Exploring perceptions of and decision-making about CFTR modulators.

INTRODUCTION: Cystic fibrosis (CF) treatment has increasingly focused on highly effective modulators. Despite measurable benefits of modulators, there is little guidance for CF care team members on providing education and support to patients regarding initiation of these therapies. We aimed to explore patient, caregiver, and clinician perceptions of modulators and influences on decisions about starting cystic fibrosis transmembrane regulator (CFTR) modulators. METHODS: We conducted semistructured interviews with CF clinicians, adults with CF, and caregivers of children with CF. We reviewed audio recordings and coded responses to identify central themes. RESULTS: We interviewed 8 CF clinicians, 9 adults with CF, and 11 caregivers of children with CF. Themes centered on emotional responses to modulator availability, influences on decision-making, concerns about side effects, impact of modulators on planning for the future, the benefits of the multidisciplinary CF care team in supporting treatment decisions, and the unique needs of people with CF who are not eligible for modulators. Clinicians described changes in conversations about modulators since the approval of elexacaftor/tezacaftor/ivacaftor, specifically greater willingness to prescribe with less nuanced conversations with patients and/or caregivers regarding their use. CONCLUSION: Based on perspectives and experiences of CF clinicians, adults with CF, and caregivers of children with CF, we suggest clinicians approach conversations about CFTR modulators thoughtfully and thoroughly, utilizing the multidisciplinary model of CF care in exploring patient and caregiver emotions while filling in knowledge gaps, asking about treatment goals beyond potential clinical benefit, and having compassionate conversations with those who are ineligible for modulators.

Vocal learning-associated convergent evolution in mammalian proteins and regulatory elements.

Vocal production learning ("vocal learning") is a convergently evolved trait in vertebrates. To identify brain genomic elements associated with mammalian vocal learning, we integrated genomic, anatomical, and neurophysiological data from the Egyptian fruit bat (Rousettus aegyptiacus) with analyses of the genomes of 215 placental mammals. First, we identified a set of proteins evolving more slowly in vocal learners. Then, we discovered a vocal motor cortical region in the Egyptian fruit bat, an emergent vocal learner, and leveraged that knowledge to identify active cis-regulatory elements in the motor cortex of vocal learners. Machine learning methods applied to motor cortex open chromatin revealed 50 enhancers robustly associated with vocal learning whose activity tended to be lower in vocal learners. Our research implicates convergent losses of motor cortex regulatory elements in mammalian vocal learning evolution.

Aniqsaaq (To Breathe): Study protocol to develop and evaluate an Alaska Native family-based financial incentive intervention for smoking cessation.

Background: Alaska Native and American Indian (ANAI) communities in Alaska are disproportionately affected by commercial tobacco use. Financial incentive interventions promote cigarette smoking cessation, but family-level incentives have not been evaluated. We describe the study protocol to adapt and evaluate the effectiveness and implementation of a remotely delivered, family-based financial incentive intervention for cigarette smoking among Alaskan ANAI people. Methods: The study has 3 phases: 1) qualitative interviews with ANAI adults who smoke, family members, and stakeholders to inform the intervention, 2) beta-test of the intervention, and 3) randomized controlled trial (RCT) evaluating intervention reach and effectiveness on verified, prolonged smoking abstinence at 6- and 12-months post-treatment. In the RCT, adult dyads (ANAI person who smokes [index participant] and family member) recruited throughout Alaska will be randomized to a no-incentives control condition (n = 328 dyads) or a 6-month incentive intervention (n = 328 dyads). All dyads will receive cessation support and family wellness materials. Smoking status will be assessed weekly for four weeks and at three and six months. Intervention index participants will receive escalating incentives for verified smoking abstinence at each time point (maximum $750 total); the family member will receive rewards of equal value. Results: A community advisory committee contributed input on the study design and methods for relevance to ANAI people, particularly emphasizing the involvement of families. Conclusion: Our study aligns with the strength and value AIAN people place on family. Findings, processes, and resources will inform how Indigenous family members can support smoking cessation within incentive interventions. Clinical Trials Registry: NCT05209451.

