ABSTRACT
Extracellular vesicles (EVs), or exosomes, play important roles in physiological and pathological cellular communication and have gained substantial traction as biological drug carriers. EVs contain both short and long non-coding RNAs that regulate gene expression and epigenetic processes. To fully capitalize on the potential of EVs as drug carriers, it is important to study and understand the intricacies of EV function and EV RNA-based communication. Here we developed a genetically encodable RNA-based biomaterial, termed EXO-Probe, for tracking EV RNAs. The EXO-Probe comprises an EV-loading RNA sequence (EXO-Code), fused to a fluorogenic RNA Mango aptamer for RNA imaging. This fusion construct allowed the visualization and tracking of EV RNA and colocalization with markers of multivesicular bodies; imaging RNA within EVs, and non-destructive quantification of EVs. Overall, the new RNA-based biomaterial provides a useful and versatile means to interrogate the role of EVs in cellular communication via RNA trafficking to EVs and to study cellular sorting decisions. The system will also help lay the foundation to further improve the therapeutic efficacy of EVs as drug carriers.
ABSTRACT
Structural variations (SVs) play a significant role in speciation and adaptation in many species, yet few studies have explored the prevalence and impact of different categories of SVs. We conducted a comparative analysis of long-read assembled reference genomes of closely related Eucalyptus species to identify candidate SVs potentially influencing speciation and adaptation. Interspecies SVs can be either fixed differences or polymorphic in one or both species. To describe SV patterns, we employed short-read whole-genome sequencing on over 600 individuals of Eucalyptus melliodora and Eucalyptus sideroxylon, along with recent high-quality genome assemblies. We aligned reads and genotyped interspecies SVs predicted between species reference genomes. Our results revealed that 49,756 of 58,025 and 39,536 of 47,064 interspecies SVs could be typed with short reads in E. melliodora and E. sideroxylon, respectively. Focusing on inversions and translocations, symmetric SVs that are readily genotyped within both populations, 24 were found to be structural divergences, 2,623 structural polymorphisms, and 928 shared structural polymorphisms. We assessed the functional significance of fixed interspecies SVs by examining differences in estimated recombination rates and genetic differentiation between species, revealing a complex history of natural selection. Shared structural polymorphisms displayed enrichment of potentially adaptive genes. Understanding how different classes of genetic mutations contribute to genetic diversity and reproductive barriers is essential for understanding how organisms enhance fitness, adapt to changing environments, and diversify. Our findings reveal the prevalence of interspecies SVs and elucidate their role in genetic differentiation, adaptive evolution, and species divergence within and between populations.
Subject(s)
Eucalyptus , Genome, Plant , Reproductive Isolation , Eucalyptus/genetics , Genomic Structural Variation , Polymorphism, Genetic , Evolution, Molecular , Adaptation, Physiological/genetics , Genetic Speciation , Whole Genome Sequencing/methods , GenotypeABSTRACT
BACKGROUND: Drought adaptation is critical to many tree species persisting under climate change, however our knowledge of the genetic basis for trees to adapt to drought is limited. This knowledge gap impedes our fundamental understanding of drought response and application to forest production and conservation. To improve our understanding of the genomic determinants, architecture, and trait constraints, we assembled a reference genome and detected ~ 6.5 M variants in 432 phenotyped individuals for the foundational tree Corymbia calophylla. RESULTS: We found 273 genomic variants determining traits with moderate heritability (h2SNP = 0.26-0.64). Significant variants were predominantly in gene regulatory elements distributed among several haplotype blocks across all chromosomes. Furthermore, traits were constrained by frequent epistatic and pleiotropic interactions. CONCLUSIONS: Our results on the genetic basis for drought traits in Corymbia calophylla have several implications for the ability to adapt to climate change: (1) drought related traits are controlled by complex genomic architectures with large haplotypes, epistatic, and pleiotropic interactions; (2) the most significant variants determining drought related traits occurred in regulatory regions; and (3) models incorporating epistatic interactions increase trait predictions. Our findings indicate that despite moderate heritability drought traits are likely constrained by complex genomic architecture potentially limiting trees response to climate change.
