Temporal dynamics and metagenomics of phosphorothioate epigenomes in the human gut microbiome.

Background: Epigenetic regulation of gene expression and host defense is well established in microbial communities, with dozens of DNA modifications comprising the epigenomes of prokaryotes and bacteriophage. Phosphorothioation (PT) of DNA, in which a chemically-reactive sulfur atom replaces a non-bridging oxygen in the sugar-phosphate backbone, is catalyzed by dnd and ssp gene families widespread in bacteria and archaea. However, little is known about the role of PTs or other microbial epigenetic modifications in the human microbiome. Here we optimized and applied fecal DNA extraction, mass spectrometric, and metagenomics technologies to characterize the landscape and temporal dynamics of gut microbes possessing PT modifications. Results: Exploiting the nuclease-resistance of PTs, mass spectrometric analysis of limit digests of PT-containing DNA reveals PT dinucleotides as part of genomic consensus sequences, with 16 possible dinucleotide combinations. Analysis of mouse fecal DNA revealed a highly uniform spectrum of 11 PT dinucleotides in all littermates, with PTs estimated to occur in 5-10% of gut microbes. Though at similar levels, PT dinucleotides in fecal DNA from 11 healthy humans possessed signature combinations and levels of individual PTs. Comparison with a widely distributed microbial epigenetic mark, m 6 dA, suggested temporal dynamics consistent with expectations for gut microbial communities based on Taylor's Power Law. Application of PT-seq for site-specific metagenomic analysis of PT-containing bacteria in one fecal donor revealed the larger consensus sequences for the PT dinucleotides in Bacteroidota, Firmicutes, Actinobacteria, and Proteobacteria, which differed from unbiased metagenomics and suggested that the abundance of PT-containing bacteria did not simply mirror the spectrum of gut bacteria. PT-seq further revealed low abundance PT sites not detected as dinucleotides by mass spectrometry, attesting to the complementarity of the technologies. Conclusions: The results of our studies provide a benchmark for understanding the behavior of an abundant and chemically-reactive epigenetic mark in the human gut microbiome, with implications for inflammatory conditions of the gut.

Translational response to mitochondrial stresses is orchestrated by tRNA modifications.

Mitochondrial stress and dysfunction play important roles in many pathologies. However, how cells respond to mitochondrial stress is not fully understood. Here, we examined the translational response to electron transport chain (ETC) inhibition and arsenite induced mitochondrial stresses. Our analysis revealed that during mitochondrial stress, tRNA modifications (namely f5C, hm5C, queuosine and its derivatives, and mcm5U) dynamically change to fine tune codon decoding, usage, and optimality. These changes in codon optimality drive the translation of many pathways and gene sets, such as the ATF4 pathway and selenoproteins, involved in the cellular response to mitochondrial stress. We further examined several of these modifications using targeted approaches. ALKBH1 knockout (KO) abrogated f5C and hm5C levels and led to mitochondrial dysfunction, reduced proliferation, and impacted mRNA translation rates. Our analysis revealed that tRNA queuosine (tRNA-Q) is a master regulator of the mitochondrial stress response. KO of QTRT1 or QTRT2, the enzymes responsible for tRNA-Q synthesis, led to mitochondrial dysfunction, translational dysregulation, and metabolic alterations in mitochondria-related pathways, without altering cellular proliferation. In addition, our analysis revealed that tRNA-Q loss led to a domino effect on various tRNA modifications. Some of these changes could be explained by metabolic profiling. Our analysis also revealed that utilizing serum deprivation or alteration with Queuine supplementation to study tRNA-Q or stress response can introduce various confounding factors by altering many other tRNA modifications. In summary, our data show that tRNA modifications are master regulators of the mitochondrial stress response by driving changes in codon decoding.

DNA Modifications Enabling Proximity Biotinylation.

Advances in peroxidase and biotin ligase-mediated signal amplification have enabled high-resolution subcellular mapping of endogenous RNA localization and protein-protein interactions. Application of these technologies has been limited to RNA and proteins because of the reactive groups required for biotinylation in each context. Here we report several novel methods for proximity biotinylation of exogenous oligodeoxyribonucleotides by application of well-established and convenient enzymatic tools. We describe approaches using simple and efficient conjugation chemistries to modify deoxyribonucleotides with "antennae" that react with phenoxy radicals or biotinoyl-5'-adenylate. In addition, we report chemical details of a previously undescribed adduct between tryptophan and a phenoxy radical group. These developments have potential application in the selection of exogenous nucleic acids capable of unaided entry into living cells.