Evolutionary constraint and innovation across hundreds of placental mammals.

Zoonomia is the largest comparative genomics resource for mammals produced to date. By aligning genomes for 240 species, we identify bases that, when mutated, are likely to affect fitness and alter disease risk. At least 332 million bases (~10.7%) in the human genome are unusually conserved across species (evolutionarily constrained) relative to neutrally evolving repeats, and 4552 ultraconserved elements are nearly perfectly conserved. Of 101 million significantly constrained single bases, 80% are outside protein-coding exons and half have no functional annotations in the Encyclopedia of DNA Elements (ENCODE) resource. Changes in genes and regulatory elements are associated with exceptional mammalian traits, such as hibernation, that could inform therapeutic development. Earth's vast and imperiled biodiversity offers distinctive power for identifying genetic variants that affect genome function and organismal phenotypes.

Relating enhancer genetic variation across mammals to complex phenotypes using machine learning.

Protein-coding differences between species often fail to explain phenotypic diversity, suggesting the involvement of genomic elements that regulate gene expression such as enhancers. Identifying associations between enhancers and phenotypes is challenging because enhancer activity can be tissue-dependent and functionally conserved despite low sequence conservation. We developed the Tissue-Aware Conservation Inference Toolkit (TACIT) to associate candidate enhancers with species' phenotypes using predictions from machine learning models trained on specific tissues. Applying TACIT to associate motor cortex and parvalbumin-positive interneuron enhancers with neurological phenotypes revealed dozens of enhancer-phenotype associations, including brain size-associated enhancers that interact with genes implicated in microcephaly or macrocephaly. TACIT provides a foundation for identifying enhancers associated with the evolution of any convergently evolved phenotype in any large group of species with aligned genomes.

Methotrexate suppresses psoriatic skin inflammation by inhibiting muropeptide transporter SLC46A2 activity.

Cytosolic innate immune sensing is critical for protecting barrier tissues. NOD1 and NOD2 are cytosolic sensors of small peptidoglycan fragments (muropeptides) derived from the bacterial cell wall. These muropeptides enter cells, especially epithelial cells, through unclear mechanisms. We previously implicated SLC46 transporters in muropeptide transport in Drosophila immunity. Here, we focused on Slc46a2, which was highly expressed in mammalian epidermal keratinocytes, and showed that it was critical for the delivery of diaminopimelic acid (DAP)-muropeptides and activation of NOD1 in keratinocytes, whereas the related transporter Slc46a3 was critical for delivering the NOD2 ligand MDP to keratinocytes. In a mouse model, Slc46a2 and Nod1 deficiency strongly suppressed psoriatic inflammation, whereas methotrexate, a commonly used psoriasis therapeutic, inhibited Slc46a2-dependent transport of DAP-muropeptides. Collectively, these studies define SLC46A2 as a transporter of NOD1-activating muropeptides, with critical roles in the skin barrier, and identify this transporter as an important target for anti-inflammatory intervention.

Machine learning sequence prioritization for cell type-specific enhancer design.

Recent discoveries of extreme cellular diversity in the brain warrant rapid development of technologies to access specific cell populations within heterogeneous tissue. Available approaches for engineering-targeted technologies for new neuron subtypes are low yield, involving intensive transgenic strain or virus screening. Here, we present Specific Nuclear-Anchored Independent Labeling (SNAIL), an improved virus-based strategy for cell labeling and nuclear isolation from heterogeneous tissue. SNAIL works by leveraging machine learning and other computational approaches to identify DNA sequence features that confer cell type-specific gene activation and then make a probe that drives an affinity purification-compatible reporter gene. As a proof of concept, we designed and validated two novel SNAIL probes that target parvalbumin-expressing (PV+) neurons. Nuclear isolation using SNAIL in wild-type mice is sufficient to capture characteristic open chromatin features of PV+ neurons in the cortex, striatum, and external globus pallidus. The SNAIL framework also has high utility for multispecies cell probe engineering; expression from a mouse PV+ SNAIL enhancer sequence was enriched in PV+ neurons of the macaque cortex. Expansion of this technology has broad applications in cell type-specific observation, manipulation, and therapeutics across species and disease models.

