DNA methylation acts in concert with restriction enzymes to protect the integrity of prokaryotic genomes. Studies in a limited number of organisms suggest that methylation also contributes to ...prokaryotic genome regulation, but the prevalence and properties of such non-restriction-associated methylation systems remain poorly understood. Here, we used single molecule, real-time sequencing to map DNA modifications including m6A, m4C, and m5C across the genomes of 230 diverse bacterial and archaeal species. We observed DNA methylation in nearly all (93%) organisms examined, and identified a total of 834 distinct reproducibly methylated motifs. This data enabled annotation of the DNA binding specificities of 620 DNA Methyltransferases (MTases), doubling known specificities for previously hard to study Type I, IIG and III MTases, and revealing their extraordinary diversity. Strikingly, 48% of organisms harbor active Type II MTases with no apparent cognate restriction enzyme. These active 'orphan' MTases are present in diverse bacterial and archaeal phyla and show motif specificities and methylation patterns consistent with functions in gene regulation and DNA replication. Our results reveal the pervasive presence of DNA methylation throughout the prokaryotic kingdoms, as well as the diversity of sequence specificities and potential functions of DNA methylation systems.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
DNA methylation is the most common form of DNA modification in prokaryotic and eukaryotic genomes. We have applied the method of single-molecule, real-time (SMRT®) DNA sequencing that is capable of ...direct detection of modified bases at single-nucleotide resolution to characterize the specificity of several bacterial DNA methyltransferases (MTases). In addition to previously described SMRT sequencing of N6-methyladenine and 5-methylcytosine, we show that N4-methylcytosine also has a specific kinetic signature and is therefore identifiable using this approach. We demonstrate for all three prokaryotic methylation types that SMRT sequencing confirms the identity and position of the methylated base in cases where the MTase specificity was previously established by other methods. We then applied the method to determine the sequence context and methylated base identity for three MTases with unknown specificities. In addition, we also find evidence of unanticipated MTase promiscuity with some enzymes apparently also modifying sequences that are related, but not identical, to the cognate site.
Alcoholic hepatitis (AH) continues to be a disease with high mortality and no efficacious medical treatment. Although severe AH is presented as acute on chronic liver failure, what underlies this ...transition from chronic alcoholic steatohepatitis (ASH) to AH is largely unknown. To address this question, unbiased RNA sequencing and proteomic analyses were performed on livers of the recently developed AH mouse model, which exhibits the shift to AH from chronic ASH upon weekly alcohol binge, and these results are compared to gene expression profiling data from AH patients. This cross‐analysis has identified Casp11 (CASP4 in humans) as a commonly up‐regulated gene known to be involved in the noncanonical inflammasome pathway. Immunoblotting confirms CASP11/4 activation in AH mice and patients, but not in chronic ASH mice and healthy human livers. Gasdermin‐D (GSDMD), which induces pyroptosis (lytic cell death caused by bacterial infection) downstream of CASP11/4 activation, is also activated in AH livers in mice and patients. CASP11 deficiency reduces GSDMD activation, bacterial load in the liver, and severity of AH in the mouse model. Conversely, the deficiency of interleukin‐18, the key antimicrobial cytokine, aggravates hepatic bacterial load, GSDMD activation, and AH. Furthermore, hepatocyte‐specific expression of constitutively active GSDMD worsens hepatocellular lytic death and polymorphonuclear leukocyte inflammation. Conclusion: These results implicate pyroptosis induced by the CASP11/4‐GSDMD pathway in the pathogenesis of AH. (Hepatology 2018;67:1737‐1753).
Synapse loss and Tau pathology are hallmarks of Alzheimer’s disease (AD) and other tauopathies, but how Tau pathology causes synapse loss is unclear. We used unbiased proteomic analysis of ...postsynaptic densities (PSDs) in Tau-P301S transgenic mice to identify Tau-dependent alterations in synapses prior to overt neurodegeneration. Multiple proteins and pathways were altered in Tau-P301S PSDs, including depletion of a set of GTPase-regulatory proteins that leads to actin cytoskeletal defects and loss of dendritic spines. Furthermore, we found striking accumulation of complement C1q in the PSDs of Tau-P301S mice and AD patients. At synapses, C1q decorated perisynaptic membranes, accumulated in correlation with phospho-Tau, and was associated with augmented microglial engulfment of synapses and decline of synapse density. A C1q-blocking antibody inhibited microglial synapse removal in cultured neurons and in Tau-P301S mice, rescuing synapse density. Thus, inhibiting complement-mediated synapse removal by microglia could be a potential therapeutic target for Tau-associated neurodegeneration.
