bluuebunny/crossref_metadata_2025_split huggingface dataset:
Re-engineering of TNFα-NF-κB signalling dynamics in cancer cells using pathogenicE. colieffectors
Authors: Zhong Qiyun , Butera Francesca , Frankel Gad , Bakal Chris 
Published: March 2023

AbstractRe-engineering NF-κB signalling towards enhancing beneficial outcomes such as tumour cell elimination, while minimising inflammatory damage, is a potential therapeutic avenue. In this study, we explored the ability of bacterial effectors injected into host cells by the type III secretion system to regulate NF-κB translocation dynamics. We used the enteropathogenicEscherichia colieffectors Tir (NF-κB activator), NleC (NF-κB protease) and NleE (TAB2/3 methyltransferase), to manipulate NF-κB translocation and cancer cell survival. We discovered that while these effectors have either limited or no cytotoxicity alone, they greatly enhanced caspase-8-dependent pancreatic cancer cell death in the presence of TNFα. Single cell analysis revealed that the sub-population of cells showing high NF-κB activation is less susceptible to cell death caused by NleC or NleE but instead is more susceptible to Tir. A combination of Tir, NleE and TNFα eliminated 95% cancer cells with limited NF-κB activation, potentially due to NleE-dependent blockage of the immediate pro-survival NF-κB activation without inhibiting Tir’s long-term NF-κB activation that promotes cell death. This work demonstrates that effector combinations could be used to re-engineer stress responses towards favourable outcomes.
Distinct horizontal transfer mechanisms for type I and type V CRISPR-associated transposons
Authors: Hu Kuang , Chou Chia-Wei , Wilke Claus O. , Finkelstein Ilya J. 
Published: March 2023

AbstractCRISPR-associated transposons (CASTs) co-opt CRISPR-Cas proteins and Tn7-family transposons for RNA-guided vertical and horizontal transmission. CASTs encode minimal CRISPR arrays but can’t acquire new spacers. Here, we show that CASTs instead co-opt defense-associated CRISPR arrays for horizontal transmission. A bioinformatic analysis shows that all CAST sub-types co-occur with defense-associated CRISPR-Cas systems. Using anE. coliquantitative transposition assay, we show that CASTs use CRISPR RNAs (crRNAs) from these defense systems for horizontal gene transfer. A high-resolution structure of the type I-F CAST-Cascade in complex with a type III-B crRNA reveals that Cas6 recognizes direct repeats via sequence-independentπ − πinteractions. In addition to using heterologous CRISPR arrays, type V CASTs can also transpose via a crRNA-independent unguided mechanism, even when the S15 co-factor is over-expressed. Over-expressing S15 and the trans-activating CRISPR RNA (tracrRNA) or a single guide RNA (sgRNA) reduces, but does not abrogate, off-target integration for type V CASTs. Exploiting new spacers in defense-associated CRISPR arrays explains how CASTs horizontally transfer to new hosts. More broadly, this work will guide further efforts to engineer the activity and specificity of CASTs for gene editing applications.
JAMS - A framework for the taxonomic and functional exploration of microbiological genomic data
Authors: McCulloch John A. , Badger Jonathan H. , Cannon Nikki , Rodrigues Richard R. , Valencia Michael , Barb Jennifer J. , Fernandes Miriam R. , Balaji Ascharya , Crowson Lisa , O’hUigin Colm , Dzutsev Amiran , Trinchieri Giorgio 
Published: March 2023

AbstractShotgun microbiome sequencing analysis presents several challenges to accurately and consistently depict sample composition and functional potential. Here we present a two-part framework – JAMS (Just a Microbiology System) – whereby with raw fastq files and metadata as input, meaningful analysis within a sample and between a sample can be performed with ease for either shotgun or 16S sequences. JAMS is the first package to provide seamless deconvolution of functions into their taxonomic contributors. We validated our JAMS framework on two human gut shotgun metagenome test datasets against the popular tool MetaPhlAn 4. We further demonstrate the application of the JAMS package, particularly the plotting functions, on a mouse shotgun dataset.
A Flexible Transgene Integration ‘Landing-Pad’ Toolkit in Human Induced Pluripotent Stem Cells Enables Facile Cellular Engineering, Gene Zygosity Control, and Parallel Transgene Integration
Authors: Rosenstein Aaron H. , Nguyen Maria , Al-Attar Rasha , Serra Danielle , Gulati Nitya , Yin Ting , Gilbert Penney M. , Garton Michael 
Published: March 2023