The absence of the queuosine tRNA modification leads to pleiotropic phenotypes revealing perturbations of metal and oxidative stress homeostasis in Escherichia coli K12.

Queuosine (Q) is a conserved hypermodification of the wobble base of tRNA containing GUN anticodons but the physiological consequences of Q deficiency are poorly understood in bacteria. This work combines transcriptomic, proteomic and physiological studies to characterize a Q-deficient Escherichia coli K12 MG1655 mutant. The absence of Q led to an increased resistance to nickel and cobalt, and to an increased sensitivity to cadmium, compared to the wild-type (WT) strain. Transcriptomic analysis of the WT and Q-deficient strains, grown in the presence and absence of nickel, revealed that the nickel transporter genes (nikABCDE) are downregulated in the Q- mutant, even when nickel is not added. This mutant is therefore primed to resist to high nickel levels. Downstream analysis of the transcriptomic data suggested that the absence of Q triggers an atypical oxidative stress response, confirmed by the detection of slightly elevated reactive oxygen species (ROS) levels in the mutant, increased sensitivity to hydrogen peroxide and paraquat, and a subtle growth phenotype in a strain prone to accumulation of ROS.

Codon Usage and mRNA Stability are Translational Determinants of Cellular Response to Canonical Ferroptosis Inducers.

Ferroptosis is a non-apoptotic cell death mechanism characterized by the generation of lipid peroxides. While many effectors in the ferroptosis pathway have been mapped, its epitranscriptional regulation is not yet fully understood. Ferroptosis can be induced via system xCT inhibition (Class I) or GPX4 inhibition (Class II). Previous works have revealed important differences in cellular response to different ferroptosis inducers. Importantly, blocking mRNA transcription or translation appears to protect cells against Class I ferroptosis inducing agents but not Class II. In this work, we examined the impact of blocking transcription (via Actinomycin D) or translation (via Cycloheximide) on Erastin (Class I) or RSL3 (Class II) induced ferroptosis. Blocking transcription or translation protected cells against Erastin but was detrimental against RSL3. Cycloheximide led to increased levels of GSH alone or when co-treated with Erastin via the activation of the reverse transsulfuration pathway. RNA sequencing analysis revealed early activation of a strong alternative splice program before observed changes in transcription. mRNA stability analysis revealed divergent mRNA stability changes in cellular response to Erastin or RSL3. Importantly, codon optimality biases were drastically different in either condition. Our data also implicated translation repression and rate as an important determinant of the cellular response to ferroptosis inducers. Given that mRNA stability and codon usage can be influenced via the tRNA epitranscriptome, we evaluated the role of a tRNA modifying enzyme in ferroptosis stress response. Alkbh1, a tRNA demethylase, led to translation repression and increased the resistance to Erastin but made cells more sensitive to RSL3.

Orbital Forcing of Martian Climate Revealed in a South Polar Outlier Ice Deposit.

Deciphering paleoclimate on Mars has been a driving goal of Martian science for decades. Most research has addressed this issue by studying Mars' large polar layered deposits (PLDs) as a paleoclimate proxy, but the certainty to which we know the link between climate and orbit is debated. Here, we instead consider the record of other, smaller ice deposits located within craters separated from the PLDs using images from NASA's High Resolution Imaging Science Experiment camera and signal processing techniques. We show that the climate record in Burroughs Crater (72.3°S, 116.6°E) contains robust evidence of orbital forcing, with periodicities that have wavelengths of 15.6 and 6.5 m. The ratio of these dominant wavelengths is 2.4, the same as the ratio between the periods of Mars' obliquity changes and orbital precession. This result suggests orbital control of recent Mars climate, and would imply an average ice accumulation rate of 0.13 mm/yr over 4.5 Myr in this region.

Strategies to Avoid Artifacts in Mass Spectrometry-Based Epitranscriptome Analyses.

