Developmental Consequences of Defective ATG7-Mediated Autophagy in Humans.

BACKGROUND: Autophagy is the major intracellular degradation route in mammalian cells. Systemic ablation of core autophagy-related (ATG) genes in mice leads to embryonic or perinatal lethality, and conditional models show neurodegeneration. Impaired autophagy has been associated with a range of complex human diseases, yet congenital autophagy disorders are rare. METHODS: We performed a genetic, clinical, and neuroimaging analysis involving five families. Mechanistic investigations were conducted with the use of patient-derived fibroblasts, skeletal muscle-biopsy specimens, mouse embryonic fibroblasts, and yeast. RESULTS: We found deleterious, recessive variants in human ATG7, a core autophagy-related gene encoding a protein that is indispensable to classical degradative autophagy. Twelve patients from five families with distinct ATG7 variants had complex neurodevelopmental disorders with brain, muscle, and endocrine involvement. Patients had abnormalities of the cerebellum and corpus callosum and various degrees of facial dysmorphism. These patients have survived with impaired autophagic flux arising from a diminishment or absence of ATG7 protein. Although autophagic sequestration was markedly reduced, evidence of basal autophagy was readily identified in fibroblasts and skeletal muscle with loss of ATG7. Complementation of different model systems by deleterious ATG7 variants resulted in poor or absent autophagic function as compared with the reintroduction of wild-type ATG7. CONCLUSIONS: We identified several patients with a neurodevelopmental disorder who have survived with a severe loss or complete absence of ATG7, an essential effector enzyme for autophagy without a known functional paralogue. (Funded by the Wellcome Centre for Mitochondrial Research and others.).

The Association Between Oncology Outreach and Timely Treatment for Rural Patients with Breast Cancer: A Claims-Based Approach.

BACKGROUND: Oncology outreach is a common strategy for increasing rural access to cancer care, where traveling oncologists commute across healthcare settings to extend specialized care. Examining the extent to which physician outreach is associated with timely treatment for rural patients is critical for informing outreach strategies. METHODS: We identified a 100% fee-for-service sample of incident breast cancer patients from 2015 to 2020 Medicare claims and apportioned them into surgery and adjuvant therapy cohorts based on treatment history. We defined an outreach visit as the provision of care by a traveling oncologist at a clinic outside of their primary hospital service area. We used hierarchical logistic regression to examine the associations between patient receipt of preoperative care at an outreach visit (preoperative outreach) and > 60-day surgical delay, and patient receipt of postoperative care at an outreach visit (postoperative outreach) and > 60-day adjuvant delay. RESULTS: We identified 30,337 rural-residing patients who received breast cancer surgery, of whom 4071 (13.4%) experienced surgical delay. Among surgical patients, 14,501 received adjuvant therapy, of whom 2943 (20.3%) experienced adjuvant delay. In adjusted analysis, we found that patient receipt of preoperative outreach was associated with reduced odds of surgical delay (odds ratio [OR] 0.75, 95% confidence interval [CI] 0.61-0.91); however, we found no association between patient receipt of postoperative outreach and adjuvant delay (OR 1.04, 95% CI 0.85-1.25). CONCLUSIONS: Our findings indicate that preoperative outreach is protective against surgical delay. The traveling oncologists who enable such outreach may play an integral role in catalyzing the coordination and timeliness of patient-centered care.

Leigh syndrome: an adult presentation of a paediatric disease.

A previously healthy 27-year-old man was admitted to the acute neurology ward with events involving his face, throat and upper limb, which video telemetry later confirmed were refractory focal seizures. He also had progressive pyramidal features, dysarthria and ataxia. MR scans of the brain identified progressive bilateral basal ganglia abnormalities, consistent with Leigh syndrome. However, extensive laboratory and genetic panels did not give a unifying diagnosis. A skeletal muscle biopsy showed no histopathological abnormalities on routine stains. Sequencing of the entire mitochondrial genome in skeletal muscle identified a well-characterised pathogenic variant (m.10191T>C in MT-ND3; NC_012920.1) at 85% heteroplasmy in skeletal muscle. We discuss the clinical and molecular diagnosis of an adult presenting with Leigh syndrome, which is more commonly a paediatric presentation of mitochondrial disease, and how early recognition of a mitochondrial cause is important to support patient care.