•Pathological Tau accumulation alters protein composition of PSD in Tau-P301S mice•Loss of postsynaptic GTPase-regulatory proteins leads to loss of dendritic spines•C1q tags Tau-affected synapses, leading to microglial engulfment and synapse loss•C1q-blocking antibody prevents microglial synapse removal and rescues synapse density
Unbiased proteomics reveals multiple molecular changes at hippocampal synapses that occur prior to neurodegeneration in a taupathy/Alzheimer’s disease mouse model. Complement C1q labels phospho-Tau-containing synapses and drives microglia-mediated synapse loss that can be rescued by a C1q-blocking antibody.
Purpose The purpose of this study was to evaluate the effect of graft size on patient-reported outcomes and revision risk after anterior cruciate ligament (ACL) reconstruction. Methods A ...retrospective chart review of prospectively collected cohort data was performed, and 263 of 320 consecutive patients (82.2%) undergoing primary ACL reconstruction with hamstring autograft were evaluated. We recorded graft size; femoral tunnel drilling technique; patient age, sex, and body mass index at the time of ACL reconstruction; Knee Injury and Osteoarthritis Outcome Score (KOOS) and International Knee Documentation Committee score preoperatively and at 2 years postoperatively; and whether each patient underwent revision ACL reconstruction during the 2-year follow-up period. Revision was used as a marker for graft failure. The relation between graft size and patient-reported outcomes was determined by multiple linear regression. The relation between graft size and risk of revision was determined by dichotomizing graft size at 8 mm and stratifying by age. Results After we controlled for age, sex, operative side, surgeon, body mass index, graft choice, and femoral tunnel drilling technique, a 1-mm increase in graft size was noted to correlate with a 3.3-point increase in the KOOS pain subscale ( P = .003), a 2.0-point increase in the KOOS activities of daily living subscale ( P = .034), a 5.2-point increase in the KOOS sport/recreation function subscale ( P = .004), and a 3.4-point increase in the subjective International Knee Documentation Committee score ( P = .026). Revision was required in 0 of 64 patients (0.0%) with grafts greater than 8 mm in diameter and 14 of 199 patients (7.0%) with grafts 8 mm in diameter or smaller ( P = .037). Among patients aged 18 years or younger, revision was required in 0 of 14 patients (0.0%) with grafts greater than 8 mm in diameter and 13 of 71 patients (18.3%) with grafts 8 mm in diameter or smaller. Conclusions Smaller hamstring autograft size is a predictor of poorer KOOS sport/recreation function 2 years after primary ACL reconstruction. A larger sample size is required to confirm the relation between graft size and risk of revision ACL reconstruction. Level of Evidence Level III, retrospective comparative study.
Bacteria are under constant assault by bacteriophages and other mobile genetic elements. As a result, bacteria have evolved a multitude of systems that protect from attack. Genes encoding bacterial ...defence mechanisms can be clustered into 'defence islands', providing a potentially synergistic level of protection against a wider range of assailants. However, there is a comparative paucity of information on how expression of these defence systems is controlled. Here, we functionally characterize a transcriptional regulator, BrxR, encoded within a recently described phage defence island from a multidrug resistant plasmid of the emerging pathogen Escherichia fergusonii. Using a combination of reporters and electrophoretic mobility shift assays, we discovered that BrxR acts as a repressor. We present the structure of BrxR to 2.15 Å, the first structure of this family of transcription factors, and pinpoint a likely binding site for ligands within the WYL-domain. Bioinformatic analyses demonstrated that BrxR-family homologues are widespread amongst bacteria. About half (48%) of identified BrxR homologues were co-localized with a diverse array of known phage defence systems, either alone or clustered into defence islands. BrxR is a novel regulator that reveals a common mechanism for controlling the expression of the bacterial phage defence arsenal.
Background:
The long-term prognosis and risk factors for quality of life and disability after anterior cruciate ligament (ACL) reconstruction remain unknown.
Hypothesis/Purpose:
Our objective was to ...identify patient-reported outcomes and patient-specific risk factors from a large prospective cohort at a minimum 10-year follow-up after ACL reconstruction. We hypothesized that meniscus and articular cartilage injuries, revision ACL reconstruction, subsequent knee surgery, and certain demographic characteristics would be significant risk factors for inferior outcomes at 10 years.
Study Design:
Therapeutic study; Level of evidence, 2.
Methods:
Unilateral ACL reconstruction procedures were identified and prospectively enrolled between 2002 and 2004 from 7 sites in the Multicenter Orthopaedic Outcomes Network (MOON). Patients preoperatively completed a series of validated outcome instruments, including the International Knee Documentation Committee (IKDC), Knee injury and Osteoarthritis Outcome Score (KOOS), and Marx activity rating scale. At the time of surgery, physicians documented all intra-articular abnormalities, treatment, and surgical techniques utilized. Patients were followed at 2, 6, and 10 years postoperatively and asked to complete the same outcome instruments that they completed at baseline. The incidence and details of any subsequent knee surgeries were also obtained. Multivariable regression analysis was used to identify significant predictors of the outcome.