Subject(s)
Droughts , Epistasis, Genetic , Genomics , Genome, Plant , Haplotypes , Quantitative Trait Loci , Phenotype , Polymorphism, Single NucleotideABSTRACT
Lipopeptides are an important class of biomolecules for drug development. Compared with conventional acylation, a chemoselective lipidation strategy offers a more efficient strategy for late-stage structural derivatisation of a peptide scaffold. It provides access to chemically diverse compounds possessing intriguing and non-native moieties. Utilising an allenamide, we report the first semisynthesis of antimicrobial lipopeptides leveraging a highly efficient thia-Michael addition of chemically diverse lipophilic thiols. Using chemoenzymatically prepared polymyxin B nonapeptide (PMBN) as a model scaffold, an optimised allenamide-mediated thia-Michael addition effected rapid and near quantitative lipidation, affording vinyl sulfide-linked lipopeptide derivatives. Harnessing the utility of this new methodology, 22â lipophilic thiols of unprecedented chemical diversity were introduced to the PMBN framework. These included alkyl thiols, substituted aromatic thiols, heterocyclic thiols and those bearing additional functional groups (e.g., amines), ultimately yielding analogues with potent Gram-negative antimicrobial activity and substantially attenuated nephrotoxicity. Furthermore, we report facile routes to transform the allenamide into a ß-keto amide on unprotected peptides, offering a powerful "jack-of-all-trades" synthetic intermediate to enable further peptide modification.
ABSTRACT
Introduction: Through the combined use of two nitrification inhibitors, Dicyandiamide (DCD) and chlorate with nitrogen amendment, this study aimed to investigate the contribution of comammox Nitrospira clade B, ammonia oxidizing bacteria (AOB) and archaea (AOA) to nitrification in a high fertility grassland soil, in a 90-day incubation study. Methods: The soil was treated with nitrogen (N) at three levels: 0 mg-N kg-1 soil, 50 mg-N kg-1 soil, and 700 mg-N kg-1 soil, with or without the two nitrification inhibitors. The abundance of comammox Nitrospira, AOA, AOB, and nitrite oxidising bacteria (NOB) was measured using qPCR. The comammox Nitrospira community structure was assessed using Illumina sequencing. Results and Discussion: The results showed that the application of chlorate inhibited the oxidation of both NH4+ and NO2- in all three nitrogen treatments. The application of chlorate significantly reduced the abundance of comammox Nitrospira amoA and nxrB genes across the 90-day experimental period. Chlorate also had a significant effect on the beta diversity (Bray-Curtis dissimilarity) of the comammox Nitrospira clade B community. Whilst AOB grew in response to the N substrate additions and were inhibited by both inhibitors, AOA showed litle or no response to either the N substrate or inhibitor treatments. In contrast, comammox Nitrospira clade B were inhibited by the high ammonium concentrations released from the urine substrates. These results demonstrate the differential and niche responses of the three ammonia oxidising communities to N substrate additions and nitrification inhibitor treatments. Further research is needed to investigate the specificity of the two inhibitors on the different ammonia oxidising communities.
ABSTRACT
Genomes have a highly organized architecture (nonrandom organization of functional and nonfunctional genetic elements within chromosomes) that is essential for many biological functions, particularly gene expression and reproduction. Despite the need to conserve genome architecture, a high level of structural variation has been observed within species. As species separate and diverge, genome architecture also diverges, becoming increasingly poorly conserved as divergence time increases. However, within plant genomes, the processes of genome architecture divergence are not well described. Here we use long-read sequencing and de novo assembly of 33 phylogenetically diverse, wild and naturally evolving Eucalyptus species, covering 1-50 million years of diverging genome evolution to measure genome architectural conservation and describe architectural divergence. The investigation of these genomes revealed that following lineage divergence, genome architecture is highly fragmented by rearrangements. As genomes continue to diverge, the accumulation of mutations and the subsequent divergence beyond recognition of rearrangements become the primary driver of genome divergence. The loss of syntenic regions also contribute to genome divergence but at a slower pace than that of rearrangements. We hypothesize that duplications and translocations are potentially the greatest contributors to Eucalyptus genome divergence.