Forecasting stroke-like episodes and outcomes in mitochondrial disease.

In this retrospective, multicentre, observational cohort study, we sought to determine the clinical, radiological, EEG, genetics and neuropathological characteristics of mitochondrial stroke-like episodes and to identify associated risk predictors. Between January 1998 and June 2018, we identified 111 patients with genetically determined mitochondrial disease who developed stroke-like episodes. Post-mortem cases of mitochondrial disease (n = 26) were identified from Newcastle Brain Tissue Resource. The primary outcome was to interrogate the clinico-radiopathological correlates and prognostic indicators of stroke-like episode in patients with mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes syndrome (MELAS). The secondary objective was to develop a multivariable prediction model to forecast stroke-like episode risk. The most common genetic cause of stroke-like episodes was the m.3243A>G variant in MT-TL1 (n = 66), followed by recessive pathogenic POLG variants (n = 22), and 11 other rarer pathogenic mitochondrial DNA variants (n = 23). The age of first stroke-like episode was available for 105 patients [mean (SD) age: 31.8 (16.1)]; a total of 35 patients (32%) presented with their first stroke-like episode ≥40 years of age. The median interval (interquartile range) between first and second stroke-like episodes was 1.33 (2.86) years; 43% of patients developed recurrent stroke-like episodes within 12 months. Clinico-radiological, electrophysiological and neuropathological findings of stroke-like episodes were consistent with the hallmarks of medically refractory epilepsy. Patients with POLG-related stroke-like episodes demonstrated more fulminant disease trajectories than cases of m.3243A>G and other mitochondrial DNA pathogenic variants, in terms of the frequency of refractory status epilepticus, rapidity of progression and overall mortality. In multivariate analysis, baseline factors of body mass index, age-adjusted blood m.3243A>G heteroplasmy, sensorineural hearing loss and serum lactate were significantly associated with risk of stroke-like episodes in patients with the m.3243A>G variant. These factors informed the development of a prediction model to assess the risk of developing stroke-like episodes that demonstrated good overall discrimination (area under the curve = 0.87, 95% CI 0.82-0.93; c-statistic = 0.89). Significant radiological and pathological features of neurodegeneration were more evident in patients harbouring pathogenic mtDNA variants compared with POLG: brain atrophy on cranial MRI (90% versus 44%, P < 0.001) and reduced mean brain weight (SD) [1044 g (148) versus 1304 g (142), P = 0.005]. Our findings highlight the often idiosyncratic clinical, radiological and EEG characteristics of mitochondrial stroke-like episodes. Early recognition of seizures and aggressive instigation of treatment may help circumvent or slow neuronal loss and abate increasing disease burden. The risk-prediction model for the m.3243A>G variant can help inform more tailored genetic counselling and prognostication in routine clinical practice.

Identification of a novel heterozygous DYSF variant in a large family with a dominantly-inherited dysferlinopathy.

