Targeted and Persistent 8-Oxoguanine Base Damage at Telomeres Promotes Telomere Loss and Crisis.

Telomeres are essential for genome stability. Oxidative stress caused by excess reactive oxygen species (ROS) accelerates telomere shortening. Although telomeres are hypersensitive to ROS-mediated 8-oxoguanine (8-oxoG) formation, the biological effect of this common lesion at telomeres is poorly understood because ROS have pleiotropic effects. Here we developed a chemoptogenetic tool that selectively produces 8-oxoG only at telomeres. Acute telomeric 8-oxoG formation increased telomere fragility in cells lacking OGG1, the enzyme that removes 8-oxoG, but did not compromise cell survival. However, chronic telomeric 8-oxoG induction over time shortens telomeres and impairs cell growth. Accumulation of telomeric 8-oxoG in chronically exposed OGG1-deficient cells triggers replication stress, as evidenced by mitotic DNA synthesis at telomeres, and significantly increases telomere losses. These losses generate chromosome fusions, leading to chromatin bridges and micronucleus formation upon cell division. By confining base damage to the telomeres, we show that telomeric 8-oxoG accumulation directly drives telomere crisis.

A mathematical model for velocity-selective arterial spin labeling.

PURPOSE: To present a theoretical framework that rigorously defines and analyzes key concepts and quantities for velocity selective arterial spin labeling (VSASL). THEORY AND METHODS: An expression for the VSASL arterial delivery function is derived based on (1) labeling and saturation profiles as a function of velocity and (2) physiologically plausible approximations of changes in acceleration and velocity across the vascular system. The dependence of labeling efficiency on the amplitude and effective bolus width of the arterial delivery function is defined. Factors that affect the effective bolus width are examined, and timing requirements to minimize quantitation errors are derived. RESULTS: The model predicts that a flow-dependent negative bias in the effective bolus width can occur when velocity selective inversion (VSI) is used for the labeling module and velocity selective saturation (VSS) is used for the vascular crushing module. The bias can be minimized by choosing a nominal labeling cutoff velocity that is lower than the nominal cutoff velocity of the vascular crushing module. CONCLUSION: The elements of the model are specified in a general fashion such that future advances can be readily integrated. The model can facilitate further efforts to understand and characterize the performance of VSASL and provide critical theoretical insights that can be used to design future experiments and develop novel VSASL approaches.

Measuring success: A comparison of ultrasound and landmark guidance for knee arthrocentesis in a cadaver model.

OBJECTIVE: Knee arthrocentesis can be performed by landmark (LM) or ultrasound (US) guidance. The goal of performing knee arthrocentesis is to obtain synovial fluid, however, it is also important to consider the number of attempts required and accidental bone contacts that occur. This study evaluates procedural success without bone contact in knee arthrocentesis and compares both LM and US guided techniques in a cadaver model. METHODS: This was a randomized crossover study comparing US vs LM guidance for arthrocentesis in a single academic center. Volunteers were randomized to perform both LM and US guided knee arthrocentesis on cadavers. The primary outcome was procedural success, defined as first attempt aspiration of synovial fluid without bone contact. Secondary outcomes included number of attempts, number of bone contacts, time to aspiration, and confidence. RESULTS: Sixty-one participants completed the study with a total of 122 procedures performed. Procedural success without bone contact was greater in the US group (84% vs 64% p = 0.02). Time to aspiration was longer for US (38.75 s vs 25.54 s p = 0.004). Participants were more confident with US compared to LM both before the procedure on a Visual Analog Scale from 1 to 100 (29 vs 21 p = 0.03) as well as after the procedure (83 vs 69 p = 0.0001). Participants had a greater median increase in confidence with US following training (44 vs 26 p = 0.01). CONCLUSIONS: Study participants had greater procedural success without bone contact when US guidance was used. The increase in confidence following training was greater for US guidance than the LM method. Use of US guidance may offer a benefit by allowing for better needle control and avoidance of sensitive structures for clinicians performing knee arthrocentesis.

TIN2 is an architectural protein that facilitates TRF2-mediated trans- and cis-interactions on telomeric DNA.