Inferring mammalian tissue-specific regulatory conservation by predicting tissue-specific differences in open chromatin.

BACKGROUND: Evolutionary conservation is an invaluable tool for inferring functional significance in the genome, including regions that are crucial across many species and those that have undergone convergent evolution. Computational methods to test for sequence conservation are dominated by algorithms that examine the ability of one or more nucleotides to align across large evolutionary distances. While these nucleotide alignment-based approaches have proven powerful for protein-coding genes and some non-coding elements, they fail to capture conservation of many enhancers, distal regulatory elements that control spatial and temporal patterns of gene expression. The function of enhancers is governed by a complex, often tissue- and cell type-specific code that links combinations of transcription factor binding sites and other regulation-related sequence patterns to regulatory activity. Thus, function of orthologous enhancer regions can be conserved across large evolutionary distances, even when nucleotide turnover is high. RESULTS: We present a new machine learning-based approach for evaluating enhancer conservation that leverages the combinatorial sequence code of enhancer activity rather than relying on the alignment of individual nucleotides. We first train a convolutional neural network model that can predict tissue-specific open chromatin, a proxy for enhancer activity, across mammals. Next, we apply that model to distinguish instances where the genome sequence would predict conserved function versus a loss of regulatory activity in that tissue. We present criteria for systematically evaluating model performance for this task and use them to demonstrate that our models accurately predict tissue-specific conservation and divergence in open chromatin between primate and rodent species, vastly out-performing leading nucleotide alignment-based approaches. We then apply our models to predict open chromatin at orthologs of brain and liver open chromatin regions across hundreds of mammals and find that brain enhancers associated with neuron activity have a stronger tendency than the general population to have predicted lineage-specific open chromatin. CONCLUSION: The framework presented here provides a mechanism to annotate tissue-specific regulatory function across hundreds of genomes and to study enhancer evolution using predicted regulatory differences rather than nucleotide-level conservation measurements.

Protected N-Acetyl Muramic Acid Probes Improve Bacterial Peptidoglycan Incorporation via Metabolic Labeling.

Metabolic glycan probes have emerged as an excellent tool to investigate vital questions in biology. Recently, methodology to incorporate metabolic bacterial glycan probes into the cell wall of a variety of bacterial species has been developed. In order to improve this method, a scalable synthesis of the peptidoglycan precursors is developed here, allowing for access to essential peptidoglycan immunological fragments and cell wall building blocks. The question was asked if masking polar groups of the glycan probe would increase overall incorporation, a common strategy exploited in mammalian glycobiology. Here, we show, through cellular assays, that E. coli do not utilize peracetylated peptidoglycan substrates but do employ methyl esters. The 10-fold improvement of probe utilization indicates that (i) masking the carboxylic acid is favorable for transport and (ii) bacterial esterases are capable of removing the methyl ester for use in peptidoglycan biosynthesis. This investigation advances bacterial cell wall biology, offering a prescription on how to best deliver and utilize bacterial metabolic glycan probes.

Addiction-Associated Genetic Variants Implicate Brain Cell Type- and Region-Specific Cis-Regulatory Elements in Addiction Neurobiology.