AIMS: Dysferlinopathy is an autosomal recessive muscular dystrophy, caused by bi-allelic variants in the gene encoding dysferlin (DYSF). Onset typically occurs in the second to third decade and is characterised by slowly progressive skeletal muscle weakness and atrophy of the proximal and/or distal muscles of the four limbs. There are rare cases of symptomatic DYSF variant carriers. Here, we report a large family with a dominantly inherited hyperCKaemia and late-onset muscular dystrophy. METHODS AND RESULTS: Genetic analysis identified a co-segregating novel DYSF variant [NM_003494.4:c.6207del p.(Tyr2070Metfs*4)]. No secondary variants in DYSF or other dystrophy-related genes were identified on whole genome sequencing and analysis of the proband's DNA. Skeletal muscle involvement was milder and later onset than typical dysferlinopathy presentations; these clinical signs manifested in four individuals, all between the fourth and sixth decades of life. All individuals heterozygous for the c.6207del variant had hyperCKaemia. Histological analysis of skeletal muscle biopsies across three generations showed clear dystrophic signs, including inflammatory infiltrates, regenerating myofibres, increased variability in myofibre size and internal nuclei. Muscle magnetic resonance imaging revealed fatty replacement of muscle in two individuals. Western blot and immunohistochemical analysis of muscle biopsy demonstrated consistent reduction of dysferlin staining. Allele-specific quantitative PCR analysis of DYSF mRNA from patient muscle found that the variant, localised to the extreme C-terminus of dysferlin, does not activate post-transcriptional mRNA decay. CONCLUSIONS: We propose that this inheritance pattern may be underappreciated and that other late-onset muscular dystrophy cases with mono-allelic DYSF variants, particularly C-terminal premature truncation variants, may represent dominant forms of disease.

Rural-Urban Differences in Breast Cancer Surgical Delays in Medicare Beneficiaries.

BACKGROUND: Delays between breast cancer diagnosis and surgery are associated with worsened survival. Delays are more common in urban-residing patients, although factors specific to surgical delays among rural and urban patients are not well understood. METHODS: We used a 100% sample of fee-for-service Medicare claims during 2007-2014 to identify 238,491 women diagnosed with early-stage breast cancer undergoing initial surgery and assessed whether they experienced biopsy-to-surgery intervals > 90 days. We employed multilevel regression to identify associations between delays and patient, regional, and surgeon characteristics, both in combined analyses and stratified by rurality of patient residence. RESULTS: Delays were more prevalent among urban patients (2.5%) than rural patients (1.9%). Rural patients with medium- or high-volume surgeons had lower odds of delay than patients with low-volume surgeons (odds ratio [OR] = 0.71, 95% confidence interval [CI] = 0.58-0.88; OR = 0.74, 95% CI = 0.61-0.90). Rural patients whose surgeon operated at ≥ 3 hospitals were more likely to experience delays (OR = 1.29, 95% CI = 1.01-1.64, Ref: 1 hospital). Patient driving times ≥ 1 h were associated with delays among urban patients only. Age, black race, Hispanic ethnicity, multimorbidity, and academic/specialty hospital status were associated with delays. CONCLUSIONS: Sociodemographic, geographic, surgeon, and facility factors have distinct associations with > 90-day delays to initial breast cancer surgery. Interventions to improve timeliness of breast cancer surgery may have disparate impacts on vulnerable populations by rural-urban status.

Ferric Heme Superoxide Reductive Transformations to Ferric Heme (Hydro)Peroxide Species: Spectroscopic Characterization and Thermodynamic Implications for H-Atom Transfer (HAT).

A new end-on low-spin ferric heme peroxide, [(PIm )FeIII -(O22- )]- (PIm -P), and subsequently formed hydroperoxide species, [(PIm )FeIII -(OOH)] (PIm -HP) are generated utilizing the iron-porphyrinate PIm with its tethered axial base imidazolyl group. Measured thermodynamic parameters, the ferric heme superoxide [(PIm )FeIII -(O2⋅- )] (PIm -S) reduction potential (E°') and the PIm -HP pKa value, lead to the finding of the OO-H bond-dissociation free energy (BDFE) of PIm -HP as 69.5 kcal mol-1 using a thermodynamic square scheme and Bordwell relationship. The results are validated by the observed oxidizing ability of PIm -S via hydrogen-atom transfer (HAT) compared to that of the F8 superoxide complex, [(F8 )FeIII -(O2.- )] (S) (F8 =tetrakis(2,6-difluorophenyl)porphyrinate, without an internally appended axial base imidazolyl), as determined from reactivity comparison of superoxide complexes PIm -S and S with the hydroxylamine (O-H) substrates TEMPO-H and ABNO-H.

Heme-FeIII Superoxide, Peroxide and Hydroperoxide Thermodynamic Relationships: FeIII-O2•- Complex H-Atom Abstraction Reactivity.