Results:
A total of 1592 patients were enrolled (57% male; median age, 24 years). Ten-year follow-up was obtained on 83% (n = 1320) of the cohort. Both IKDC and KOOS scores significantly improved at 2 years and were maintained at 6 and 10 years. Conversely, Marx scores dropped markedly over time, from a median score of 12 points at baseline to 9 points at 2 years, 7 points at 6 years, and 6 points at 10 years. The patient-specific risk factors for inferior 10-year outcomes were lower baseline scores; higher body mass index; being a smoker at baseline; having a medial or lateral meniscus procedure performed before index ACL reconstruction; undergoing revision ACL reconstruction; undergoing lateral meniscectomy; grade 3 to 4 articular cartilage lesions in the medial, lateral, or patellofemoral compartments; and undergoing any subsequent ipsilateral knee surgery after index ACL reconstruction.
Conclusion:
Patients were able to perform sports-related functions and maintain a relatively high knee-related quality of life 10 years after ACL reconstruction, although activity levels significantly declined over time. Multivariable analysis identified several key modifiable risk factors that significantly influence the outcome.
Enzyme polymerization (also known as filamentation) has emerged as a new layer of enzyme regulation. SgrAI is a sequence-dependent DNA endonuclease that forms polymeric filaments with enhanced DNA ...cleavage activity as well as altered DNA sequence specificity. To better understand this unusual regulatory mechanism, full global kinetic modeling of the reaction pathway, including the enzyme filamentation steps, has been undertaken. Prior work with the primary DNA recognition sequence cleaved by SgrAI has shown how the kinetic rate constants of each reaction step are tuned to maximize activation and DNA cleavage while minimizing the extent of DNA cleavage to the host genome. In the current work, we expand on our prior study by now including DNA cleavage of a secondary recognition sequence, to understand how the sequence of the bound DNA modulates filamentation and activation of SgrAI. The work shows that an allosteric equilibrium between low and high activity states is modulated by the sequence of bound DNA, with primary sequences more prone to activation and filament formation, while SgrAI bound to secondary recognition sequences favor the low (and nonfilamenting) state by up to 40-fold. In addition, the degree of methylation of secondary sequences in the host organism, Streptomyces griseus, is now reported for the first time and shows that as predicted, these sequences are left unprotected from the SgrAI endonuclease making sequence specificity critical in this unusual filament-forming enzyme.
Mitochondrial dysfunction contributes to numerous health problems, including neurological and muscular degeneration, cardiomyopathies, cancer, diabetes, and pathologies of aging. Severe mitochondrial ...defects can result in childhood disorders such as Leigh syndrome, for which there are no effective therapies. We found that rapa my ein, a specific inhibitor of the mechanistic target of rapamycin (mTOR) signaling pathway, robustly enhances survival and attenuates disease progression in a mouse model of Leigh syndrome. Administration of rapamycin to these mice, which are deficient in the mitochondrial respiratory chain subunit Ndufs4 NADH dehydrogenase (ubiquinone) Fe-S protein 4, delays onset of neurological symptoms, reduces neuroinflammation, and prevents brain lesions. Although the precise mechanism of rescue remains to be determined, rapamycin induces a metabolic shift toward amino acid catabolism and away from glycolysis, alleviating the buildup of glycolytic intermediates. This therapeutic strategy may prove relevant for a broad range of mitochondrial diseases.
Bacteriophage exclusion ('BREX') phage restriction systems are found in a wide range of bacteria. Various BREX systems encode unique combinations of proteins that usually include a site-specific ...methyltransferase; none appear to contain a nuclease. Here we describe the identification and characterization of a Type I BREX system from Acinetobacter and the effect of deleting each BREX ORF on growth, methylation, and restriction. We identified a previously uncharacterized gene in the BREX operon that is dispensable for methylation but involved in restriction. Biochemical and crystallographic analyses of this factor, which we term BrxR ('BREX Regulator'), demonstrate that it forms a homodimer and specifically binds a DNA target site upstream of its transcription start site. Deletion of the BrxR gene causes cell toxicity, reduces restriction, and significantly increases the expression of BrxC. In contrast, the introduction of a premature stop codon into the BrxR gene, or a point mutation blocking its DNA binding ability, has little effect on restriction, implying that the BrxR coding sequence and BrxR protein play independent functional roles. We speculate that elements within the BrxR coding sequence are involved in cis regulation of anti-phage activity, while the BrxR protein itself plays an additional regulatory role, perhaps during horizontal transfer.
PDF