Recent large genome-wide association studies have identified multiple confident risk loci linked to addiction-associated behavioral traits. Most genetic variants linked to addiction-associated traits lie in noncoding regions of the genome, likely disrupting cis-regulatory element (CRE) function. CREs tend to be highly cell type-specific and may contribute to the functional development of the neural circuits underlying addiction. Yet, a systematic approach for predicting the impact of risk variants on the CREs of specific cell populations is lacking. To dissect the cell types and brain regions underlying addiction-associated traits, we applied stratified linkage disequilibrium score regression to compare genome-wide association studies to genomic regions collected from human and mouse assays for open chromatin, which is associated with CRE activity. We found enrichment of addiction-associated variants in putative CREs marked by open chromatin in neuronal (NeuN+) nuclei collected from multiple prefrontal cortical areas and striatal regions known to play major roles in reward and addiction. To further dissect the cell type-specific basis of addiction-associated traits, we also identified enrichments in human orthologs of open chromatin regions of female and male mouse neuronal subtypes: cortical excitatory, D1, D2, and PV. Last, we developed machine learning models to predict mouse cell type-specific open chromatin, enabling us to further categorize human NeuN+ open chromatin regions into cortical excitatory or striatal D1 and D2 neurons and predict the functional impact of addiction-associated genetic variants. Our results suggest that different neuronal subtypes within the reward system play distinct roles in the variety of traits that contribute to addiction.SIGNIFICANCE STATEMENT We combine statistical genetic and machine learning techniques to find that the predisposition to for nicotine, alcohol, and cannabis use behaviors can be partially explained by genetic variants in conserved regulatory elements within specific brain regions and neuronal subtypes of the reward system. Our computational framework can flexibly integrate open chromatin data across species to screen for putative causal variants in a cell type- and tissue-specific manner for numerous complex traits.

Cell Type-Specific Oxidative Stress Genomic Signatures in the Globus Pallidus of Dopamine-Depleted Mice.

Neuron subtype dysfunction is a key contributor to neurologic disease circuits, but identifying associated gene regulatory pathways is complicated by the molecular complexity of the brain. For example, parvalbumin-expressing (PV+) neurons in the external globus pallidus (GPe) are critically involved in the motor deficits of dopamine-depleted mouse models of Parkinson's disease, where cell type-specific optogenetic stimulation of PV+ neurons over other neuron populations rescues locomotion. Despite the distinct roles these cell types play in the neural circuit, the molecular correlates remain unknown because of the difficulty of isolating rare neuron subtypes. To address this issue, we developed a new viral affinity purification strategy, Cre-Specific Nuclear Anchored Independent Labeling, to isolate Cre recombinase-expressing (Cre+) nuclei from the adult mouse brain. Applying this technology, we performed targeted assessments of the cell type-specific transcriptomic and epigenetic effects of dopamine depletion on PV+ and PV- cells within three brain regions of male and female mice: GPe, striatum, and cortex. We found GPe PV+ neuron-specific gene expression changes that suggested increased hypoxia-inducible factor 2α signaling. Consistent with transcriptomic data, regions of open chromatin affected by dopamine depletion within GPe PV+ neurons were enriched for hypoxia-inducible factor family binding motifs. The gene expression and epigenomic experiments performed on PV+ neurons isolated by Cre-Specific Nuclear Anchored Independent Labeling identified a transcriptional regulatory network mediated by the neuroprotective factor Hif2a as underlying neural circuit differences in response to dopamine depletion.SIGNIFICANCE STATEMENT Cre-Specific Nuclear Anchored Independent Labeling is an enhanced, virus-based approach to isolate nuclei of a specific cell type for transcriptome and epigenome interrogation that decreases dependency on transgenic animals. Applying this technology to GPe parvalbumin-expressing neurons in a mouse model of Parkinson's disease, we discovered evidence for an upregulation of the oxygen homeostasis maintaining pathway involving Hypoxia-inducible factor 2α. These results provide new insight into how neuron subtypes outside the substantia nigra pars compacta may be compensating at a molecular level for differences in the motor production neural circuit during the progression of Parkinson's disease. Furthermore, they emphasize the utility of cell type-specific technologies, such as Cre-Specific Nuclear Anchored Independent Labeling, for isolated assessment of specific neuron subtypes in complex systems.

Chemical Biology Tools for Examining the Bacterial Cell Wall.

