bluuebunny/crossref_metadata_2025_split huggingface dataset:
Tools for Genetic Engineering and Gene Expression Control inNovosphingobium aromaticivoransandRhodobacter sphaeroides
Authors: Hall Ashley N. , Hall Benjamin W. , Kinney Kyle J. , Olsen Gabby G. , Banta Amy B. , Noguera Daniel R. , Donohue Timothy J. , Peters Jason M. 
Published: August 2023

ABSTRACTAlphaproteobacteria have a variety of cellular and metabolic features that provide important insights into biological systems and enable biotechnologies. For example, some species are capable of converting plant biomass into valuable biofuels and bioproducts have the potential to form the backbone of the sustainable bioeconomy. Among the Alphaproteobacteria,Novosphingobium aromaticivorans,Rhodobacter sphaeroides, andZymomonas mobilis, show particular promise as organisms that can be engineered to convert extracted plant lignin or sugars into bioproducts and biofuels. Genetic manipulation of these bacteria is needed to introduce engineered pathways and modulate expression of native genes with the goal of enhancing bioproduct output. Although recent work has expanded the genetic toolkit forZ. mobilis,N. aromaticivoransandR. sphaeroidesstill need facile, reliable approaches to deliver genetic payloads to the genome and to control gene expression. Here, we expand the platform of genetic tools forN. aromaticivoransandR. sphaeroidesto address these issues. We demonstrate that Tn7transposition is an effective approach for introducing engineered DNA into the chromosome ofN. aromaticivoransandR. sphaeroides. We screen a synthetic promoter library to identify inducible promoters with strong, regulated activity in both organisms. Combining Tn7integration with promoters from our library, we establish CRISPR interference systems forN. aromaticivoransandR. sphaeroidesthat can target essential genes and modulate engineered pathways. We anticipate that these systems will greatly facilitate both genetic engineering and gene function discovery efforts in these industrially important species and other Alphaproteobacteria.IMPORTANCEIt is important to increase our understanding of the microbial world to improve health, agriculture, the environment and biotechnology. For example, building a sustainable bioeconomy depends on the efficient conversion of plant material to valuable biofuels and bioproducts by microbes. One limitation in this conversion process is that microbes with otherwise excellent properties for conversion are challenging to genetically engineer. Here, we report systems to overcome that barrier in the Alphaproteobacteria,Novosphingobium aromaticivoransandRhodobacter sphaeroides, by producing genetic tools that allow easy insertion of engineered pathways into their genomes and to precisely control gene expression by inducing genes with synthetic promoters or repressing genes using CRISPR interference. These tools can be used in future work to gain additional insight into these and other Alphaproteobacteria and to optimize yield of biofuels and bioproducts.
How Should Eukaryotic Chemotaxis be Measured?
Authors: Tweedy Luke , Thomason Peter A. , Insall Robert H. 
Published: August 2023

AbstractChemotaxis and directed cell migration are fundamentally important to eukaryotic biology. To understand the mechanisms that drive such complex processes, informative and robust measurements are essential, but the field does not always agree what these should be. Here we identify the most dependable measures of chemotactic steering and the underlying cell migration, and provide tools to test them. Some widely-used metrics can end up misleading, in particular “cos 8” for directional accuracy. We suggest that chemotactic efficiency should be used as a primary metric. Mean squared displacement and directional autocorrelation can be used to unpick different models of random and directed cell migration. Transition matrices are another useful tool for understanding migration mechanisms and avoiding artefacts, and provide a graphical illustration of how well cells maintain each direction. Unexpectedly, the choice of start and end points of tracks strongly affects the measurements and can seriously bias the measured results. This is particularly clear when cells are not homogeneously distributed at the start of an assay. To support straightforward adoption of these methods, we provide a suite of tools as a plugin for the open-source ImageJ program, and describe how they can be used to understand complex scenarios like self-generated chemotactic gradients.
CaMKII autophosphorylation but not downstream kinase activity is required for synaptic memory
Authors: Chen Xiumin , Cai Qixu , Zhou Jing , Pleasure Samuel J. , Schulman Howard , Zhang Mingjie , Nicoll Roger A. 
Published: August 2023