AbstractDevelopment of a repeatable method for delivering transgene payloads to human induced pluripotent stem cells (hiPSCs) without risking unintended off-target effects is not fully realized. Yet, such methods are indispensable to fully unlocking the potential for applying synthetic biological approaches to regenerative medicine, delivering quantum impacts to cell-based therapeutics development. Here we present a toolkit for engineering hiPSCs centred on the development of two core ‘landing-pad’ cell-lines, facilitating rapid high-efficiency delivery of transgenes to theAAVS1safe-harbour locus using the Bxb1 large-serine recombinase. We developed two landing-pad cell lines expressing green and red fluorescent reporters respectively, both retaining stemness whilst fully capable of differentiation into all three germ layers. A fully selected hiPSC population can be isolated within 1-2 weeks after landing-pad recombinase-mediated cassette exchange. We demonstrate the capability for investigator-controlled homozygous or heterozygous transgene configurations in these cells. As such, the toolkit of vectors and protocols associated with this landing-pad hiPSC system has the potential to accelerate engineering workflows for researchers in a variety of disciplines.
Computational Scoring and Experimental Evaluation of Enzymes Generated by Neural Networks
Authors: Johnson Sean R. , Fu Xiaozhi , Viknander Sandra , Goldin Clara , Monaco Sarah , Zelezniak Aleksej , Yang Kevin K. 
Published: March 2023

AbstractIn recent years, generative protein sequence models have been developed to sample novel sequences. However, predicting whether generated proteins will fold and function remains challenging. We evaluate computational metrics to assess the quality of enzyme sequences produced by three contrasting generative models: ancestral sequence reconstruction, a generative adversarial network, and a protein language model. Focusing on two enzyme families, we expressed and purified over 440 natural and generated sequences with 70-90% identity to the most similar natural sequences to benchmark computational metrics for predictingin vitroenzyme activity. Over three rounds of experiments, we developed a computational filter that improved experimental success rates by 44-100%. Surprisingly, neither sequence identity to natural sequences nor AlphaFold2 residue-confidence scores were predictive of enzyme activity. The proposed metrics and models will drive protein engineering research by serving as a benchmark for generative protein sequence models and helping to select active variants to test experimentally.
SUNi mutagenesis: scalable and uniform nicking for efficient generation of variant libraries
Authors: Mighell Taylor L. , Toledano Ignasi , Lehner Ben 
Published: March 2023

AbstractMultiplexed assays of variant effects (MAVEs) have made possible the functional assessment of all possible mutations to genes and regulatory sequences. A core pillar of the approach is generation of variant libraries, but current methods are either difficult to scale or not uniform enough to enable MAVEs at the scale of gene families or beyond. We present an improved method called Scalable and Uniform Nicking (SUNi) mutagenesis that combines massive scalability with high uniformity to enable cost-effective MAVEs of gene families and eventually genomes.
Conformational coupling of the sialic acid TRAP transporter HiSiaQM with its substrate binding protein HiSiaP
Authors: Peter Martin F. , Ruland Jan A. , Kim Yeojin , Hendricks Philipp , Siebrasse Jan Peter , Thomas Gavin H. , Kubitscheck Ulrich , Hagelueken Gregor 
Published: March 2023

AbstractThe tripartite ATP-independent periplasmic (TRAP) transporters use an extra cytoplasmic substrate binding protein (SBP) to transport a wide variety of substrates in bacteria and archaea. The SBP can adopt an ‘open’ or ‘closed’ state depending on the presence of substrate. The two transmembrane domains of TRAP transporters form a monomeric elevator whose function is strictly dependent on the presence of a sodium ion gradient. Insights from experimental structures, structural predictions and molecular modeling have suggested a conformational coupling between the membrane elevator and the substrate binding protein. Here, we use a disulfide engineering approach to lock the TRAP transporter HiSiaPQM fromHaemophilus influenzaein different conformational states. The SBP, HiSiaP, was locked in its substrate-bound form and the transmembrane elevator, HiSiaQM, was locked in either its predicted inward- or outward-facing states. We characterized the disulfide-locked variants and used single-molecule total internal reflection fluorescence (TIRF) microscopy to study their interactions. Our experiments demonstrate that the SBP and the transmembrane elevator are indeed ‘conformationally coupled’, meaning that the open and closed state of the SBP recognize specific conformational states of the transporter and vice versa.
Nodule-specific Cu+-chaperone NCC1 is required for symbiotic nitrogen fixation inMedicago truncatularoot nodules
Authors: Navarro-Gómez Cristina , León-Mediavilla Javier , Küpper Hendrik , Rodríguez-Simón Mario , Paganelli-López Alba , Wen Jiangqi , Burén Stefan , Mysore Kirankumar S. , Hussain Bokhari Syed Nadeem , Imperial Juan , Escudero Viviana , González-Guerrero Manuel 
Published: March 2023