Subject(s)
Eucalyptus , Evolution, Molecular , Genome, Plant , Eucalyptus/genetics , Synteny , Gene Rearrangement , Phylogeny , Chromosomes, Plant/genetics , Genetic VariationABSTRACT
The brevicidines represent a novel class of nonribosomal antimicrobial peptides that possess remarkable potency and selectivity toward highly problematic and resistant Gram-negative pathogenic bacteria. A recently discovered member of the brevicidine family, coined brevicidine B (2), comprises a single amino acid substitution (from d-Tyr2 to d-Phe2) in the amino acid sequence of the linear moiety of brevicidine (1) and was reported to exhibit broader antimicrobial activity against both Gram-negative (MIC = 2-4 µgmL-1) and Gram-positive (MIC = 2-8 µgmL-1) pathogens. Encouraged by this, we herein report the first total synthesis of the proposed structure of brevicidine B (2), building on our previously reported synthetic strategy to access brevicidine (1). In agreement with the original isolation paper, pleasingly, synthetic 2 demonstrated antimicrobial activity toward Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae (MIC = 4-8 µgmL-1). Interestingly, however, synthetic 2 was inactive toward all of the tested Gram-positive pathogens, including methicillin-resistant Staphylococcus aureus strains. Substitution of d-Phe2 with its enantiomer, and other hydrophobic residues, yields analogues that were either inactive or only exhibited activity toward Gram-negative strains. The striking difference in the biological activity of our synthetic 2 compared to the reported natural compound warrants the re-evaluation of the original natural product for purity or possible differences in relative configuration. Finally, the evaluation of synthetic 1 and 2 in a human kidney organoid model of nephrotoxicity revealed substantial toxicity of both compounds, although 1 was less toxic than 2 and polymyxin B. These results indicate that modification to position 2 may afford a strategy to mitigate the nephrotoxicity of brevicidine.
Subject(s)
Anti-Bacterial Agents , Microbial Sensitivity Tests , Structure-Activity Relationship , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/chemical synthesis , Anti-Bacterial Agents/chemistry , Molecular Structure , Pseudomonas aeruginosa/drug effects , Humans , Depsipeptides/pharmacology , Depsipeptides/chemistry , Depsipeptides/chemical synthesis , Klebsiella pneumoniae/drug effects , Escherichia coli/drug effects , Gram-Negative Bacteria/drug effects , Gram-Positive Bacteria/drug effects , Peptides, Cyclic/pharmacology , Peptides, Cyclic/chemical synthesis , Peptides, Cyclic/chemistryABSTRACT
Chloroplasts are photosynthetic organelles in algal and plant cells that contain their own genome. Chloroplast genomes are commonly used in evolutionary studies and taxonomic identification and are increasingly becoming a target for crop improvement studies. As DNA sequencing becomes more affordable, researchers are collecting vast swathes of high-quality whole-genome sequence data from laboratory and field settings alike. Whole tissue read libraries sequenced with the primary goal of understanding the nuclear genome will inadvertently contain many reads derived from the chloroplast genome. These whole-genome, whole-tissue read libraries can additionally be used to assemble chloroplast genomes with little to no extra cost. While several tools exist that make use of short-read second generation and third-generation long-read sequencing data for chloroplast genome assembly, these tools may have complex installation steps, inadequate error reporting, poor expandability, and/or lack scalability. Here, we present CLAW (Chloroplast Long-read Assembly Workflow), an easy to install, customise, and use Snakemake tool to assemble chloroplast genomes from chloroplast long-reads found in whole-genome read libraries (https://github.com/aaronphillips7493/CLAW). Using 19 publicly available reference chloroplast genome assemblies and long-read libraries from algal, monocot and eudicot species, we show that CLAW can rapidly produce chloroplast genome assemblies with high similarity to the reference assemblies. CLAW was designed such that users have complete control over parameterisation, allowing individuals to optimise CLAW to their specific use cases. We expect that CLAW will provide researchers (with varying levels of bioinformatics expertise) with an additional resource useful for contributing to the growing number of publicly available chloroplast genome assemblies.