The telomere specific shelterin complex, which includes TRF1, TRF2, RAP1, TIN2, TPP1 and POT1, prevents spurious recognition of telomeres as double-strand DNA breaks and regulates telomerase and DNA repair activities at telomeres. TIN2 is a key component of the shelterin complex that directly interacts with TRF1, TRF2 and TPP1. In vivo, the large majority of TRF1 and TRF2 are in complex with TIN2 but without TPP1 and POT1. Since knockdown of TIN2 also removes TRF1 and TRF2 from telomeres, previous cell-based assays only provide information on downstream effects after the loss of TRF1/TRF2 and TIN2. Here, we investigated DNA structures promoted by TRF2-TIN2 using single-molecule imaging platforms, including tracking of compaction of long mouse telomeric DNA using fluorescence imaging, atomic force microscopy (AFM) imaging of protein-DNA structures, and monitoring of DNA-DNA and DNA-RNA bridging using the DNA tightrope assay. These techniques enabled us to uncover previously unknown unique activities of TIN2. TIN2S and TIN2L isoforms facilitate TRF2-mediated telomeric DNA compaction (cis-interactions), dsDNA-dsDNA, dsDNA-ssDNA and dsDNA-ssRNA bridging (trans-interactions). Furthermore, TIN2 facilitates TRF2-mediated T-loop formation. We propose a molecular model in which TIN2 functions as an architectural protein to promote TRF2-mediated trans and cis higher-order nucleic acid structures at telomeres.

Structure, dynamics, and regulation of TRF1-TIN2-mediated trans- and cis-interactions on telomeric DNA.

TIN2 is a core component of the shelterin complex linking double-stranded telomeric DNA-binding proteins (TRF1 and TRF2) and single-strand overhang-binding proteins (TPP1-POT1). In vivo, the large majority of TRF1 and TRF2 exist in complexes containing TIN2 but lacking TPP1/POT1; however, the role of TRF1-TIN2 interactions in mediating interactions with telomeric DNA is unclear. Here, we investigated DNA molecular structures promoted by TRF1-TIN2 interaction using atomic force microscopy (AFM), total internal reflection fluorescence microscopy (TIRFM), and the DNA tightrope assay. We demonstrate that the short (TIN2S) and long (TIN2L) isoforms of TIN2 facilitate TRF1-mediated DNA compaction (cis-interactions) and DNA-DNA bridging (trans-interactions) in a telomeric sequence- and length-dependent manner. On the short telomeric DNA substrate (six TTAGGG repeats), the majority of TRF1-mediated telomeric DNA-DNA bridging events are transient with a lifetime of ~1.95 s. On longer DNA substrates (270 TTAGGG repeats), TIN2 forms multiprotein complexes with TRF1 and stabilizes TRF1-mediated DNA-DNA bridging events that last on the order of minutes. Preincubation of TRF1 with its regulator protein Tankyrase 1 and the cofactor NAD+ significantly reduced TRF1-TIN2 mediated DNA-DNA bridging, whereas TIN2 protected the disassembly of TRF1-TIN2 mediated DNA-DNA bridging upon Tankyrase 1 addition. Furthermore, we showed that TPP1 inhibits TRF1-TIN2L-mediated DNA-DNA bridging. Our study, together with previous findings, supports a molecular model in which protein assemblies at telomeres are heterogeneous with distinct subcomplexes and full shelterin complexes playing distinct roles in telomere protection and elongation.

Optic nerve sheath diameter in severe preeclampsia with neurologic features versus controls.

BACKGROUND: Optic nerve sheath diameters (ONSD) have been validated as an accurate screening tool to detect elevated intracranial pressure in hypertensive encephalopathy. The neurologic manifestations of preeclampsia and/or eclampsia mimic those of hypertensive encephalopathy. This study was performed to assess the incidence of elevated optic nerve sheath diameters of patients with severe preeclampsia and neurologic criteria compared to non-preeclamptic patients. The secondary objective was to determine baseline optic nerve sheath diameters in patients with severe preeclampsia without neurologic criteria and preeclampsia without severe features. METHODS: Single site cohort study including 62 pregnant women 18 years or older and 20 weeks or further gestation. Patients with preeclampsia without severe features, preeclampsia with severe features by non-neurologic criteria, preeclampsia with severe features with neurologic criteria, and patients without preeclampsia were enrolled via convenience sampling. One blinded reviewer measured sheath diameters; baseline demographics and pregnancy data were collected by chart review. Statistical analysis was completed with STATA/IC 16. Categorical variables were compared by the χ2 test. Continuous variables were presented as mean ± standard deviation, and discrete variables were presented as medians and compared by Kruskal-Wallis testing. Normality was confirmed by Shapiro-Wilk testing. Linear and logistic regression were used to test the association between the preeclampsia groups and optic nerve sheath diameters. Models were presented as unadjusted and adjusted for BMI, gestation, hypertension, diabetes, parity, and gravidity. RESULTS: The incidence of optic nerve sheath diameters > 5.8 mm was 43.8% in the severe preeclampsia with neurologic features cohort, and 42.1% in the control cohort, with a relative risk of 1.04. Patients with severe preeclampsia without neurologic features had sheath diameters of 5.75 mm ± 1.09 mm; non-severe preeclampsia patients had sheath diameters of 5.54 mm ± 1.26 mm. CONCLUSIONS: We did not find a significant elevated optic nerve sheath diameter relative risk between severe preeclampsia patients with neurologic features and non-preeclampsia control patients. This is the first study to assess a North American population utilizing ACOG criteria for severe and non-severe preeclampsia, with severe cohorts additionally stratified by neurologic criteria.