ABSTRACTCu+-chaperones are a diverse group of proteins that allocate Cu+ions to specific copper-proteins, creating different copper pools targeted to specific physiological processes. Symbiotic nitrogen fixation carried out in legume root nodules indirectly requires relatively large amounts of copper e.g. for energy delivery via respiration, for which targeted copper deliver systems would be required. MtNCC1 is a nodule-specific Cu+-chaperone encoded in theMedicago truncatulagenome, with a N-terminus Atx1-like domain that can bind Cu+with picomolar affinities. This gene is expressed primarily from the late infection zone to the early fixation zone, and is located in the cytosol, associated to plasma and symbiosome membranes, and within nuclei. Consistent with its key role in nitrogen fixation,ncc1mutants have a severe reduction of nitrogenase activity, and a 50% reduction in copper-dependent cytochromecoxidase activity. A subset of the copper-proteome is also affected in the mutant nodules. Many of these proteins can be pulled-down when using a Cu+-loaded N-terminal MtNCC1 moiety as a bait, indicating a role in nodule copper homeostasis and in copper-dependent physiological processes. Overall, these data suggest a pleiotropic role of MtNCC1 in copper delivery for symbiotic nitrogen fixation.
Beyond analytic solution: analysis of FRAP experiments by spatial simulation of the forward problem
Authors: Cowan Ann E. , Loew Leslie M. 
Published: March 2023

AbstractFluorescence redistribution after photobleaching (FRAP) is a commonly used method to understand the dynamic behavior of molecules within cells. Analytic solutions have been developed for specific, well-defined models of dynamic behavior in idealized geometries, but these solutions are inaccurate in complex geometries or when complex binding and diffusion behaviors exist. We demonstrate the use of numerical reaction-diffusion simulation approaches using the easily accessible Virtual Cell (VCell) software, to establish methods for analyzing photobleaching data. We show how multiple simulations employing parameter scans and varying bleaching locations and sizes can help to bracket diffusion coefficients and kinetic rate constants. This approach is applied to problems in membrane surface diffusion, diffusion and binding in cytosolic volumes in complex cell geometries, and analysis of diffusion and binding in intracellular liquid droplets.Statement of SignificanceFluorescence Redistribution After Photobleaching (FRAP) is a widely used experimental method that can reveal important parameters for reaction/diffusion events within cells. However, analytic methods to analyze FRAP experiments are limited to specific geometries and conditions. We demonstrate how spatial numerical simulation methods using the freely available software Virtual Cell can be used to obtain parameter information from FRAP experiments in situations that are not amenable to analytic solutions and that are accessible to most bench biologists.
ATP1A1-linked diseases require a malfunctioning protein product from one allele
Authors: Spontarelli Kerri , Young Victoria C. , Sweazey Ryan , Padro Alexandria , Lee Jeannie , Bueso Tulio , Hernandez Roberto M. , Kim Jongyeol , Katz Alexander , Rossignol Francis , Turner Clesson , Wilczewski Caralynn M. , Maxwell George L. , Holmgren Miguel , Bailoo Jeremy D. , Yano Sho T. , Artigas Pablo 
Published: March 2023

AbstractHeterozygous germline variants inATP1A1, the gene encoding the α1 subunit of the Na+/K+-ATPase (NKA), have been linked to diseases including primary hyperaldosteronism and the peripheral neuropathy Charcot-Marie-Tooth disease (CMT).ATP1A1variants that cause CMT induce loss-of-function of NKA. This heterodimeric (αβ) enzyme hydrolyzes ATP to establish transmembrane electrochemical gradients of Na+and K+that are essential for electrical signaling and cell survival. Of the 4 catalytic subunit isoforms, α1 is ubiquitously expressed and is the predominant paralog in peripheral axons. Human population sequencing datasets indicate strong negative selection against both missense and protein-nullATP1A1variants. To test whether haploinsufficiency generated by heterozygous protein-null alleles are sufficient to cause disease, we tested the neuromuscular characteristics of heterozygousAtp1a1+/-knockout mice and their wildtype littermates, while also evaluating if exercise increased CMT penetrance. We found thatAtp1a1+/-mice were phenotypically normal up to 18 months of age. Consistent with the observations in mice, we report clinical phenotyping of a healthy adult human who lacks any clinical features of knownATP1A1-related diseases despite carrying a protein-null early truncation variant, p.Y148*. Taken together, these results suggest that a malfunctioning gene product is required for disease induction byATP1A1variants and that if any pathology is associated with protein-null variants, they may display low penetrance or high age of onset.
Induction of T-DNA amplification by retrotransposon-derived sequences
Authors: Dickinson Lauren , Yuan Wenxin , LeBlanc Chantal , Thomson Geoffrey , Wang Siyuan , Jacob Yannick 
Published: March 2023