AbstractCaMKII plays a critical role in long-term potentiation (LTP), a well-established model for learning and memory through the enhancement of synaptic transmission. Biochemical studies indicate that CaMKII catalyzes a phosphotransferase (kinase) reaction of both itself (autophosphorylation) and of multiple downstream target proteins. However, whether either type of phosphorylation plays any role in the synaptic enhancing action of CaMKII remains hotly contested. We have designed a series of experiments to define the minimal requirements for the synaptic enhancement by CaMKII. We find that autophosphorylation of T286 and further binding of CaMKII to the GluN2B subunit are required both for initiating LTP and for its maintenance (synaptic memory). Once bound to the NMDA receptor, the synaptic action of CaMKII occurs in the absence of kinase activity. Thus, autophosphorylation, together with binding to the GluN2B subunit, are the only two requirements for CaMKII in synaptic memory.
A glucanotransferase that uses two sub-sites and four catalytic aspartates/glutamates to disproportionate oligosaccharides ranging in length from maltotriose to starch
Authors: Sarkar Arpita , Kaila Pallavi , Tiwari Prince , Guptasarma Purnananda 
Published: August 2023

AbstractPF0272 (PfuAmyGT) fromPyrococcus furiosusis a 656 residues-long, homodimeric, three-domain GH57 glycoside hydrolase [homologous to TLGT fromThermococcus litoralis(PDB ID: 1K1X)]. It is proposed to be either an α-amylase (EC 3.2.1.1), or a 4-α-glucanotransferase (EC 2.4.1.25). We demonstrate that PfuAmyGT is an exo-amylase-cum-glucanotransferase capable of transferring glucose, and dis-proportionating oligosaccharides, by excising glucose from malto-oligosaccharides (ranging in length from maltotriose to amylose/starch), and transferring it to malto-oligosaccharides (ranging in length from glucose to maltoheptaose and, possibly, even longer lengths). Convention holds that glucanotransferases transfer sugars through the serial and alternating binding of donors and acceptors to the same site, with covalent retention of excised sugars between such bindings. We present evidence of multiple behaviors in PfuAmyGT that challenge this view: (i) Production of free glucose, indicating scope for release of excised glucose; (ii) Higher activity with longer donors, indicating processivity; (iii) Accelerated activity with shorter acceptors, indicating a dependence upon rapid acceptor turnover; and (iv) Evidence of four catalytic glutamates/aspartates (E131, D222, E224, D362), indicating possible separation of excision and ligation functions. These behaviours collectively indicate the binding of donors and acceptors to separate sub-sites that have different substrate ‘length’ preferences, supporting our own previous proposal regarding separate tunnel (internal) and groove (surface) binding sites. Although PfuAmyGT’s mechanism of function remains to be fully elucidated, this paper definitively demonstrate ‘coupling’ of exo-amylase and glucanotransferase functions involving separate sub-sites for donor and acceptor binding.
Kinetic Proofreading can Enhance Specificity in a Non-enzymatic DNA Strand Displacement Network
Authors: Mukherjee Rakesh , Sengar Aditya , Cabello-García Javier , Ouldridge Thomas E. 
Published: August 2023

ABSTRACTKinetic proofreading is used throughout natural systems to enhance the specificity of molecular recognition. At its most basic level, kinetic proofreading uses a supply of chemical fuel to drive a recognition interaction out of equilibrium, allowing a single free-energy difference between correct and incorrect targets to be exploited two or more times. Despite its importance in biology, there has been little effort to incorporate kinetic proofreading into synthetic systems in which molecular recognition is important, such as nucleic acid nanotechnology. In this article, we introduce a DNA strand displacement-based kinetic proofreading motif, showing that the consumption of a DNA-based fuel can be used to enhance molecular recognition during a templated dimerization reaction. We then show that kinetic proofreading can enhance the specificity with which a probe discriminates single nucleotide mutations, both in terms of the initial rate with which the probe reacts and the long-time behaviour.
Differences in the activities of domain-swapped chimeras of two homologous GH57 glucanotransferases suggest that a glucan-binding DUF could influence donor substrate specificity
Authors: Sarkar Arpita , Kaila Pallavi , Guptasarma Purnananda 
Published: August 2023