Subject(s)
Genome, Chloroplast , Humans , Genome, Chloroplast/genetics , Workflow , Sequence Analysis, DNA , Computational Biology , Chloroplasts/genetics , High-Throughput Nucleotide SequencingABSTRACT
Soil application of Ca- and Mg-rich silicates can capture and store atmospheric carbon dioxide as inorganic carbon but could also have the potential to stabilise soil organic matter (SOM). Synergies between these two processes have not been investigated. Here, we apply finely ground silicate rock mining residues (basalt and granite blend) to a loamy sand in a pot trial at a rate of 4% (equivalent to 50 t ha-1 ) and investigate the effects of a wheat plant and two watering regimes on soil carbon sequestration over the course of 6 months. Rock dust addition increased soil pH, electric conductivity, inorganic carbon content and soil-exchangeable Ca and Mg contents, as expected for weathering. However, it decreased exchangeable levels of micronutrients Mn and Zn, likely related to the elevated soil pH. Importantly, it increased mineral-associated organic matter by 22% due to the supply of secondary minerals and associated sites for SOM sorption. Additionally, in the nonplanted treatments, rock supply of Ca and Mg increased soil microaggregation that subsequently stabilised labile particulate organic matter as organic matter occluded in aggregates by 46%. Plants, however, reduced soil-exchangeable Mg and Ca contents and hence counteracted the silicate rock effect on microaggregates and carbon within. We suggest this cation loss might be attributed to plant exudates released to solubilise micronutrients and hence neutralise plant deficiencies. The effect of enhanced silicate rock weathering on SOM stabilisation could substantially boost its carbon sequestration potential.
Subject(s)
Dust , Soil , Soil/chemistry , Silicates , Minerals , Plants , MicronutrientsABSTRACT
The oxygen partial pressure within the interstitial space (PO2is; mmHg) provides the driving force for oxygen diffusion into the myocyte thereby supporting oxidative phosphorylation. We tested the hypothesis that potentiation of the nitric oxide pathway with sildenafil (phosphodiesterase type 5 inhibitor) would enhance PO2is during muscle metabolic transitions, thereby slowing PO2is on- and accelerating PO2is off-kinetics. The rat spinotrapezius muscle (n = 17) was exposed for PO2is measurements via phosphorescence quenching under control (CON), low-dose sildenafil (1 mg/kg i.a., SIL1) and high-dose sildenafil (7 mg/kg i.a., SIL7). Data were collected at rest and during submaximal twitch contractions (1 Hz, 4-6 V, 3 min) and recovery (3 min). Mean arterial blood pressure (MAP; mmHg) was reduced with both SIL1 (pre:132 ± 5; post:99 ± 5) and SIL7 (pre:111 ± 6; post:99 ± 4) (p < 0.05). SIL7 elevated resting PO2is (18.4 ± 1.1) relative to both CON (15.7 ± 0.7) and SIL1 (15.2 ± 0.7) (p < 0.05). In addition, SIL7 increased end-recovery PO2is (17.7 ± 1.6) compared to CON (12.8 ± 0.9) and SIL1 (13.4 ± 0.8) (p < 0.05). The overall PO2is response during recovery (i.e., area under the PO2is curve) was greater in SIL7 (4107 ± 444) compared to CON (3493 ± 222) and SIL1 (3114 ± 205 mmHg s) (p < 0.05). Contrary to our hypothesis, there was no impact of acute SIL (1 or 7 mg/kg) on the speed of the PO2is response during contractions or recovery (p > 0.05). However, sildenafil lowered MAP and improved skeletal muscle interstitial oxygenation in healthy rats. Specifically, SIL7 enhanced PO2is at rest and during recovery from submaximal muscle contractions. Potentiation of the nitric oxide pathway with sildenafil enhances microvascular blood-myocyte O2 transport and is expected to improve repeated bouts of contractile activity.
