The Mre11-Rad50-Nbs1 (MRN) complex is providing paradigm-shifting results of exceptional biomedical interest. MRN is among the earliest respondents to DNA double-strand breaks (DSBs), and MRN ...mutations cause the human cancer predisposition diseases Nijmegen breakage syndrome and ataxia telangiectasia-like disorder (ATLD). MRN's 3-protein multidomain composition promotes its central architectural, structural, enzymatic, sensing, and signaling functions in DSB responses. To organize the MRN complex, the Mre11 exonuclease directly binds Nbs1, DNA, and Rad50. Rad50, a structural maintenance of chromosome (SMC) related protein, employs its ATP-binding cassette (ABC) ATPase, Zn hook, and coiled coils to bridge DSBs and facilitate DNA end processing by Mre11. Contributing to MRN regulatory roles, Nbs1 harbors N-terminal phosphopeptide interacting FHA and BRCT domains, as well as C-terminal ataxia telangiectasia mutated (ATM) kinase and Mre11 interaction domains. Current emerging structural and biological evidence suggests that MRN has 3 coupled critical roles in DSB sensing, stabilization, signaling, and effector scaffolding: (1) expeditious establishment of protein--nucleic acid tethering scaffolds for the recognition and stabilization of DSBs; (2) initiation of DSB sensing, cell-cycle checkpoint signaling cascades, and establishment of epigenetic marks via the ATM kinase; and (3) functional regulation of chromatin remodeling in the vicinity of a DSB.
Background:
Biomechanical data and contributions to ankle joint stability have been previously reported for the individual distal tibiofibular ligaments. These results have not yet been validated ...based on recent anatomic descriptions or using current biomechanical testing devices.
Methods:
Eight matched-pair, lower leg specimens were tested using a dynamic, biaxial testing machine. The proximal tibiofibular joint and the medial and lateral ankle ligaments were left intact. After fixation, specimens were preconditioned and then biomechanically tested following sequential cutting of the tibiofibular ligaments to assess the individual ligamentous contributions to syndesmotic stability. Matched paired specimens were randomly divided into 1 of 2 cutting sequences: (1) anterior-to-posterior: intact, anterior inferior tibiofibular ligament (AITFL), interosseous tibiofibular ligament (ITFL), deep posterior inferior tibiofibular ligament (PITFL), superficial PITFL, and complete interosseous membrane; (2) posterior-to-anterior: intact, superficial PITFL, deep PITFL, ITFL, AITFL, and complete interosseous membrane. While under a 750-N axial compressive load, the foot was rotated to 15 degrees of external rotation and 10 degrees of internal rotation for each sectioned state. Torque (Nm), rotational position (degrees), and 3-dimensional data were recorded continuously throughout testing.
Results:
Testing of the intact ankle syndesmosis under simulated physiologic conditions revealed 4.3 degrees of fibular rotation in the axial plane and 3.3 mm of fibular translation in the sagittal plane. Significant increases in fibular sagittal translation and axial rotation were observed after syndesmotic injury, particularly after sectioning of the AITFL and superficial PITFL. Sequential sectioning of the syndesmotic ligaments resulted in significant reductions in resistance to both internal and external rotation. Isolated injuries to the AITFL resulted in the most substantial reduction of resistance to external rotation (average of 24%). However, resistance to internal rotation was not significantly diminished until the majority of the syndesmotic structures had been sectioned.
Conclusion:
The ligaments of the syndesmosis provide significant contributions to rotary stability of the distal tibiofibular joint within the physiologic range of motion.
Clinical Relevance:
This study defined normal motion of the syndesmosis and the biomechanical consequences of injury. The degree of instability was increased with each additional injured structure; however, isolated injuries to the AITFL alone may lead to significant external rotary instability.
Background:
Significant debate exists regarding optimal repair for unstable syndesmosis injuries. Techniques range from screw fixation, suture-button fixation, or a combination of the two. In this ...study, 3 common repairs were compared using a simulated weightbearing protocol with internal and external rotation of the foot.