AbstractTransformation viaAgrobacterium tumefaciens(Agrobacterium) is the predominant method used to introduce exogenous DNA into plants. Transfer DNA (T-DNA) originating from Agrobacterium can be integrated as a single copy or in concatenated forms in plant genomes, but the mechanisms affecting final T-DNA structure remain unknown. In this study, we demonstrate that the inclusion of retrotransposon (RT)-derived sequences in T-DNA can increase transgene copy number by more than 50-fold inArabidopsis thaliana(Arabidopsis). RT-mediated amplification of T-DNA results in large concatemers in the Arabidopsis genome, which are primarily induced by the long terminal repeats (LTRs) of RTs. T-DNA amplification is dependent on the activity of DNA repair proteins associated with theta-mediated end joining (TMEJ). Finally, we show that T-DNA amplification can increase the frequency of targeted mutagenesis and gene targeting. Overall, this work uncovers molecular determinants that modulate T-DNA copy number in Arabidopsis and demonstrates the utility of inducing T-DNA amplification for plant gene editing.
Movements and habitat selection of a marsupial carnivore in a modified landscape
Authors: Jones Evie M. , Koch Amelia J. , Pay James M. , Mann Dydee F. , Jones Menna E. , Hamede Rodrigo K. 
Published: March 2023

AbstractLandscape modification is a major threat to carnivores worldwide, but modified landscapes can also provide important habitat for these species, as protected areas alone are insufficient. Understanding how carnivores use modified landscapes, such as production forests, can inform management strategies to improve the value of these landscapes to carnivores. Little is known about habitat selection by marsupial carnivores in production forests, where they occupy a similar ecological niche to their more well-studied eutherian counterparts. We used GPS tracking, Hidden Markov Models, and Manly’s selection ratios to identify the habitat selected in 3 behavioral states by the largest marsupial carnivore, the Tasmanian devil (Sarcophilus harrisii), in a timber plantation-dominated landscape. Behavioral states were approximated as: state 1, resting or feeding at a carcass; state 2, foraging; and state 3, travel. Devils did not show preferences for any of native forest, native grassland, and plantation in any behavioral state. Within plantations, devils preferred a plantation age of 4–7 years (selection ratio [wi] = 1.52). Devils preferred roads (state 1:wi =2.71, state 2:wi= 2.48, state 3:wi= 2.97) and plantation edges (state 1:wi =2.38, state 2:wi= 2.24, state 3:wi= 2.78) in all behavioral states, and moved faster on roads and edges than away from them. Together, our results indicate devils use road and edges for foraging (scavenging and hunting) and travel. No measured habitat variables influenced devil home range size. To support devils in plantation landscapes, we recommend maintaining a heterogeneous landscape of different plantation ages and native remnants and reducing the risk of vehicle collisions by minimizing forestry traffic at night. Tasmanian devils share similar adaptable traits to generalist eutherian carnivore species in their use of modified landscapes. Plantations can provide valuable habitat for this and other threatened predator species.
Common And Distinct Patterns Of Task-Related Neural Activation Abnormalities In Patients With Remitted And Current Major Depressive Disorder: A Systematic Review And Coordinate-Based Meta-Analysis
Authors: Wang Zuxing , Wang Peijia , He Danmei , Yang Lin , Zhu Hongru 
Published: March 2023