Establishing redox and thermodynamic relationships between metal-ion-bound O2 and its reduced (and protonated) derivatives is critically important for a full understanding of (bio)chemical processes involving dioxygen processing. Here, a ferric heme peroxide complex, [(F8)FeIII-(O22-)]- (P) (F8 = tetrakis(2,6-difluorophenyl)porphyrinate), and a superoxide complex, [(F8)FeIII-(O2•-)] (S), are shown to be redox interconvertible. Using Cr(η-C6H6)2, an equilibrium state where S and P are present is established in tetrahydrofuran (THF) at -80 °C, allowing determination of the reduction potential of S as -1.17 V vs Fc+/0. P could be protonated with 2,6-lutidinium triflate, yielding the low-spin ferric hydroperoxide species, [(F8)FeIII-(OOH)] (HP). Partial conversion of HP back to P using a derivatized phosphazene base gave a P/HP equilibrium mixture, leading to the determination of pKa = 28.8 for HP (THF, -80 °C). With the measured reduction potential and pKa, the O-H bond dissociation free energy (BDFE) of hydroperoxide species HP was calculated to be 73.5 kcal/mol, employing the thermodynamic square scheme and Bordwell relationship. This calculated O-H BDFE of HP, in fact, lines up with an experimental demonstration of the oxidizing ability of S via hydrogen atom transfer (HAT) from TEMPO-H (2,2,6,6-tetramethylpiperdine-N-hydroxide, BDFE = 66.5 kcal/mol in THF), forming the hydroperoxide species HP and TEMPO radical. Kinetic studies carried out with TEMPO-H(D) reveal second-order behavior, kH = 0.5, kD = 0.08 M-1 s-1 (THF, -80 °C); thus, the hydrogen/deuterium kinetic isotope effect (KIE) = 6, consistent with H-atom abstraction by S being the rate-determining step. This appears to be the first case where experimentally derived thermodynamics lead to a ferric heme hydroperoxide OO-H BDFE determination, that FeIII-OOH species being formed via HAT reactivity of the partner ferric heme superoxide complex.

Surveillance imaging for patients with head and neck cancer treated with definitive radiotherapy: A partially observed Markov decision process model.

BACKGROUND: A possible surveillance model for patients with head and neck cancer (HNC) who received definitive radiotherapy was created using a partially observed Markov decision process. The goal of this model is to guide surveillance imaging policies after definitive radiotherapy. METHODS: The partially observed Markov decision process model was formulated to determine the optimal times to scan patients. Transition probabilities were computed using a data set of 1508 patients with HNC who received definitive radiotherapy between the years 2000 and 2010. Kernel density estimation was used to smooth the sample distributions. The reward function was derived using cost estimates from the literature. Additional model parameters were estimated using either data from the literature or clinical expertise. RESULTS: When considering all forms of relapse, the model showed that the optimal time between scans was longer than the time intervals used in the institutional guidelines. The optimal policy dictates that there should be less time between surveillance scans immediately after treatment compared with years after treatment. Comparable results also held when only locoregional relapses were considered as relapse events in the model. Simulation results for the inclusive relapse cases showed that <15% of patients experienced a relapse over a simulated 36-month surveillance program. CONCLUSIONS: This model suggests that less frequent surveillance scan policies can maintain adequate information on relapse status for patients with HNC treated with radiotherapy. This model could potentially translate into a more cost-effective surveillance program for this group of patients.

Identification of a novel heterozygous guanosine monophosphate reductase (GMPR) variant in a patient with a late-onset disorder of mitochondrial DNA maintenance.