Subject(s)
Nitric Oxide , Oxygen Consumption , Rats , Animals , Rats, Sprague-Dawley , Nitric Oxide/metabolism , Cyclic Nucleotide Phosphodiesterases, Type 5/metabolism , Sildenafil Citrate/pharmacology , Muscle, Skeletal/metabolism , Muscle Contraction , Oxygen/metabolism , MicrocirculationABSTRACT
Currently approved blood biomarkers detect intracranial lesions in adult patients with mild to moderate traumatic brain injury (TBI) acutely post-injury. However, blood biomarkers are still needed to help with a differential diagnosis of mild TBI (mTBI) and post-traumatic stress disorder (PTSD) at chronic post-injury time points. Owing to the association between phospholipid (PL) dysfunction and chronic consequences of TBI, we hypothesized that examining bioactive PL metabolites (oxylipins and ethanolamides) would help identify long-term lipid changes associated with mTBI and PTSD. Lipid extracts of plasma from active-duty soldiers deployed to the Iraq/Afghanistan wars (control = 52, mTBI = 21, PTSD = 34, and TBI + PTSD = 13) were subjected to liquid chromatography/mass spectrometry analysis to examine oxylipins and ethanolamides. Linear regression analyses followed by post hoc comparisons were performed to assess the association of these lipids with diagnostic classifications. Significant differences were found in oxylipins derived from arachidonic acid (AA) between controls and mTBI, PTSD, and mTBI + PTSD groups. Levels of AA-derived oxylipins through the cytochrome P450 pathways and anandamide were significantly elevated among mTBI + PTSD patients who were carriers of the apolipoprotein E E4 allele. These studies demonstrate that AA-derived oxylipins and anandamide may be unique blood biomarkers of PTSD and mTBI + PTSD. Further, these AA metabolites may be indicative of an underlying inflammatory process that warrants further investigation. Future validation studies in larger cohorts are required to determine a potential application of this approach in providing a differential diagnosis of mTBI and PTSD in a clinical setting.
ABSTRACT
Traumatic optic neuropathy (TON) is a condition in which acute injury to the optic nerve from direct or indirect trauma results in vision loss. The most common cause of TON is indirect injury to the optic nerve caused by concussive forces that are transmitted to the optic nerve. TON occurs in up to 5% of closed-head trauma patients and there is currently no known effective treatment. One potential treatment option for TON is ST266, a cell-free biological solution containing the secretome of amnion-derived multipotent progenitor (AMP) cells. We investigated the efficacy of intranasal ST266 in a mouse model of TON induced by blunt head trauma. Injured mice treated with a 10-day regimen of ST266 showed an improvement in spatial memory and learning, a significant preservation of retinal ganglion cells, and a decrease in neuropathological markers in the optic nerve, optic tract, and dorsal lateral geniculate nucleus. ST266 treatment effectively downregulated the NLRP3 inflammasome-mediated neuroinflammation pathway after blunt trauma. Overall, treatment with ST266 was shown to improve functional and pathological outcomes in a mouse model of TON, warranting future exploration of ST266 as a cell-free therapeutic candidate for testing in all optic neuropathies.
ABSTRACT
Exosomes and extracellular vesicles (EV) are increasingly being explored as circulating biomarkers, but their heterogenous composition will likely mandate the development of multiplexed EV technologies. Iteratively multiplexed analyses of near single EVs have been challenging to implement beyond a few colors during spectral sensing. Here we developed a multiplexed analysis of EV technique (MASEV) to interrogate thousands of individual EVs during 5 cycles of multi-channel fluorescence staining for 15 EV biomarkers. Contrary to the common belief, we show that: several markers proposed to be ubiquitous are less prevalent than believed; multiple biomarkers concur in single vesicles but only in small fractions; affinity purification can lead to loss of rare EV subtypes; and deep profiling allows detailed analysis of EV, potentially improving the diagnostic content. These findings establish the potential of MASEV for uncovering fundamental EV biology and heterogeneity and increasing diagnostic specificity.