Point-of-Care Ultrasound Facilitates Management of Ruptured Ectopic Pregnancy.

ABSTRACT: Abdominal pain in the pregnant adolescent presents a diagnostic dilemma with potential life-threatening etiologies. We present a case where point-of-care ultrasound was used to facilitate diagnosis and expedite lifesaving management of a ruptured ectopic pregnancy. We further review the technique and literature for first-trimester transabdominal point-of-care ultrasound.

Ultrasound-Guided Posterior Tibial Nerve Block for Plantar Foot Foreign Body Removal.

Ultrasound-guided regional anesthesia is a growing modality within the pediatric emergency department. Here we present a case where a posterior tibial nerve block was used for anesthesia during foreign body removal from the plantar foot. We further review the technique and literature regarding this straightforward and highly effective procedure.

Heterogeneity of Network Structures and Water Dynamics in κ-Carrageenan Gels Probed by Nanoparticle Diffusometry.

A set of functionalized nanoparticles (PEGylated dendrimers, d = 2.8-11 nm) was used to probe the structural heterogeneity in Na+/K+ induced κ-carrageenan gels. The self-diffusion behavior of these nanoparticles as observed by 1H pulsed-field gradient NMR, fluorescence recovery after photobleaching, and raster image correlation spectroscopy revealed a fast and a slow component, pointing toward microstructural heterogeneity in the gel network. The self-diffusion behavior of the faster nanoparticles could be modeled with obstruction by a coarse network (average mesh size <100 nm), while the slower-diffusing nanoparticles are trapped in a dense network (lower mesh size limit of 4.6 nm). Overhauser dynamic nuclear polarization-enhanced NMR relaxometry revealed a reduced local solvent water diffusivity near 2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPO)-labeled nanoparticles trapped in the dense network, showing that heterogeneity in the physical network is also reflected in heterogeneous self-diffusivity of water. The observed heterogeneity in mesh sizes and in water self-diffusivity is of interest for understanding and modeling of transport through and release of solutes from heterogeneous biopolymer gels.

Spatially Heterogeneous Surface Water Diffusivity around Structured Protein Surfaces at Equilibrium.

Hydration water on the surface of a protein is thought to mediate the thermodynamics of protein-ligand interactions. For hydration water to play a role beyond modulating global protein solubility or stability, the thermodynamic properties of hydration water must reflect on the properties of the heterogeneous protein surface and thus spatially vary over the protein surface. A potent read-out of local variations in thermodynamic properties of hydration water is its equilibrium dynamics spanning picosecond to nanosecond time scales. In this study, we employ Overhauser dynamic nuclear polarization (ODNP) to probe the equilibrium hydration water dynamics at select sites on the surface of Chemotaxis Y (CheY) in dilute solution. ODNP reports on site-specific hydration water dynamics within 5-10 Å of a label tethered to the biomolecular surface on two separate time scales of motion, corresponding to diffusive water (DW) and protein-water coupled motions, referred to as bound water (BW). We find DW dynamics to be highly heterogeneous across the surface of CheY. We identify a significant correlation between DW dynamics and the local hydropathy of the CheY protein surface, empirically determined by molecular dynamics (MD) simulations, and find the more hydrophobic sites to be hydrated with slower diffusing water. Furthermore, we compare the hydration water dynamics on different polypeptides and liposome surfaces and find the DW dynamics on globular proteins to be significantly more heterogeneous than on intrinsically disordered proteins (IDPs), peptides, and liposomes. The heterogeneity in the hydration water dynamics suggests that structured proteins have the capacity to encode information into the surrounding hydration shell.

Mango polyphenolics reduce inflammation in intestinal colitis-involvement of the miR-126/PI3K/AKT/mTOR axis in vitro and in vivo.