AbstractWhether remitted major depressive disorder (rMDD) and MDD present common or distinct neuropathological mechanisms remains unclear. We performed a meta-analysis of task-related whole-brain functional magnetic resonance imaging (fMRI) using anisotropic effect-size signed differential mapping software to compare brain activation between rMDD/MDD patients and healthy controls (HCs). We included 18 rMDD studies (458 patients and 476 HCs) and 120 MDD studies (3746 patients and 3863 HCs). The results showed that MDD and rMDD patients shared increased neural activation in the right temporal pole and right superior temporal gyrus. Several brain regions, including the right middle temporal gyrus, left inferior parietal, prefrontal cortex, left superior frontal gyrus and striatum, differed significantly between MDD and rMDD. Meta-regression analyses revealed that the percentage of females with MDD was positively associated with brain activity in the right lenticular nucleus/putamen. Our meta-analysis provides useful insights for understanding the potential neuropathology of brain dysfunction in MDD, developing more targeted and efficacious treatment and intervention strategies, and more importantly, providing potential neuroimaging targets for early screening of MDD.
Association between types of physical activity and risk of ischemic heart disease based on US Guidelines: A systematic review and meta-analysis
Authors: Cha Jaewoo , Kim Jeehyun , Hong Kwan 
Published: March 2023

AbstractBackgroundMortality from ischemic heart disease (IHD) is increasing worldwide. There is no available literature regarding the validity of US guidelines for IHD risk reduction through physical exercise. In this meta-analysis, we aimed to measure the effectiveness of US guidelines for physical activity in reducing IHD risk based on the types of exercise.MethodsSix databases, including MEDLINE, EMBASE, Cochrane Library, CINAHL, Scopus, and Web of Science, were searched from January 1, 2000, to November 6, 2022. The most recent literature search was conducted on November 6, 2022, and only English-language articles were included. Studies designed and conducted on humans on any type of IHD-related physical activity were included. Study outcomes included heterogeneity among the studies, overall effects of all types of physical activity, and IHD risk. The random effects model was measured. A funnel plot was used to assess publication bias.ResultsWhen the US guidelines for physical activity were not applied, there was a high level of heterogeneity in the effects of physical activity by type, with overall effects of 0.764 (odds ratio [OR], 95% confidence interval [CI]: 0.737–0.795). The effect of physical activity on IHD was 0.593 (OR, 95% CI: 0.489–0.720). No publication bias was observed. After applying the US guidelines for physical activity, there was a low level of heterogeneity in the effects of physical activity by type and an observed OR of 0.515 (95% CI: 0.401–0.662) for myocardial infarction.DiscussionEach type of exercise had different effects on reducing IHD, and there were certain beneficial results if the US guidelines for physical activity were satisfied.
Unraveling the Interactions between Human DPP4 Receptor, SARS-CoV-2 Variants, and MERS-CoV, converged for Pulmonary Disorders Integrating through Immunoinformatics and Molecular Dynamics
Authors: Roy Arpan Narayan , Mallick Gupta Aayatti , Banerjee Deboshmita , Chakraborty Jaydeb , Raghavendra Pongali B 
Published: March 2023

AbstractHuman coronaviruses like MERS CoV are known to utilize dipeptidyl peptidase 4 (DPP4), apart from angiotensin-converting enzyme 2(ACE2) as potential co-receptor for viral cell entry. DPP4, ubiquitous membrane-bound aminopeptidase is closely associated with elevation of disease severity in comorbidities. In SARS-CoV-2, there is inadequate evidence for combination of spike protein variants with DPP4, and underlying adversity in COVID19. To elucidate this mechanistic basis, we have investigated interaction of spike protein variants with DPP4 through molecular docking and simulation studies. The possible binding interactions between receptor binding domain (RBD) of different spike variants of SARS-CoV-2 and DPP4 have been compared with interactions observed in experimentally determined structure of complex of MERS-CoV with DPP4. Comparative binding affinity confers that Delta-CoV-2:DPP4 shows close proximity with MERS-CoV:DPP4, as depicted from accessible surface area, radius of gyration, number of hydrogen bonding and energy of interactions. Mutation in delta variant, L452R and T478K, directly participate in DPP4 interaction enhancing DPP4 binding. E484K in alpha and gamma variant of spike protein is also found to interact with DPP4. Hence, DPP4 interaction with spike protein gets more suitable due to mutation especially due to L452R, T478K and E484K. Furthermore, perturbation in the nearby residues Y495, Q474 and Y489 is evident due to L452R, T478K and E484K respectively. Virulent strains of spike protein are more susceptible to DPP4 interaction and are prone to be victimized in patients due to comorbidities. Our results will aid the rational optimization of DPP4 as a potential therapeutic target to manage COVID-19 disease severity.