AbstractFive enzymes of the archaeal hyperthermophilic family of disproportionating GH57 4-α-glucanotransferases have been studied till date. Our focus here lies upon three homologous members of this family: (i) PfuAmyGT fromPyrococcus furiosus(PF0272), (ii) TonAmyGT fromThermococcus onnurenius(B6YUX8), and (iii) TliAmyGT (TLGT) fromThermococcus litoralis(O32462). The polypeptide chain of each of these enzymes is approximately 655 residues long, folded into three distinct domains (1, 2 and 3), and assembled into a homodimer. Domain 1 is a beta/alpha barrel containing an aspartate known to function as a catalytic nucleophile in TLGT. Domain 2 (which is helical) and domain 3 (made up of beta sheets) are thought to be domains of unknown function (or DUFs). In PfuAmyGT and TonAmyGT, we have recently identified a catalytically-important aspartate upon a loop in domain 2. In PfuAmyGT, we demonstrate the presence of two additional catalytically-important (glutamate) residues in domain 1, in a companion paper. In this paper, our focus lies upon domain 3 which hosts a second binding site (SBS) for a glucan, at its domain-domain interface with domain 2. Using strategies involving studies of both (a) domains (or pairs of contiguous domains) extracted from PfuAmyGT, and (b) chimeric three-domain enzymes recombining analogous domains between PfuAmyGT and TonAmyGT, we demonstrate that domain 3 determines the choice of the preferred glucan that acts as a donor in the glucan transfer reaction.
Forensic Facial Approximation of Achondroplastic Dwarf from Medieval Cemetery in Central Europe
Authors: Moraes Cicero , Krenz-Niedbała Marta , Łukasik Sylwia , Prada Camilo Serrano 
Published: August 2023

AbstractAchondroplasia (ACH, achondroplastic dwarfism) represents the most common form of skeletal dysplasia, occurring in c. 4 out of every 100,000 births. This study presents a computer-based facial approximation of the skull of a male individual suffering from ACH, who died at 30-45 years of age and was buried in Łekno, Poland between the 9th and 11th centuries AD. For the approximation procedure, soft tissue data from CT scans and ultrasonic measurements performed on living individuals were used. Additionally, the anatomical deformation technique was applied to arrive at the most reliable reconstruction of the dwarf’s appearance. To our knowledge, this is the first recreation of a person with achondroplasia, and one of the few showing a head of an individual suffering from a hereditary disease, with dimensions and shape differing from the population average values.Highlights-Forensic facial approximation of an achondroplastic dwarf from 9th–11th century AD has been performed as the first in the world-The applied procedure included CT of a virtual donor-Anatomical deformation technique allowed to extract the endocast, revealing a large volume of the endocranium-Few measurements applied to the facial skeleton proved successful in identification of a person suffering from achondroplasia
An amplicon panel for high-throughput and low-cost genotyping of Pacific oyster
Authors: Sutherland Ben J. G. , Thompson Neil F. , Surry Liam B. , Gujjula Krishna Reddy , Carrasco Claudio D. , Chadaram Srinivas , Lunda Spencer L. , Langdon Christopher J. , Chan Amy M. , Suttle Curtis A. , Green Timothy J. 
Published: August 2023

AbstractMaintaining genetic diversity in cultured shellfish can be challenging due to high variance in individual reproductive success, founder effects, and rapid genetic drift, but is important to retain adaptive potential and avoid inbreeding depression. To support broodstock management and selective breeding in cultured Pacific oysters (Crassostrea(Magallana)gigas), we developed an amplicon panel targeting 592 genomic regions and SNP variants with an average of 50 amplicons per chromosome. Target SNPs were selected based on elevated observed heterozygosity or differentiation in Pacific oyster populations in British Columbia (BC), Canada. The use of the panel for parentage applications was evaluated by genotyping three generations of oysters from a breeding program in BC (n = 181) and a set of families that were selected for Ostreid herpesvirus-1 (OSHV-1) resistance from the Molluscan Broodstock Program, a Pacific oyster breeding program in Oregon, USA (n = 136). Population characterization was evaluated using collections of wild, naturalized, farmed, or hatchery oysters sampled throughout the Northern Hemisphere (n = 190). Technical replicate samples showed high genotype concordance (97.5%; n = 68 replicates). Initial parentage analysis found instances of suspected pedigree and sample handling errors, demonstrating the panel’s value for quality control in breeding programs. Suspected null alleles were identified in parentage datasets and were found to reduce assignment success. Null alleles were largely population dependent, suggesting population-specific variation impacts target amplification. By taking an iterative approach, null alleles were identified using existing data without the need for pedigree information, and once null alleles were removed, assignments increased to 93.0% and 86.0% of possible assignments in the two breeding program datasets. A pipeline for analyzing the amplicon sequence data from sequencer output,amplitools, is also provided.
Unravelling ATP processing by the AAA+ protein p97 at the atomic level
Authors: Shein Mikhail , Hitzenberger Manuel , Cheng Tat Cheung , Rout Smruti R. , Leitl Kira D. , Sato Yusuke , Zacharias Martin , Sakata Eri , Schütz Anne K. 
Published: August 2023