This study sought to elucidate the mechanisms underlying the anti-inflammatory effect of mango (Mangifera Indica L.) polyphenolics containing gallic acid and gallotanins, and the role of the miR-126/PI3K/AKT/mTOR signaling axis in vitro and in vivo. Polyphenolics extracted from mango (var. Keitt) were investigated in lipopolysaccharide (LPS)-treated CCD-18Co cells. Rats received either a beverage with mango polyphenolics or a control beverage, and were exposed to three cycles of 3% dextran sodium sulfate (DSS) followed by a 2-wk recovery period. The mango extract (10 mg GAE/L) suppressed the protein expression of NF-κB, p-NF-κB, PI3K (p85ß), HIF-1α, p70S6K1, and RPS6 in LPS-treated CCD-18Co cells. LPS reduced miR-126 expression, whereas, the mango extract induced miR-126 expression in a dose-dependent manner. The relationship between miR-126 and its target, PI3K (p85ß), was confirmed by treating cells with miR-126 antagomiR where mango polyphenols reversed the effects of the antagomiR. In vivo, mango beverage protected against DSS-induced colonic inflammation (47%, P = 0.05) and decreased the Ki-67 labeling index in the central and basal regions compared to the control. Mango beverage significantly attenuated the expression of pro-inflammatory cytokines such as TNF-α, IL-1ß, and iNOS at the mRNA and protein level. Moreover, the expression of PI3K, AKT, and mTOR was reduced, whereas, miR-126 was upregulated by the mango treatment. These results suggest that mango polyphenols attenuated inflammatory response by modulating the PI3K/AKT/mTOR pathway at least in part through upregulation of miRNA-126 expression both in vitro and in vivo; thus, mango polyphenolics might be relevant as preventive agents in ulcerative colitis. © 2016 Wiley Periodicals, Inc.

Human PrimPol is a highly error-prone polymerase regulated by single-stranded DNA binding proteins.

PrimPol is a recently identified polymerase involved in eukaryotic DNA damage tolerance, employed in both re-priming and translesion synthesis mechanisms to bypass nuclear and mitochondrial DNA lesions. In this report, we investigate how the enzymatic activities of human PrimPol are regulated. We show that, unlike other TLS polymerases, PrimPol is not stimulated by PCNA and does not interact with it in vivo. We identify that PrimPol interacts with both of the major single-strand binding proteins, RPA and mtSSB in vivo. Using NMR spectroscopy, we characterize the domains responsible for the PrimPol-RPA interaction, revealing that PrimPol binds directly to the N-terminal domain of RPA70. In contrast to the established role of SSBs in stimulating replicative polymerases, we find that SSBs significantly limit the primase and polymerase activities of PrimPol. To identify the requirement for this regulation, we employed two forward mutation assays to characterize PrimPol's replication fidelity. We find that PrimPol is a mutagenic polymerase, with a unique error specificity that is highly biased towards insertion-deletion errors. Given the error-prone disposition of PrimPol, we propose a mechanism whereby SSBs greatly restrict the contribution of this enzyme to DNA replication at stalled forks, thus reducing the mutagenic potential of PrimPol during genome replication.

Hydration Dynamics of a Peripheral Membrane Protein.

Water dynamics in the hydration shell of the peripheral membrane protein annexin B12 were studied using MD simulations and Overhauser DNP-enhanced NMR. We show that retardation of water motions near phospholipid bilayers is extended by the presence of a membrane-bound protein, up to around 10 Å above that protein. Near the membrane surface, electrostatic interactions with the lipid head groups strongly slow down water dynamics, whereas protein-induced water retardation is weaker and dominates only at distances beyond 10 Å from the membrane surface. The results can be understood from a simple model based on additive contributions from the membrane and the protein to the activation free energy barriers of water diffusion next to the biomolecular surfaces. Furthermore, analysis of the intermolecular vibrations of the water network reveals that retarded water motions near the membrane shift the vibrational modes to higher frequencies, which we used to identify an entropy gradient from the membrane surface toward the bulk water. Our results have implications for processes that take place at lipid membrane surfaces, including molecular recognition, binding, and protein-protein interactions.

Repair of Full-Thickness Gluteus Maximus Tear With Suture Anchors.

Gluteal tendon tears are a common cause of hip pain. Most commonly, tears occur in the gluteus medius and minimus, and there are well-established nonoperative or operative treatment pathways. In this Technical Note, we describe our technique for repair of a full-thickness gluteus maximus tendon tear using anchor fixation.

Oxidative guanine base damage plays a dual role in regulating productive ALT-associated homology-directed repair.

