides. Unlike exopolysaccharides, the matrix protein TasA induced bacillaene but inhibited the expression of the biosynthetic clusters for surfactin, and therefore its overall effect on microbial competition during floating biofilm formation was neutral. Thus, the exopolysaccharides provide a dual fitness advantage for biofilm-forming cells, as it acts to promote co-aggregation of related species, as well as, a secreted cue for chemical interference with non-compatible partners. These results experimentally demonstrate a general assembly principle of complex communities and provides an appealing explanation for how closely related species are favored during community assembly. Furthermore, the differential regulation of surfactin and bacillaene by the extracellular matrix may explain the spatio-temporal gradients of antibiotic production within biofilms.
The short term High Fat Diet sparks gut microbiota associated metabolic syndrome pathogenesis and impact of Amoxicillin intervention in mice
Authors: Kumar Suresh , Saini Vikram , Raj V. Samuel 
Published: September 2021

AbstractBackgroundKnowledge about alteration in gut microbiota on a high-fat diet (HFD) and common antibiotics for a short period is little known. The study was undertaken to evaluate the changes in the microbiota profile in HFD fed mice followed by amoxicillin intervention.ResultsFor two weeks, the mice were fed with HFD followed by amoxicillin for one week. Animals were evaluated for haemato-biochemical, histopathological and 16S rRNA sequencing followed by bioinformatics analysis to know any changes in gut microbiota ecology. Amoxicillin treatment for a short duration had marked the remarkable remodelling of gut microbiota that directly linked to beneficial impact by countering the pathogenesis of the metabolic syndrome besides overgrowth of particular opportunistic pathogens of gut inflammatory diseases. Amoxicillin treatment significantly decreased blood glucose and a slight elevation in cholesterol levels on blood biochemistry analysis. No marked pathological changes were observed in HFD fed mice.ConclusionsOur results suggest that Amoxicillin treatment had a beneficial influence on the obese related metabolic syndrome with the risk of hypercholesterolemia and some gut-resistant intestinal pathobionts.
A different mechanism of C-type inactivation in the Kv-like KcsA mutant E71V
Authors: Rohaim Ahmed , Vermeulen Bram J.A. , Li Jing , Kümmerer Felix , Napoli Federico , Blachowicz Lydia , Medeiros-Silva João , Roux Benoit , Weingarth Markus 
Published: September 2021

ABSTRACTA large class of K+ channels display a time-dependent phenomenon called C-type inactivation whereby prolonged activation by an external stimulus leads to a non-conductive conformation of the selectivity filter. C-type inactivation is of great physiological importance particularly in voltage-activated K+ channels (Kv), affecting the firing patterns of neurons and shaping cardiac action potentials. While understanding the molecular basis of inactivation has a direct impact on human health, its structural basis remains unresolved. Knowledge about C-type inactivation has been largely deduced from the pH-activated bacterial K+ channel KcsA, whose selectivity filter under inactivating conditions adopts a constricted conformation at the level of the central glycine (TTVGYGD) that is stabilized by tightly bound water molecules. However, C-type inactivation is highly sensitive to the molecular environment surrounding the selectivity filter in the pore domain, which is different in Kv channels than in the model KcsA. In particular, a glutamic acid residue at position 71 along the pore helix in KcsA is consistently substituted by a nonpolar valine in most Kv channels, suggesting that this side chain is an important molecular determinant of function. Here, a combination of X-ray crystallography, solid-state NMR and molecular dynamics simulations of the E71V mutant of KcsA is undertaken to explore the features associated with this Kv-like construct. In both X-ray and ssNMR data, it is observed that the filter of the Kv-like KcsA mutant does not adopt the familiar constricted conformation under inactivating conditions. Rather, the filter appears to adopt a conformation that is slightly narrowed and rigidified over its entire length. No structural inactivation water molecules are present. On the other hand, molecular dynamics simulations indicate that the familiar constricted conformation can nonetheless be stably established in the mutant channel. Together, these findings suggest that the Kv-like E71V mutation in the KcsA channel may be associated with different modes of C-type inactivation, showing that distinct selectivity filter environments entail distinct C-type inactivation mechanisms.
Deleterious Mutations Accumulate Faster in Allopolyploid than Diploid Cotton (Gossypium) and Unequally Between Subgenomes
Authors: Conover Justin L. , Wendel Jonathan F. 
Published: September 2021