AbstractThe human enzyme p97 regulates various cellular pathways by unfolding hundreds of protein substrates in an ATP-dependent manner, making it an essential component of homeostasis and impactful pharmacological target.The hexameric complex undergoes substantial conformational changes in the course of its catalytic cycle. Here, we elucidate the molecular motions that occur at the active site in the temporal window immediately before and after ATP hydrolysis by merging cryo-EM, NMR spectroscopy and MD simulations. p97 populates a metastable reaction intermediate, the ADP.Pi state, which is poised between hydrolysis and product release. Detailed snapshots reveal that the active site is finely tuned to trap and eventually discharge the cleaved phosphate. Signalling pathways originating at the active site coordinate the action of the hexamer subunits and couple hydrolysis with allosteric conformational changes.Our multidisciplinary approach enables a glimpse into the sophisticated spatial and temporal orchestration of ATP handling by a prototype AAA+ protein.
FRET-GP – A Local Measure of the Impact of Transmembrane Peptide on Lipids
Authors: Thakur Garima C. N. , Uday Arunima , Jurkiewicz Piotr 
Published: August 2023

ABSTRACTReconstitution of a transmembrane protein in model lipid systems allows studying its structure and dynamics in isolation from the complexity of the natural environment. This approach also provides a well-defined environment for studying the interactions of the protein with lipids. In this work we describe the FRET-GP method, which utilizes Förster resonance energy transfer (FRET) to specifically probe nanoenvironment of a transmembrane domain. The tryptophan residues flanking this domain act as efficient FRET donors, while Laurdan acts as acceptor. The fluorescence of this solvatochromic probe, is quantified using generalized polarization (GP) to reports on lipid fluidity in the vicinity of the transmembrane domain. We applied FRET-GP to study the transmembrane peptide WALP incorporated in liposomes. We found that the direct excitation of Laurdan to its second singlet state strongly contribute to GP values measured in FRET conditions. Removal of this parasitic contribution was essential for proper determination ofGPFRET– the local analogue of classicalGPparameter. The presence of WALP significantly increased both parameters, but the local effects were considerably stronger (GPFRET≫GP). We conclude that WALP restricts lipid movement in its vicinity, inducing lateral inhomogeneity in membrane fluidity. WALP was also found to influence lipid phase transition. Our findings demonstrated that FRET-GP simultaneously provides local and global results, thereby increasing comprehensibility of the measurement. We highlight the simplicity and sensitivity of the method, but also discuss its potential and limitations in studying protein-lipid interactions.TOC graphics
TRPM7 regulates phagocytosis and clearance ofCandida albicans
Authors: Stremska Marta E. , Stipes Eric J. , Jang Jessica J. , Busey Gregory W. , Iobst Wesley H. , Seegren Philip V. , Kennedy Joel , Desai Bimal N. 
Published: August 2023