Cancer cells maintain telomeres by upregulating telomerase or alternative lengthening of telomeres (ALT) via homology-directed repair at telomeric DNA breaks. 8-Oxoguanine (8oxoG) is a highly prevalent endogenous DNA lesion in telomeric sequences, altering telomere structure and telomerase activity, but its impact on ALT is unclear. Here, we demonstrate that targeted 8oxoG formation at telomeres stimulates ALT activity and homologous recombination specifically in ALT cancer cells. Mechanistically, an acute 8oxoG induction increases replication stress, as evidenced by increased telomere fragility and ATR kinase activation at ALT telomeres. Furthermore, ALT cells are more sensitive to chronic telomeric 8oxoG damage than telomerase-positive cancer cells, consistent with increased 8oxoG-induced replication stress. However, telomeric 8oxoG production in G2 phase, when ALT telomere elongation occurs, impairs telomeric DNA synthesis. Our study demonstrates that a common oxidative base lesion has a dual role in regulating ALT depending on when the damage arises in the cell cycle.

Tobramycin and Vancomycin in an In Vitro Model of Anterior Cruciate Ligament Allograft Decontamination.

BACKGROUND: Approximately 100,000 anterior cruciate ligament (ACL) reconstructions (ACLRs) occur annually in the United States, and postoperative surgical-site infection is a relatively rare but devastating complication, often leading to graft failure or septic arthritis of the knee, necessitating repeat surgery. Wrapping allografts in vancomycin-soaked gauze has been adopted as a common sterilization technique in the operating room to reduce surgical-site infection; however, identifying effective alternatives to vancomycin has not been extensively pursued. HYPOTHESIS: Tobramycin would be as effective as vancomycin in reducing the concentrations of Staphylococcus epidermidis bacteria on tendon allografts. STUDY DESIGN: Controlled laboratory study. METHODS: S. epidermidis strain ATCC 12228 was inoculated onto the human cadaveric gracilis tendon. The tendons were wrapped in sterile gauze saturated with tobramycin or vancomycin at various experimental concentrations. Bacteria remaining on the tendon were dislodged, serially diluted, and plated for colony counting. Statistical analysis was performed utilizing 2-way analysis of variance testing. Results were considered statistically significant when P < .05. RESULTS: Vancomycin (P = .0001) and tobramycin (P < .0001) reduced bacterial concentration. Tobramycin was found to produce a statistically significant reduction in bacterial concentration at concentrations as low as 0.1 mg/mL (P < .0001 and P = .01 at 10 and 20 minutes), while vancomycin produced a statistically significant reduction at a concentration as low as 2.5 mg/mL (P < .0001 at both 10 and 20 minutes). CONCLUSION: This study demonstrates that tobramycin is as effective as vancomycin in bacterial concentration reduction but can achieve this reduction level at lower doses. Further studies clarifying the biomechanical and cytotoxic effects of tobramycin on tendon tissue are indicated to solidify its use as a clinical alternative to vancomycin in ACLR. CLINICAL RELEVANCE: These results will begin establishing tobramycin as an alternative to vancomycin in ACL graft decontamination. Because of relatively frequent shortages of vancomycin, establishing tobramycin as an alternative agent is a useful option for the orthopaedic surgeon.

Modified Loop Suture Technique in Arthroscopic Labral Repair of the Hip.

Hip arthroscopy is rapidly increasing in case volume annually in the United States. However, it remains a challenging surgery with a steep learning curve. Labral repair is commonly performed to preserve the labrum rather than labral debridement or reconstruction. Many techniques have been described for labral repair. In this technical note, we describe our technique for a modified loop suture technique for arthroscopic labral repair of the hip.

Telomere Fragility and MiDAS: Managing the Gaps at the End of the Road.

Telomeres present inherent difficulties to the DNA replication machinery due to their repetitive sequence content, formation of non-B DNA secondary structures, and the presence of the nucleo-protein t-loop. Especially in cancer cells, telomeres are hot spots for replication stress, which can result in a visible phenotype in metaphase cells termed "telomere fragility". A mechanism cells employ to mitigate replication stress, including at telomeres, is DNA synthesis in mitosis (MiDAS). While these phenomena are both observed in mitotic cells, the relationship between them is poorly understood; however, a common link is DNA replication stress. In this review, we will summarize what is known to regulate telomere fragility and telomere MiDAS, paying special attention to the proteins which play a role in these telomere phenotypes.

Posterior Meniscal Root Repair With Transtibial Double Tunnel Pullout Technique and Anchor Fixation.

Meniscus root tears are tears that occur within 1 cm of the meniscus root attachment to the tibia. If not repaired, root tears will lead to altered knee biomechanics and accelerated degenerative changes in the knee. In this surgical technique, we demonstrate repair of a meniscus root tear with an all-inside suture repair of the posterior meniscus that is then passed through 2 transtibial tunnels and then fixed with an anchor. After surgery, patients are non-weightbearing for 4 weeks and can return to activity by 6 months.