Autosomal dominant progressive external ophthalmoplegia (adPEO) is a late-onset, Mendelian mitochondrial disorder characterised by paresis of the extraocular muscles, ptosis, and skeletal-muscle restricted multiple mitochondrial DNA (mtDNA) deletions. Although dominantly inherited, pathogenic variants in POLG, TWNK and RRM2B are among the most common genetic defects of adPEO, identification of novel candidate genes and the underlying pathomechanisms remains challenging. We report the clinical, genetic and molecular investigations of a patient who presented in the seventh decade of life with PEO. Oxidative histochemistry revealed cytochrome c oxidase-deficient fibres and occasional ragged red fibres showing subsarcolemmal mitochondrial accumulation in skeletal muscle, while molecular studies identified the presence of multiple mtDNA deletions. Negative candidate screening of known nuclear genes associated with PEO prompted diagnostic exome sequencing, leading to the prioritisation of a novel heterozygous c.547G>C variant in GMPR (NM_006877.3) encoding guanosine monophosphate reductase, a cytosolic enzyme required for maintaining the cellular balance of adenine and guanine nucleotides. We show that the novel c.547G>C variant causes aberrant splicing, decreased GMPR protein levels in patient skeletal muscle, proliferating and quiescent cells, and is associated with subtle changes in nucleotide homeostasis protein levels and evidence of disturbed mtDNA maintenance in skeletal muscle. Despite confirmation of GMPR deficiency, demonstrating marked defects of mtDNA replication or nucleotide homeostasis in patient cells proved challenging. Our study proposes that GMPR is the 19th locus for PEO and highlights the complexities of uncovering disease mechanisms in late-onset PEO phenotypes.

Pathogenic variants in MT-ATP6: A United Kingdom-based mitochondrial disease cohort study.

Distinct clinical syndromes have been associated with pathogenic MT-ATP6 variants. In this cohort study, we identified 125 individuals (60 families) including 88 clinically affected individuals and 37 asymptomatic carriers. Thirty-one individuals presented with Leigh syndrome and 7 with neuropathy ataxia retinitis pigmentosa. The remaining 50 patients presented with variable nonsyndromic features including ataxia, neuropathy, and learning disability. We confirmed maternal inheritance in 39 families and demonstrated that tissue segregation patterns and phenotypic threshold are variant dependent. Our findings suggest that MT-ATP6-related mitochondrial DNA disease is best conceptualized as a mitochondrial disease spectrum disorder and should be routinely included in genetic ataxia and neuropathy gene panels. ANN NEUROL 2019;86:310-315.

Patient and physician factors associated with Oncotype DX and adjuvant chemotherapy utilization for breast cancer patients in New Hampshire, 2010-2016.

BACKGROUND: Oncotype DX® (ODX) is used to assess risk of disease recurrence in hormone receptor positive, HER2-negative breast cancer and to guide decisions regarding adjuvant chemotherapy. Little is known about how physician factors impact treatment decisions. The purpose of this study was to examine patient and physician factors associated with ODX testing and adjuvant chemotherapy for breast cancer patients in New Hampshire. METHODS: We examined New Hampshire State Cancer Registry data on 5630 female breast cancer patients diagnosed from 2010 to 2016. We performed unadjusted and adjusted hierarchical logistic regression to identify factors associated with a patient's receipt of ODX, being recommended and receiving chemotherapy, and refusing chemotherapy. We calculated intraclass correlation coefficients (ICCs) to examine the proportion of variance in clinical decisions explained by between-physician and between-hospital variation. RESULTS: Over the study period, 1512 breast cancer patients received ODX. After adjustment for patient and tumor characteristics, we found that patients seen by a male medical oncologist were less likely to be recommended chemotherapy following ODX (OR = 0.50 (95% CI = 0.34-0.74), p < 0.01). Medical oncologists with more clinical experience (reference: less than 10 years) were more likely to recommend chemotherapy (20-29 years: OR = 4.05 (95% CI = 1.57-10.43), p < 0.01; > 29 years: OR = 4.48 (95% CI = 1.68-11.95), p < 0.01). A substantial amount of the variation in receiving chemotherapy was due to variation between physicians, particularly among low risk patients (ICC = 0.33). CONCLUSIONS: In addition to patient clinicopathologic characteristics, physician gender and clinical experience were associated with chemotherapy treatment following ODX testing. The significant variation between physicians indicates the potential for interventions to reduce variation in care.