ABSTRACTSentinel phagocytes of the innate immune system have a critical role in detecting and eliminating fungal pathogens. We used patch clamp electrophysiology to explore the electrical signals elicited when macrophages engulfCandida albicans. In the perforated patch configuration, which is least disruptive to intracellular signaling, we detected a composite outwardly rectifying current during the engulfment ofC. albicansor zymosan. FTY720, a known inhibitor of ion channel TRPM7, suppressed the current. We then tested the hypothesis that TRPM7 regulates the engulfment and clearance ofC. albicans. We found thatTrpm7-/-macrophages are highly deficient in the engulfment ofC. albicans. Trpm7-/-macrophages initiate phagocytosis of yeast but are defective in sealing the phagocytic cups. While the precise mechanism through which TRPM7 regulates phagosome sealing is not clear, we tested the immunological significance of this discovery using a mouse model of systemic candidiasis. We show that in mice, wherein TRPM7 is deleted selectively in the myeloid cells, infection byC. albicansresults in significantly higher lethality, increased colonization of vital organs and increased inflammatory cytokines in the blood. Our study establishes TRPM7 as an ion channel critical for the innate immune responses against fungal pathogens and sets the stage for cell biological studies that define the mechanisms through which TRPM7 regulates phagosome sealing.Significance statementThe worldwide increase in deadly or persistent fungal infections has prompted the research for alternative ways of treatment. We applied the specialized, perforated patch clamp technique to track and identify electrical currents elicited during the detection and engulfment of fungi by macrophages. The ion channel TRPM7 emerged as an important determinant of anti-fungal host defense as its deletion in the murine myeloid cells made the host mice highly susceptible to lethal candidiasis. Ion channels are attractive drug targets whose activation and inhibition can be manipulated with pharmacological therapeutics. This study raises the possibility of enhancing fungal clearance using activators of TRPM7. Such pharmacological strategy may benefit patients of persistent fungal infections that are recalcitrant to anti-fungal drugs.
Enterobacterial common antigen biosynthesis inYersinia pestisis tied to antimicrobial peptide resistance
Authors: Aoyagi Kari L. , Mathew Basil , Fisher Mark A. 
Published: August 2023

AbstractResistance to antimicrobial peptides (AMPs) plays an important role in allowingYersinia pestisto maintain a successful infection in the flea vectorXenopsylla cheopis. Mutants that are unable to modify lipid A in their outer membrane with aminoarabinose (Ara4N), showed increased sensitivity to AMPs such as polymyxin B (PB), as well as decreased survival in fleas. A deletion mutant ofwecE, a gene involved in biosynthesis of enterobacterial common antigen (ECA), also displayed hypersusceptibility to PB in vitro. Additional mutants in the ECA biosynthetic pathway were generated, some designed to cause accumulation of intermediate products that sequester undecaprenyl phosphate (Und-P), a lipid carrier that is also used in numerous other pathways, including for peptidoglycan, O-antigen, and Ara4N biosynthesis. Mutants that accumulate Und-PP-linked intermediates (ECA-lipid II) showed increased susceptibility to PB, reduced Ara4N-modified lipid A, altered cell morphology, and decreased ability to maintain flea infections. These effects are consistent with a model whereY. pestishas a sufficiently limited free Und-P pool such that sequestration of Und-P as ECA-lipid II prevents adequate Ara4N biosynthesis, ultimately resulting in AMP hypersusceptibility.
Using transition density models to interpret experimental optical spectra of exciton-coupled cyanine (iCy3)2dimer probes of local DNA conformations at or near functional protein binding sites
Authors: Heussman Dylan , Enkhbaatar Lulu , Sorour Mohammed I. , Kistler Kurt A. , von Hippel Peter H. , Matsika Spiridoula , Marcus Andrew H. 
Published: August 2023

AbstractExciton-coupled chromophore dimers are an emerging class of optical probes for studies of site-specific biomolecular interactions. Applying accurate theoretical models for the electrostatic coupling of a molecular dimer probe is a key step for simulating its optical properties and analyzing spectroscopic data. In this work, we compare experimental absorbance and circular dichroism (CD) spectra of ‘internally-labeled’ (iCy3)2dimer probes inserted site-specifically into DNA fork constructs to theoretical calculations of the structure and geometry of these exciton-coupled dimers. We compare transition density models of varying levels of approximation to determine conformational parameters of the (iCy3)2dimer-labeled DNA fork constructs. By applying an atomistically detailed transition charge (TQ) model, we can distinguish between dimer conformations in which the stacking and tilt angles between planar iCy3 monomers are varied. A major strength of this approach is that the local conformations of the (iCy3)2dimer probes that we determined can be used to infer information about the structures of the DNA framework immediately surrounding the probes at various positions within the constructs, both deep in the duplex DNA sequences and at sites at or near the DNA fork junctions where protein complexes bind to discharge their biological functions.