Subject(s)
Exosomes , Extracellular Vesicles , Biomarkers , Chromatography, Affinity , Staining and LabelingABSTRACT
Massively parallel, second-generation short-read DNA sequencing has become an integral tool in biology for genomic studies. Offering highly accurate base-pair resolution at the most competitive price, the technology has become widespread. However, high-throughput generation of multiplexed DNA libraries can be costly and cumbersome. Here, we present a cost-conscious protocol for generating multiplexed short-read DNA libraries using a bead-linked transposome from Illumina. We prepare libraries in high-throughput with small reaction volumes that use 1/50th the amount of transposome compared to Illumina DNA Prep tagmentation protocols. By reducing transposome usage and optimising the protocol to circumvent magnetic bead-based clean-ups between steps, we reduce costs, labour time and DNA input requirements. Developing our own dual index primers further reduced costs and enables up to nine 96-well microplate combinations. This facilitates efficient usage of large-scale sequencing platforms, such as the Illumina NovaSeq 6000, which offers up to three terabases of sequencing per S4 flow cell. The protocol presented substantially reduces the cost per library by approximately 1/20th compared to conventional Illumina methods.
Subject(s)
DNA , Genome , Gene Library , DNA/genetics , Sequence Analysis, DNA/methods , Genomics , High-Throughput Nucleotide Sequencing/methodsABSTRACT
Synteny, the ordering of sequences within homologous chromosomes, must be maintained within the genomes of sexually reproducing species for the sharing of alleles and production of viable, reproducing offspring. However, when the genomes of closely related species are compared, a loss of synteny is often observed. Unequal homologous recombination is the primary mechanism behind synteny loss, occurring more often in transposon rich regions, and resulting in the formation of chromosomal rearrangements. To examine patterns of synteny among three closely related, interbreeding, and wild Eucalyptus species, we assembled their genomes using long-read DNA sequencing and de novo assembly. We identify syntenic and rearranged regions between these genomes and estimate that ~48% of our genomes remain syntenic while ~36% is rearranged. We observed that rearrangements highly fragment microsynteny. Our results suggest that synteny between these species is primarily lost through small-scale rearrangements, not through sequence loss, gain, or sequence divergence. Further examination of identified rearrangements suggests that rearrangements may be altering the phenotypes of Eucalyptus species. Our study also underscores that the use of single reference genomes in genomic variation studies could lead to reference bias, especially given the scale at which we show potentially adaptive loci have highly diverged, deleted, duplicated and/or rearranged. This study provides an unbiased framework to look at potential speciation and adaptive loci among a rapidly radiating foundation species of woodland trees that are free from selective breeding seen in most crop species.
Subject(s)
Eucalyptus , Eucalyptus/genetics , Genome , Synteny/genetics , Chromosomes , Sequence Analysis, DNA/methodsABSTRACT
The recent discovery of comammox Nitrospira, a complete ammonia oxidizer, capable of completing the nitrification on their own has presented tremendous challenges to our understanding of the nitrification process. There are two divergent clades of comammox Nitrospira, Clade A and B. However, their population abundance, community structure and role in ammonia and nitrite oxidation are poorly understood. We conducted a 94-day microcosm study using a grazed dairy pasture soil amended with urea fertilizers, synthetic cow urine, and the nitrification inhibitor, dicyandiamide (DCD), to investigate the growth and community structure of comammox Nitrospira spp. We discovered that comammox Nitrospira Clade B was two orders of magnitude more abundant than Clade A in this fertile dairy pasture soil and the most abundant subcluster was a distinctive phylogenetic uncultured subcluster Clade B2. We found that comammox Nitrospira Clade B might not play a major role in nitrite oxidation compared to the role of canonical Nitrospira nitrite-oxidizers, however, comammox Nitrospira Clade B is active in nitrification and the growth of comammox Nitrospira Clade B was inhibited by a high ammonium concentration (700 kg synthetic urine-N ha-1) and the nitrification inhibitor DCD. We concluded that comammox Nitrospira Clade B: (1) was the most abundant comammox in the dairy pasture soil; (2) had a low tolerance to ammonium and can be inhibited by DCD; and (3) was not the dominant nitrite-oxidizer in the soil. This is the first study discovering a new subcluster of comammox Nitrospira Clade B2 from an agricultural soil.