In this report, we perform structure validation of recently reported RNA phosphorothioate (PT) modifications, a new set of epitranscriptome marks found in bacteria and eukaryotes including humans. By comparing synthetic PT-containing diribonucleotides with native species in RNA hydrolysates by high-resolution mass spectrometry (MS), metabolic stable isotope labeling, and PT-specific iodine-desulfurization, we disprove the existence of PTs in RNA from E. coli, S. cerevisiae, human cell lines, and mouse brain. Furthermore, we discuss how an MS artifact led to the initial misidentification of 2'-O-methylated diribonucleotides as RNA phosphorothioates. To aid structure validation of new nucleic acid modifications, we present a detailed guideline for MS analysis of RNA hydrolysates, emphasizing how the chosen RNA hydrolysis protocol can be a decisive factor in discovering and quantifying RNA modifications in biological samples.

Active Mars: A Dynamic World.

Mars exhibits diverse surface changes at all latitudes and all seasons. Active processes include impact cratering, aeolian sand and dust transport, a variety of slope processes, changes in polar ices, and diverse effects of seasonal CO2 frost. The extent of surface change has been surprising and indicates that the present climate is capable of reshaping the surface. Activity has important implications for the Amazonian history of Mars: understanding processes is a necessary step before we can understand their implications and variations over time.

Phenoxy Radical Reactivity of Nucleic Acids: Practical Implications for Biotinylation.

Recent advances in peroxidase-mediated biotin tyramide (BT) signal amplification technology have resulted in high-resolution and subcellular compartment-specific mapping of protein and RNA localization. Horseradish peroxidase (HRP) in the presence of H2 O2 is known to activate phenolic compounds for phenoxy radical reaction with nucleic acids, where biotinylation by BT is a practical example. BT reactivity with RNA and DNA is not understood in detail. We report that BT phenoxy radicals react in a sequence-independent manner with guanosine bases in RNA. In contrast, DNA reactivity with BT cannot be detected by our methods under the same conditions. Remarkably, we show that fluorescein conjugates DNA rapidly and selectively reacts with BT phenoxy radicals, allowing convenient and practical biotinylation of DNA on fluorescein with retention of fluorescence.

Unraveling Reversible DNA Cross-Links with a Biological Machine.

The reversible generation and capture of certain electrophilic quinone methide intermediates support dynamic reactions with DNA that allow for migration and transfer of alkylation and cross-linking. This reversibility also expands the possible consequences that can be envisioned when confronted by DNA repair processes and biological machines. To begin testing the response to such an encounter, quinone methide-based modification of DNA has now been challenged with a helicase (T7 bacteriophage gene protein four, T7gp4) that promotes 5' to 3' translocation and unwinding. This model protein was selected based on its widespread application, well characterized mechanism and detailed structural information. Little over one-half of the cross-linking generated by a bisfunctional quinone methide remained stable to T7gp4 and did not suppress its activity. The helicase likely avoids the topological block generated by this fraction of cross-linking by its ability to shift from single- to double-stranded translocation. The remaining fraction of cross-linking was destroyed during T7gp4 catalysis. Thus, this helicase is chemically competent to promote release of the quinone methide from DNA. The ability of T7gp4 to act as a Brownian ratchet for unwinding DNA may block recapture of the QM intermediate by DNA during its transient release from a donor strand. Most surprisingly, T7gp4 releases the quinone methide from both the translocating strand that passes through its central channel and the excluded strand that was typically unaffected by other lesions. The ability of T7gp4 to reverse the cross-link formed by the quinone methide does not extend to that formed irreversibly by the nitrogen mustard mechlorethamine.

Self-collection: An appropriate alternative during the SARS-CoV-2 pandemic.

OBJECTIVES: To evaluate the reliability of self-collection for SARS-CoV-2 and other respiratory viruses because swab collections for SARS-CoV-2 put health workers at risk of infection and require use of personal protective equipment (PPE). METHODS: In a prospective study, patients from two states in Australia attending dedicated COVID-19 collection clinics were offered the option to first self-collect (SC) nasal and throat swabs (SCNT) prior to health worker collect (HC) using throat and nasal swabs (Site 1) or throat and nasopharyngeal swabs (Site 2). Samples were analysed for SARS-CoV-2 as well as common respiratory viruses. Concordance of results between methods was assessed using Cohen's kappa (κ) and Cycle threshold (Ct) values were recorded for all positive results as a surrogate measure for viral load. RESULTS: Of 236 patients sampled by HC and SC, 25 had SARS-CoV-2 (24 by HC and 25 by SC) and 63 had other respiratory viruses (56 by HC and 58 by SC). SC was highly concordant with HC (κ = 0.890) for all viruses including SARS-CoV-2 and more concordant than HC to positive results by any method (κ = 0.959 vs 0.933). Mean SARS-CoV-2 E-gene and N-gene, rhinovirus and parainfluenza Ct values did not differ between HC and SCNT. CONCLUSIONS: Self-collection of nasal and throat swabs offers a reliable alternative to health worker collection for the diagnosis of SARS-CoV-2 and other respiratory viruses and provides patients with easier access to testing, reduces exposure of the community and health workers to those being tested and reduces requirement for PPE.