Methods:
Twenty-four lower leg specimens with mean age 54 years (range, 38-68 years) were used for testing. Following creation of a complete syndesmotic injury (AITFL, ITFL, PITFL, interosseous membrane), specimens were repaired using 1 of 3 randomly assigned techniques: (1) one 3.5-mm syndesmotic screw, (2) 1 suture-button construct, and (3) 2 divergent suture-button constructs. Repairs were cycled for 500 cycles between 7.5 Nm of internal/external rotation torque under a constant 750 N axial compressive load in a neutral dorsiflexion position. At 0, 10, 100, and 500 cycles, torsional cyclic loading was interrupted to assess torsional resistance to rotation within a physiologic range of motion (15 degrees external rotation to 10 degrees internal rotation). Torque (Nm), rotational position (degrees), and 3-dimensional data were collected throughout the testing to characterize relative spatial relationships of the tibiofibular articulation.
Results:
There were no significant differences between repair techniques in resistance to internal and external rotation with respect to the intact syndesmosis. Three-dimensional analysis revealed significant differences between repair techniques for sagittal fibular translation with external rotation of the foot. Screw fixation had the smallest magnitude of posterior sagittal translation (2.5 mm), and a single suture-button construct demonstrated the largest magnitude of posterior sagittal translation (4.6 mm). Screw fixation also allowed for significantly less anterior sagittal translation with internal rotation of the foot (0.1 mm) when compared to both 1 (2.7 mm) and 2 (2.9 mm) suture-button constructs.
Conclusion:
All repairs provided comparable rotational stability to the syndesmosis; however, no repair technique completely restored rotational stability and tibiofibular anatomic relationships of the preinjury state.
Clinical Relevance:
Constructs were comparable across most conditions; however, when repairing injuries with a suture-button construct, a single suture-button construct may not provide sufficient resistance to sagittal translation of the fibula.
Mre11 forms the core of the multifunctional Mre11-Rad50-Nbs1 (MRN) complex that detects DNA double-strand breaks (DSBs), activates the ATM checkpoint kinase, and initiates homologous recombination ...(HR) repair of DSBs. To define the roles of Mre11 in both DNA bridging and nucleolytic processing during initiation of DSB repair, we combined small-angle X-ray scattering (SAXS) and crystal structures of Pyrococcus furiosus Mre11 dimers bound to DNA with mutational analyses of fission yeast Mre11. The Mre11 dimer adopts a four-lobed U-shaped structure that is critical for proper MRN complex assembly and for binding and aligning DNA ends. Further, mutations blocking Mre11 endonuclease activity impair cell survival after DSB induction without compromising MRN complex assembly or Mre11-dependant recruitment of Ctp1, an HR factor, to DSBs. These results show how Mre11 dimerization and nuclease activities initiate repair of DSBs and collapsed replication forks, as well as provide a molecular foundation for understanding cancer-causing Mre11 mutations in ataxia telangiectasia-like disorder (ATLD).
Idiopathic pulmonary fibrosis (IPF) is a chronic disease for which novel approaches are urgently required. We reported increased sphingosine kinase 1 (SPHK1) in IPF lungs and that SPHK1 inhibition ...using genetic and pharmacologic approaches reduces murine bleomycin-induced pulmonary fibrosis. We determined whether PF543, a specific SPHK1 inhibitor post bleomycin or asbestos challenge mitigates lung fibrosis by reducing mitochondrial (mt) DNA damage and pro-fibrotic monocyte recruitment-both are implicated in the pathobiology of pulmonary fibrosis. Bleomycin (1.5 U/kg), crocidolite asbestos (100 µg/50 µL) or controls was intratracheally instilled in Wild-Type (
mice. PF543 (1 mg/kg) or vehicle was intraperitoneally injected once every two days from day 7-21 following bleomycin and day 14-21 or day 30-60 following asbestos. PF543 reduced bleomycin- and asbestos-induced pulmonary fibrosis at both time points as well as lung expression of profibrotic markers, lung mtDNA damage, and fibrogenic monocyte recruitment. In contrast to human lung fibroblasts, asbestos augmented lung epithelial cell (MLE) mtDNA damage and PF543 was protective. Post-exposure PF543 mitigates pulmonary fibrosis in part by reducing lung epithelial cell mtDNA damage and monocyte recruitment. We reason that SPHK1 signaling may be an innovative therapeutic target for managing patients with IPF and other forms of lung fibrosis.