ABSTRACT
BACKGROUND: Long-read sequencing platforms offered by Oxford Nanopore Technologies (ONT) allow native DNA containing epigenetic modifications to be directly sequenced, but can be limited by lower per-base accuracies. A key step post-sequencing is basecalling, the process of converting raw electrical signals produced by the sequencing device into nucleotide sequences. This is challenging as current basecallers are primarily based on mixtures of model species for training. Here we utilise both ONT PromethION and higher accuracy PacBio Sequel II HiFi sequencing on two plants, Phebalium stellatum and Xanthorrhoea johnsonii, to train species-specific basecaller models with the aim of improving per-base accuracy. We investigate sequencing accuracies achieved by ONT basecallers and assess accuracy gains by training single-species and species-specific basecaller models. We also evaluate accuracy gains from ONT's improved flowcells (R10.4, FLO-PRO112) and sequencing kits (SQK-LSK112). For the truth dataset for both model training and accuracy assessment, we developed highly accurate, contiguous diploid reference genomes with PacBio Sequel II HiFi reads. RESULTS: Basecalling with ONT Guppy 5 and 6 super-accurate gave almost identical results, attaining read accuracies of 91.96% and 94.15%. Guppy's plant-specific model gave highly mixed results, attaining read accuracies of 91.47% and 96.18%. Species-specific basecalling models improved read accuracy, attaining 93.24% and 95.16% read accuracies. R10.4 sequencing kits also improve sequencing accuracy, attaining read accuracies of 95.46% (super-accurate) and 96.87% (species-specific). CONCLUSIONS: The use of a single mixed-species basecaller model, such as ONT Guppy super-accurate, may be reducing the accuracy of nanopore sequencing, due to conflicting genome biology within the training dataset and study species. Training of single-species and genome-specific basecaller models improves read accuracy. Studies that aim to do large-scale long-read genotyping would primarily benefit from training their own basecalling models. Such studies could use sequencing accuracy gains and improving bioinformatics tools to improve study outcomes.
ABSTRACT
With the post-antibiotic era rapidly approaching, many have turned their attention to developing new treatments, often by structural modification of existing antibiotics. Polymyxins, a family of lipopeptide antibiotics that are used as a last line of defense in the clinic, have recently developed resistance and exhibit significant nephrotoxicity issues. Using thiol-ene chemistry, the facile preparation of six unique S-lipidated building blocks was demonstrated and used to generate lipopeptide mimetics upon incorporation into solid-phase peptide synthesis (SPPS). We then designed and synthesized 38 polymyxin analogues, incorporating these unique building blocks at the N-terminus, or to replace hydrophobic residues at positions 6 and 7 of the native lipopeptides. Several polymyxin analogues bearing one or more S-linked lipids were found to be equipotent to polymyxin, showed minimal kidney nephrotoxicity, and demonstrated activity against several World Health Organisation (WHO) priority pathogens. The S-lipidation strategy has demonstrated potential as a novel approach to prepare innovative new lipopeptide antibiotics.
Subject(s)
Anti-Bacterial Agents , Polymyxin B , Anti-Bacterial Agents/pharmacologyABSTRACT
Chemical and pharmaceutical exposures have been associated with the development of Gulf War Illness (GWI), but how these factors interact with the pathophysiology of traumatic brain injury (TBI) remains an area of study that has received little attention thus far. We studied the effects of pyridostigmine bromide (an anti-nerve agent) and permethrin (a pesticide) exposure in a mouse model of repetitive mild TBI (r-mTBI), with 5 impacts over a 9-day period, followed by Gulf War (GW) toxicant exposure for 10 days beginning 30 days after the last head injury. We then assessed the chronic behavioral and pathological sequelae 5 months after GW agent exposure. We observed that r-mTBI and GWI cumulatively affect the spatial memory of mice in the Barnes maze and result in a shift of search strategies employed by r-mTBI/GW exposed mice. GW exposure also produced anxiety-like behavior in sham animals, but r-mTBI produced disinhibition in both the vehicle and GW treated mice. Pathologically, GW exposure worsened r-mTBI dependent axonal degeneration and neuroinflammation, increased oligodendrocyte cell counts, and increased r-mTBI dependent phosphorylated tau, which was found to colocalize with oligodendrocytes in the corpus callosum. These results suggest that GW exposures may worsen TBI-related deficits. Veterans with a history of both GW chemical exposures as well as TBI may be at higher risk for worse symptoms and outcomes. Subsequent exposure to various toxic substances can influence the chronic nature of mTBI and should be considered as an etiological factor influencing mTBI recovery.