Bacteria surround themselves with cell walls to maintain cell rigidity and protect against environmental insults. Here we review chemical and biochemical techniques employed to study bacterial cell wall biogenesis. Recent advances including the ability to isolate critical intermediates, metabolic approaches for probe incorporation, and isotopic labeling techniques have provided critical insight into the biochemistry of cell walls. Fundamental manuscripts that have used these techniques to discover cell wall-interacting proteins, flippases, and cell wall stoichiometry are discussed in detail. The review highlights that these powerful methods and techniques have exciting potential to identify and characterize new targets for antibiotic development.

Utility of bacterial peptidoglycan recycling enzymes in the chemoenzymatic synthesis of valuable UDP sugar substrates.

Uridine diphosphate (UDP) sugars are essential precursors for glycosylation reactions in all forms of life. Reactions that transfer the carbohydrate from the UDP donor are catalyzed by glycosyltransferases (Gtfs). While the stereochemistry and negative physiological charge of UDP-sugars are essential for their biochemical function in the cell, these characteristics make them challenging molecules to synthesize and purify on scale in the laboratory. This chapter focuses on the utilization of a chemoenzymatic synthesis of muramyl UDP-sugars, key building blocks in the bacterial cell peptidoglycan. A scalable strategy to obtain UDP-N-acetyl muramic acid derivatives (UDP-NAM), the first committed intermediate used solely in peptidoglycan biosynthesis, is described herein. This methodology utilizes two enzymes involving the cell wall recycling enzymes MurNAc/GlcNAc anomeric kinase (AmgK) and NAM α-1-phosphate uridylyl transferase (MurU), respectively. The promiscuity of these enzymes allows for the unique chemical functionality to be embedded in bacterial peptidoglycan both in vitro and in whole bacterial cells for subsequent structural and functional studies of this important biopolymer.

Decreases in Iron Oxide Reducibility during Microbial Reductive Dissolution and Transformation of Ferrihydrite.

Ferrous iron formed during microbial ferric iron reduction induces phase transformations of poorly crystalline into more crystalline and thermodynamically more stable iron (oxyhydr)oxides. Yet, characterizing the resulting decreases in the reactivity of the remaining oxide ferric iron toward reduction (i.e., its reducibility) has been challenging. Here, we used the reduction of six-line ferrihydrite by Shewanella oneidensis MR-1 as a model system to demonstrate that mediated electrochemical reduction (MER) allows directly following decreases in oxide ferric iron reducibility during the transformation of ferrihydrite into goethite and magnetite which we characterized by X-ray diffraction analysis and transmission electron microscopy imaging. Ferrihydrite was fully reducible in MER at both pHMER of 5.0 and 7.5. Decreases in iron oxide reducibility associated with ferrihydrite transformation into magnetite were accessible at both pHMER because the formed magnetite was not reducible under either of these conditions. Conversely, decreases in iron oxide reducibility associated with goethite formation were apparent only at the highest tested pHMER of 7.5 and thus the thermodynamically least favorable conditions for iron oxide reductive dissolution. The unique capability to adjust the thermodynamic boundary conditions in MER to the specific reducibilities of individual iron (oxyhydr)oxides makes this electrochemical approach broadly applicable for studying changes in iron oxide reducibility in heterogeneous environmental samples such as soils and sediments.

Modulation of the NOD-like receptors NOD1 and NOD2: A chemist's perspective.

The innate immune system is the body's first defense against invading microorganisms, relying on the recognition of bacterial-derived small molecules by host protein receptors. This recognition event and downstream immune response rely heavily on the specific chemical features of both the innate immune receptors and their bacterial derived ligands. This review presents a chemist's perspective on some of the most crucial and complex components of two receptors (NOD1 and NOD2): starting from the structural and chemical characteristics of bacterial-derived small molecules, to the specific proposed models of molecular recognition of these molecules by immune receptors, to the subsequent post-translational modifications that ultimately dictate downstream immune signaling. Recent advances in the field are discussed, as well as the potential for the development of targeted therapeutics.

A Novel Adaptation Mechanism Underpinning Algal Colonization of a Nuclear Fuel Storage Pond.