AbstractWhole genome duplication (polyploidization) is among the most dramatic mutational processes in nature, so understanding how natural selection differs in polyploids relative to diploids is an important goal. Population genetics theory predicts that recessive deleterious mutations accumulate faster in allopolyploids than diploids due to the masking effect of redundant gene copies, but this prediction is hitherto unconfirmed. Here, we use the cotton genus (Gossypium), which contains seven allopolyploids derived from a single polyploidization event 1-2 million years ago, to investigate deleterious mutation accumulation. We use two methods of identifying deleterious mutations at the nucleotide and amino acid level, along with whole-genome resequencing of 43 individuals spanning six allopolyploid species and their two diploid progenitors, to demonstrate that deleterious mutations accumulate faster in allopolyploids than in their diploid progenitors. We find that, unlike what would be expected under models of demographic changes alone, strongly deleterious mutations show the biggest difference between ploidy levels, and this effect diminishes for moderately and mildly deleterious mutations. We further show that the proportion of nonsynonymous mutations that are deleterious differs between the two co-resident subgenomes in the allopolyploids, suggesting that homoeologous masking acts unequally between subgenomes. Our results provide a genome-wide perspective on classic notions of the significance of gene duplication that likely are broadly applicable to allopolyploids, with implications for our understanding of the evolutionary fate of deleterious mutations. Finally, we note that some measures of selection (e.g. dN/dS, πN/πS) may be biased when species of different ploidy levels are compared.
A phylogenetically-restricted essential cell cycle progression factor in the human pathogen Candida albicans
Authors: Jaitly Priya , Legrand Mélanie , Das Abhijit , Patel Tejas , Chauvel Murielle , d’Enfert Christophe , Sanyal Kaustuv 
Published: September 2021

AbstractChromosomal instability in fungal pathogens caused by cell division errors is associated with antifungal drug resistance. To identify mechanisms underlying such instability and to uncover new potential antifungal targets, we conducted an overexpression screen monitoring chromosomal stability in the human fungal pathogen Candida albicans. Analysis of ~1000 genes uncovered six chromosomal stability (CSA) genes, five of which are related to cell division genes in other organisms. The sixth gene, CSA6, is selectively present in the CUG-Ser clade species that includes C. albicans and other human fungal pathogens. The protein encoded by CSA6 localizes to the spindle pole bodies, is required for exit from mitosis, and induces a checkpoint-dependent metaphase arrest upon overexpression. Together, Csa6 defines an essential CUG-Ser fungal clade-specific cell cycle progression factor, highlighting the existence of phylogenetically-restricted cell division genes which may serve as potential unique therapeutic targets.TeaserCsa6 is essential for mitotic progression and mitotic exit in the human fungal pathogen Candida albicans.
RIG-I uses its intrinsically disordered CARDs-Helicase linker in RNA proofreading functions
Authors: Schweibenz Brandon D. , Devarkar Swapnil C. , Solotchi Mihai , Zhang Jie , Pascal Bruce D. , Griffin Patrick R. , Patel Smita S. 
Published: September 2021

AbstractThe innate immune receptor RIG-I provides the first line of defense against viral infections. Viral RNAs are recognized by the C-terminal domain (CTD) of RIG-I, but the RNA must engage the helicase domain to release the signaling domain CARDs from their autoinhibitory CARD2:Hel2i interactions. Because the helicase lacks RNA specificity, there must be mechanisms to proofread RNAs entering the helicase domain. Although such mechanisms are crucial in preventing aberrant immune responses by non-specific RNAs, they remain largely unknown. This study reveals a previously unknown proofreading mechanism that RIG-I uses to ensure the helicase engages RNAs chosen explicitly by the CTD. A crucial part of this mechanism involves the intrinsically disordered CARDs-Helicase Linker (CHL), which uses its negatively charged regions to electrostatically antagonize incoming RNAs. In addition to RNA gating, CHL is essential in stabilizing the CARD2:Hel2i interface. The CHL and CARD2:Hel2i interface work together, establishing a tunable gating mechanism that allows CTD-chosen RNAs to bind into the helicase while blocking non-specific RNAs. With its critical regulatory functions, CHL represents a novel target for RIG-I-based therapeutics.
Molecular mechanisms associated with clustered lesion-induced impairment of 8-oxoG recognition by the human glycosylase OGG1
Authors: Jiang Tao , Monari Antonio , Dumont Elise , Bignon Emmanuelle 
Published: September 2021

AbstractThe 8-oxo-7,8-dihydroguanine, referred to as 8-oxoG, is a highly mutagenic DNA lesion that can provoke the appearance of mismatches if it escapes the DNA Damage Response. The specific recognition of its structural signature by the hOGG1 glycosylase is the first step along the Base Excision Repair pathway, that ensures the integrity of the genome by preventing the emergence of mutations. 8-oxoG formation, structural features and repair have been the matter of extensive research and more recently this active field of research expended to the more complicated case of 8-oxoG within clustered lesions. Indeed, the presence of a second lesion within 1 or 2 helix turns can dramatically impact the repair yields of 8-oxoG by glycosylases. In this work, we use μs-range molecular dynamics simulations and machine learning-based post-analysis to explore the molecular mechanisms associated with the recognition of 8-oxoG by hOGG1 when embedded in a multiple lesions site with a mismatch in 5’ or 3’. We delineate the stiffening of the DNA-protein interactions upon the presence of the mismatches, and rationalize the much lower repair yields reported with a 5’ mismatch by describing the perturbation of 8-oxoG structural features upon addition of an adjacent lesion.