Directing Quinone Methide-Dependent Alkylation and Cross-Linking of Nucleic Acids with Quaternary Amines.

Polyamine and polyammonium ion conjugates are often used to direct reagents to nucleic acids based on their strong electrostatic attraction to the phosphoribose backbone. Such nonspecific interactions do not typically alter the specificity of the attached reagent, but polyammonium ions dramatically redirected the specificity of a series of quinone methide precursors. Replacement of a relatively nonspecific intercalator based on acridine with a series of polyammonium ions resulted in a surprising change of DNA products. Piperidine stable adducts were generated in duplex DNA that lacked the ability to support a dynamic cross-linking observed previously with acridine conjugates. Minor reaction at guanine N7, the site of reversible reaction, was retained by a monofunctional quinone methide-polyammonium ion conjugate, but a bisfunctional analogue designed for tandem quinone methide formation modified guanine N7 in only single-stranded DNA. The resulting intrastrand cross-links were sufficiently dynamic to rearrange to interstrand cross-links. However, no further transfer of adducts was observed in duplex DNA. An alternative design that spatially and temporally decoupled the two quinone methide equivalents neither restored the dynamic reaction nor cross-linked DNA efficiently. While di- and triammonium ion conjugates successfully enhanced the yields of cross-linking by a bisquinone methide relative to a monoammonium equivalent, alternative ligands will be necessary to facilitate the migration of cross-linking and its potential application to disrupt DNA repair.

Effect of Nucleosome Assembly on Alkylation by a Dynamic Electrophile.

Quinone methides are reactive electrophiles that are generated during metabolism of various drugs, natural products, and food additives. Their chemical properties and cellular effects have been described previously, and now their response to packaging DNA in a nucleosome core is described. A model bisquinone methide precursor (bisQMP) was selected based on its ability to form reversible adducts with guanine N7 that allow for their redistribution and transfer after quinone methide regeneration. Assembly of Widom's 601 DNA with the histone octamer of H2A, H2B, H3, and H4 from Xenopus laevis significantly suppressed alkylation of the DNA. This result is a function of DNA packaging since addition of the octamer without nucleosome reconstitution only mildly protected DNA from alkylation. The lack of competition between nucleophiles of DNA and the histones was consistent with the limited number of adducts formed by the histones as detected by tryptic digestion and ultraperformance liquid chromatography-mass spectrometry. Only three peptide adducts were observed after reaction with a monofunctional analogue of bisQMP, and only two peptide adducts were observed after reaction with bisQMP. Histone reaction was also suppressed when reconstituted into the nucleosome core particle. However, bisQMP was capable of cross-linking the DNA and histones in moderate yields (∼20%) that exceeded expectations derived from reaction of cisplatin, nitrogen mustards, and diepoxybutane. The core histones also demonstrated a protective function against dynamic alkylation by trapping the reactive quinone methide after its spontaneous regeneration from DNA adducts.

Exposed subsurface ice sheets in the Martian mid-latitudes.

Thick deposits cover broad regions of the Martian mid-latitudes with a smooth mantle; erosion in these regions creates scarps that expose the internal structure of the mantle. We investigated eight of these locations and found that they expose deposits of water ice that can be >100 meters thick, extending downward from depths as shallow as 1 to 2 meters below the surface. The scarps are actively retreating because of sublimation of the exposed water ice. The ice deposits likely originated as snowfall during Mars' high-obliquity periods and have now compacted into massive, fractured, and layered ice. We expect the vertical structure of Martian ice-rich deposits to preserve a record of ice deposition and past climate.

Detection of local H2O exposed at the surface of Ceres.