Geochemical analyses alongside molecular techniques were used to characterize the microbial ecology and biogeochemistry of an outdoor spent nuclear fuel storage pond at Sellafield, United Kingdom, that is susceptible to seasonal algal blooms that cause plant downtime. 18S rRNA gene profiling of the filtered biomass samples showed the increasing dominance of a species closely related to the alga Haematococcus pluvialis, alongside 16S rRNA genes affiliated with a diversity of freshwater bacteria, including Proteobacteria and Cyanobacteria High retention of 137Cs and 90Sr on pond water filters coincided with high levels of microbial biomass in the pond, suggesting that microbial colonization may have an important control on radionuclide fate in the pond. To interpret the unexpected dominance of Haematococcus species during bloom events in this extreme environment, the physiological response of H. pluvialis to environmentally relevant ionizing radiation doses was assessed. Irradiated laboratory cultures produced significant quantities of the antioxidant astaxanthin, consistent with pigmentation observed in pond samples. Fourier transform infrared (FT-IR) spectroscopy suggested that radiation did not have a widespread impact on the metabolic fingerprint of H. pluvialis in laboratory experiments, despite the 80-Gy dose. This study suggests that the production of astaxanthin-rich encysted cells may be related to the preservation of the Haematococcus phenotype, potentially allowing it to survive oxidative stress arising from radiation doses associated with the spent nuclear fuel. The oligotrophic and radiologically extreme conditions in this environment do not prevent extensive colonization by microbial communities, which play a defining role in controlling the biogeochemical fate of major radioactive species present.IMPORTANCE Spent nuclear fuel is stored underwater in large ponds prior to processing and disposal. Such environments are intensively radioactive but can be colonized by microorganisms. Colonization of such inhospitable radioactive ponds is surprising, and the survival mechanisms that microbes use is of fundamental interest. It is also important to study these unusual ecosystems, as microbes growing in the pond waters may accumulate radionuclides present in the waters (for bioremediation applications), while high cell loads can hamper management of the ponds due to poor visibility. In this study, an outdoor pond at the U.K. Sellafield facility was colonized by a seasonal bloom of microorganisms, able to accumulate high levels of 137Cs and 90Sr and dominated by the alga Haematococcus This organism is not normally associated with deep water bodies, but it can adapt to radioactive environments via the production of the pigment astaxanthin, which protects the cells from radiation damage.

Crosstalk between mismatch repair and base excision repair in human gastric cancer.

DNA repair gene expression in a set of gastric cancers suggested an inverse association between the expression of the mismatch repair (MMR) gene MLH1 and that of the base excision repair (BER) gene DNA polymerase ß (Polß). To gain insight into possible crosstalk of these two repair pathways in cancer, we analysed human gastric adenocarcinoma AGS cells over-expressing Polß or Polß active site mutants, alone or in combination with MLH1 silencing. Next, we investigated the cellular response to the alkylating agent methyl methanesulfonate (MMS) and the purine analogue 6-thioguanine (6-TG), agents that induce lesions that are substrates for BER and/or MMR. AGS cells over-expressing Polß were resistant to 6-TG to a similar extent as when MLH1 was inactivated while inhibition of O6-methylguanine-DNA methyltransferase (MGMT) was required to detect resistance to MMS. Upon either treatment, the association with MLH1 down-regulation further amplified the resistant phenotype. Moreover, AGS cells mutated in Polß were hypersensitive to both 6-TG and MMS killing and their sensitivity was partially rescued by MLH1 silencing. We provide evidence that the critical lethal lesions in this new pathway are double strand breaks that are exacerbated when Polß is defective and relieved when MLH1 is silenced. In conclusion, we provide evidence of crosstalk between MLH1 and Polß that modulates the response to alkylation damage. These studies suggest that the Polß/MLH1 status should be taken into consideration when designing chemotherapeutic approaches for gastric cancer.

Phenotypic Characterisation of Shewanella oneidensis MR-1 Exposed to X-Radiation.