Recovering endangered species is a difficult and often controversial task that challenges status quo land uses. Southern Mountain caribou are a threatened ecotype of caribou that historically ranged ...in southwestern Canada and northwestern USA and epitomize the tension between resource extraction, biodiversity conservation, and Indigenous Peoples' treaty rights. Human‐induced habitat alteration is considered the ultimate cause of caribou population declines, whereby an increased abundance of primary prey—such as moose and deer—elevates predator populations and creates unsustainable caribou mortality. Here we focus on the Klinse‐Za and Quintette subpopulations, part of the endangered Central Group of Southern Mountain caribou in British Columbia. These subpopulations were trending toward immediate extirpation until a collaborative group initiated recovery by implementing two short‐term recovery actions. We test the effectiveness of these recovery actions—maternity penning of adult females and their calves, and the reduction of a primary predator, wolves—in increasing vital rates and population growth. Klinse‐Za received both recovery actions, whereas Quintette only received wolf reductions, providing an opportunity to test efficacy between recovery actions. Between 1995 and 2021, we followed 162 collared female caribou for 414 animal‐years to estimate survival and used aerial counts to estimate population abundance and calf recruitment. We combined these data in an integrated population model to estimate female population growth, total population abundance, and recovery action effectiveness. Results suggest that the subpopulations were declining rapidly (λ = 0.90–0.93) before interventions and would have been functionally extirpated (<10 animals) within 10–15 years. Wolf reduction increased population growth rates by ~0.12 for each subpopulation. Wolf reduction halted the decline of Quintette caribou and allowed them to increase (λ = 1.05), but alone would have only stabilized the Klinse‐Za (λ = 1.02). However, maternity penning in the Klinse‐Za increased population growth by a further ~0.06, which when combined with wolf reductions, allowed populations to grow (λ = 1.08). Taken together, the recovery actions in these subpopulations increased adult female survival, calf recruitment, and overall population growth, more than doubling abundance. Our results suggest that maternity penning and wolf reductions can be effective at increasing caribou numbers in the short term, while long‐term commitments to habitat protection and restoration are made.
Abstract Background Prescription opioid-associated abuse and overdose is a significant cause of morbidity and mortality in the United States. Opioid prescriptions generated from emergency departments ...(EDs) nationwide have increased dramatically over the past 20 years, and opioid-related overdose deaths have become an epidemic, according to the Centers for Disease Control and Prevention. Objective Our aim was to determine the effectiveness of implementing a prescription policy for opioids on overall opioid prescribing patterns in a hospital ED. Methods The ED provider group of an academic, non-university–affiliated urban hospital with 23,000 annual patient visits agreed to opioid prescribing guidelines for chronic pain with the goal of limiting prescriptions that may be used for abuse or diversion. These guidelines were instituted in the ED through collaborative staff meetings and educational and training sessions. We used the electronic medical record to analyze the number and type of opioid discharge prescriptions during the study period from 2006–2014, before and after the prescribing guidelines were instituted in the ED. Results The number of patients discharged with a prescription for opioids decreased 39.6% (25.7% to 15.6%; absolute decrease 10.2%; 95% confidence interval CI 9.6–10.7; p < 0.001) after the intervention. The improvements were sustained 2.5 years after the intervention. Decreases were seen in all major opioids (hydrocodone, oxycodone, hydromorphone, and codeine). The number of pills per prescription also decreased 14.8%, from 19.5% to 16.6% (absolute decrease 2.9; 95% CI 2.6–3.1; p < 0.001). Conclusions Implementation of an ED prescription opioid policy was associated with a significant reduction in total opioid prescriptions and in the number of pills per prescription.