The surface of dwarf planet Ceres contains hydroxyl-rich materials. Theories predict a water ice-rich mantle, and water vapor emissions have been observed, yet no water (H2O) has been previously identified. The Visible and InfraRed (VIR) mapping spectrometer onboard the Dawn spacecraft has now detected water absorption features within a low-illumination, highly reflective zone in Oxo, a 10-kilometer, geologically fresh crater, on five occasions over a period of 1 month. Candidate materials are H2O ice and mineral hydrates. Exposed H2O ice would become optically undetectable within tens of years under current Ceres temperatures; consequently, only a relatively recent exposure or formation of H2O would explain Dawn's findings. Some mineral hydrates are stable on geological time scales, but their formation would imply extended contact with ice or liquid H2O.

Creating the nursing associate role is a waste of time and money.

There has been a lot of talk about the new 'nursing associate' role Health Education England (HEE) is looking to create.

Advances in molecular diagnosis of parasitic enteropathogens.

Here, recent developments in the detection and identification of parasites causing enteric infection are reviewed including the utility and challenges of multi-target molecular assays. Difficulties in clinical interpretation arising from increased detection of parasites, of co-infection with other enteropathogens and of asymptomatic carriage are discussed. Published approaches for detection across a broad range of organisms are described, including commercial assays available to Australian laboratories. Using local data, the impact of introduction of modern molecular assays is assessed. In addition, recent advances in high density multi-target molecular platforms are discussed as potential platforms for increasing the scope of enteric pathogens to be detected whilst maintaining appropriate costs.

IHG-1 increases mitochondrial fusion and bioenergetic function.

Induced in high glucose-1 (IHG-1) is a conserved mitochondrial protein associated with diabetic nephropathy (DN) that amplifies profibrotic transforming growth factor (TGF)-ß1 signaling and increases mitochondrial biogenesis. Here we report that inhibition of endogenous IHG-1 expression results in reduced mitochondrial respiratory capacity, ATP production, and mitochondrial fusion. Conversely, overexpression of IHG-1 leads to increased mitochondrial fusion and also protects cells from reactive oxygen species-induced apoptosis. IHG-1 forms complexes with known mediators of mitochondrial fusion-mitofusins (Mfns) 1 and 2-and enhances the GTP-binding capacity of Mfn2, suggesting that IHG-1 acts as a guanine nucleotide exchange factor. IHG-1 must be localized to mitochondria to interact with Mfn1 and Mfn2, and this interaction is necessary for increased IHG-1-mediated mitochondrial fusion. Together, these findings indicate that IHG-1 is a novel regulator of both mitochondrial dynamics and bioenergetic function and contributes to cell survival following oxidant stress. We propose that in diabetic kidney disease increased IHG-1 expression protects cell viability and enhances the actions of TGF-ß, leading to renal proximal tubule dedifferentiation, an important event in the pathogenesis of this devastating condition.

Seasonal flows on warm Martian slopes.

Water probably flowed across ancient Mars, but whether it ever exists as a liquid on the surface today remains debatable. Recurring slope lineae (RSL) are narrow (0.5 to 5 meters), relatively dark markings on steep (25° to 40°) slopes; repeat images from the Mars Reconnaissance Orbiter High Resolution Imaging Science Experiment show them to appear and incrementally grow during warm seasons and fade in cold seasons. They extend downslope from bedrock outcrops, often associated with small channels, and hundreds of them form in some rare locations. RSL appear and lengthen in the late southern spring and summer from 48°S to 32°S latitudes favoring equator-facing slopes, which are times and places with peak surface temperatures from ~250 to 300 kelvin. Liquid brines near the surface might explain this activity, but the exact mechanism and source of water are not understood.

Massive CO2 ice deposits sequestered in the south polar layered deposits of Mars.

Shallow Radar soundings from the Mars Reconnaissance Orbiter reveal a buried deposit of carbon dioxide (CO(2)) ice within the south polar layered deposits of Mars with a volume of 9500 to 12,500 cubic kilometers, about 30 times that previously estimated for the south pole residual cap. The deposit occurs within a stratigraphic unit that is uniquely marked by collapse features and other evidence of interior CO(2) volatile release. If released into the atmosphere at times of high obliquity, the CO(2) reservoir would increase the atmospheric mass by up to 80%, leading to more frequent and intense dust storms and to more regions where liquid water could persist without boiling.