Biogeochemical processes mediated by Fe(III)-reducing bacteria such as Shewanella oneidensis have the potential to influence the post-closure evolution of a geological disposal facility for radioactive wastes and to affect the solubility of some radionuclides. Furthermore, their potential to reduce both Fe(III) and radionuclides can be harnessed for the bioremediation of radionuclide-contaminated land. As some such sites are likely to have significant radiation fluxes, there is a need to characterise the impact of radiation stress on such microorganisms. There have, however, been few global cell analyses on the impact of ionizing radiation on subsurface bacteria, so here we address the metabolic response of S. oneidensis MR-1 to acute doses of X-radiation. UV/Vis spectroscopy and CFU counts showed that although X-radiation decreased initial viability and extended the lag phase of batch cultures, final biomass yields remained unchanged. FT-IR spectroscopy of whole cells indicated an increase in lipid associated vibrations and decreases in vibrations tentatively assigned to nucleic acids, phosphate, saccharides and amines. MALDI-TOF-MS detected an increase in total protein expression in cultures exposed to 12 Gy. At 95 Gy, a decrease in total protein levels was generally observed, although an increase in a putative cold shock protein was observed, which may be related to the radiation stress response of this organism. Multivariate statistical analyses applied to these FT-IR and MALDI-TOF-MS spectral data suggested that an irradiated phenotype developed throughout subsequent generations. This study suggests that significant alteration to the metabolism of S. oneidensis MR-1 is incurred as a result of X-irradiation and that dose dependent changes to specific biomolecules characterise this response. Irradiated S. oneidensis also displayed enhanced levels of poorly crystalline Fe(III) oxide reduction, though the mechanism underpinning this phenomenon is unclear.

The impact of gamma radiation on sediment microbial processes.

Microbial communities have the potential to control the biogeochemical fate of some radionuclides in contaminated land scenarios or in the vicinity of a geological repository for radioactive waste. However, there have been few studies of ionizing radiation effects on microbial communities in sediment systems. Here, acetate and lactate amended sediment microcosms irradiated with gamma radiation at 0.5 or 30 Gy h(-1) for 8 weeks all displayed NO3 (-) and Fe(III) reduction, although the rate of Fe(III) reduction was decreased in 30-Gy h(-1) treatments. These systems were dominated by fermentation processes. Pyrosequencing indicated that the 30-Gy h(-1) treatment resulted in a community dominated by two Clostridial species. In systems containing no added electron donor, irradiation at either dose rate did not restrict NO3 (-), Fe(III), or SO4 (2-) reduction. Rather, Fe(III) reduction was stimulated in the 0.5-Gy h(-1)-treated systems. In irradiated systems, there was a relative increase in the proportion of bacteria capable of Fe(III) reduction, with Geothrix fermentans and Geobacter sp. identified in the 0.5-Gy h(-1) and 30-Gy h(-1) treatments, respectively. These results indicate that biogeochemical processes will likely not be restricted by dose rates in such environments, and electron accepting processes may even be stimulated by radiation.

HSP90 regulates DNA repair via the interaction between XRCC1 and DNA polymerase ß.

Cellular DNA repair processes are crucial to maintain genome stability and integrity. In DNA base excision repair, a tight heterodimer complex formed by DNA polymerase ß (Polß) and XRCC1 is thought to facilitate repair by recruiting Polß to DNA damage sites. Here we show that disruption of the complex does not impact DNA damage response or DNA repair. Instead, the heterodimer formation is required to prevent ubiquitylation and degradation of Polß. In contrast, the stability of the XRCC1 monomer is protected from CHIP-mediated ubiquitylation by interaction with the binding partner HSP90. In response to cellular proliferation and DNA damage, proteasome and HSP90-mediated regulation of Polß and XRCC1 alters the DNA repair complex architecture. We propose that protein stability, mediated by DNA repair protein complex formation, functions as a regulatory mechanism for DNA repair pathway choice in the context of cell cycle progression and genome surveillance.