Three-dimensional variably saturated flow and multicomponent biogeochemical reactive transport modeling, based on published and newly generated data, is used to better understand the interplay of ...hydrology, geochemistry, and biology controlling the cycling of carbon, nitrogen, oxygen, iron, sulfur, and uranium in a shallow floodplain. In this system, aerobic respiration generally maintains anoxic groundwater below an oxic vadose zone until seasonal snowmelt-driven water table peaking transports dissolved oxygen (DO) and nitrate from the vadose zone into the alluvial aquifer. The response to this perturbation is localized due to distinct physico-biogeochemical environments and relatively long time scales for transport through the floodplain aquifer and vadose zone. Naturally reduced zones (NRZs) containing sediments higher in organic matter, iron sulfides, and non-crystalline U(IV) rapidly consume DO and nitrate to maintain anoxic conditions, yielding Fe(II) from FeS oxidative dissolution, nitrite from denitrification, and U(VI) from nitrite-promoted U(IV) oxidation. Redox cycling is a key factor for sustaining the observed aquifer behaviors despite continuous oxygen influx and the annual hydrologically induced oxidation event. Depth-dependent activity of fermenters, aerobes, nitrate reducers, sulfate reducers, and chemolithoautotrophs (e.g., oxidizing Fe(II), S compounds, and ammonium) is linked to the presence of DO, which has higher concentrations near the water table.
MAGE proteins are cancer testis antigens (CTAs) that are characterized by highly conserved MAGE homology domains (MHDs) and are increasingly being found to play pivotal roles in promoting aggressive ...cancer types. MAGE-A4, in particular, increases DNA damage tolerance and chemoresistance in a variety of cancers by stabilizing the E3-ligase RAD18 and promoting trans-lesion synthesis (TLS). Inhibition of the MAGE-A4:RAD18 axis could sensitize cancer cells to chemotherapeutics like platinating agents. We use an mRNA display of thioether cyclized peptides to identify a series of potent and highly selective macrocyclic inhibitors of the MAGE-A4:RAD18 interaction. Co-crystal structure indicates that these inhibitors bind in a pocket that is conserved across MHDs but take advantage of A4-specific residues to achieve high isoform selectivity. Cumulatively, our data represent the first reported inhibitor of the MAGE-A4:RAD18 interaction and establish biochemical tools and structural insights for the future development of MAGE-A4-targeted cellular probes.
The decline in approval of new drugs during the past decade has led to a close analysis of the drug discovery process. One of the main reasons for attrition is preclinical toxicity, frequently ...attributed to the generation of protein‐reactive drug metabolites. In this review, we present a critique of such reactive metabolites and evaluate the evidence linking them to observed toxic effects. Methodology for the characterization of reactive metabolites has advanced greatly in recent years, and is summarized first. Next, we consider the inhibition of key metabolic enzymes by electrophilic metabolites, as well as unfavorable drug–drug interactions that may ensue. One important class of protein‐reactive metabolites, not linked conclusively to a toxic event, is acyl glucuronides. Their properties are discussed in light of the safety characteristics of carboxylic acid containing drugs. Many adverse drug reactions (ADRs) are known collectively as idiosyncratic events, that is, not predictable from knowledge of the pharmacology and pharmacokinetics of the parent compound. Observed ADRs may take various forms. Specific organ injury, particularly of the liver, is the most direct: we examine this in some detail. Moving to the cellular level, we also consider the upregulation of induced cellular processes. The related, but distinct, issue of hypersensitivity or allergic reactions to drugs and their metabolites, possibly via the immune system, is considered next. Finally, we discuss the impact of such data on the drug discovery process, both through early detection of reactive metabolites and informed synthetic design, which eliminates unfavorable functionality from drug candidates.
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