A biomechanical comparison of conventional classifications of bowling action-types in junior fast bowlers.

Fast bowling is categorised into four action types: side-on, front-on, semi-open and mixed; however, little biomechanical comparison exists between action types in junior fast bowlers. This study investigated whether there are significant differences between action-type mechanics in junior fast bowlers. Three-dimensional kinematic and kinetic analyses were completed on 60 junior male fast bowlers bowling a five-over spell. Mixed-design factorial analyses of variance were used to test for differences between action-type groups across the phases of the bowling action. One kinetic difference was observed between groups, with a higher vertical ground reaction force loading rate during the front-foot contact phase in mixed and front-on compared to semi-open bowlers; no other significant group differences in joint loading occurred. Significant kinematic differences were observed between the front-on, semi-open and mixed action types during the front-foot contact phase for the elbow and trunk. Significant kinematic differences were also present for the ankle, T12-L1, elbow, trunk and pelvis during the back-foot phase. Overall, most differences in action types for junior fast bowlers occurred during the back-foot contact phase, particularly trunk rotation and T12-L1 joint angles/ranges of motion, where after similar movement patterns were utilized across groups during the front-foot contact phase.

Geometric and Electronic Structure Contributions to O-O Cleavage and the Resultant Intermediate Generated in Heme-Copper Oxidases.

This study investigates the mechanism of O-O bond cleavage in heme-copper oxidase (HCO) enzymes, combining experimental and computational insights from enzyme intermediates and synthetic models. It is determined that HCOs undergo a proton-initiated O-O cleavage mechanism where a single water molecule in the active site enables proton transfer (PT) from the cross-linked tyrosine to a peroxo ligand bridging the heme FeIII and CuII, and multiple H-bonding interactions lower the tyrosine p Ka. Due to sterics within the active site, the proton must either transfer initially to the O(Fe) (a high-energy intermediate), or from another residue over a ∼10 Å distance to reach the O(Cu) atom directly. While the distance between the H+ donor (Tyr) and acceptor (O(Cu)) results in a barrier to PT, this separation is critical for the low barrier to O-O cleavage as it enhances backbonding from Fe into the O22- σ* orbital. Thus, PT from Tyr precedes O-O elongation and is rate-limiting, consistent with available kinetic data. The electron transfers from tyrosinate after the barrier via a superexchange pathway provided by the cross-link, generating intermediate PM. PM is evaluated using available experimental data. The geometric structure contains an FeIV═O that is H-bonded to the CuII-OH. The electronic structure is a singlet, where the FeIV and CuII are antiferromagnetically coupled through the H-bond between the oxo(Fe) and hydroxo(Cu) ligands, while the CuII and Tyr• are ferromagnetically coupled due their delocalization into orthogonal magnetic orbitals on the cross-linked His residue. These findings provide critical insights into the mechanism of efficient O2 reduction in HCOs, and the nature of the PM intermediate that couples this reaction to proton pumping.

Ligand Identity-Induced Generation of Enhanced Oxidative Hydrogen Atom Transfer Reactivity for a CuII2(O2•-) Complex Driven by Formation of a CuII2(-OOH) Compound with a Strong O-H Bond.

A superoxide-bridged dicopper(II) complex, [CuII2(XYLO)(O2•-)]2+ (1) (XYLO = binucleating m-xylyl derivative with a bridging phenolate ligand donor and two bis(2-{2-pyridyl}ethyl)amine arms), was generated from chemical oxidation of the peroxide-bridged dicopper(II) complex [CuII2(XYLO)(O22-)]+ (2), using ferrocenium (Fc+) derivatives, in 2-methyltetrahydrofuran (MeTHF) at -125 °C. Using Me10Fc+, a 1 ⇆ 2 equilibrium was established, allowing for calculation of the reduction potential of 1 as -0.525 ± 0.01 V vs Fc+/0. Addition of 1 equiv of strong acid to 2 afforded the hydroperoxide-bridged dicopper(II) species [CuII2(XYLO)(OOH)]2+ (3). An acid-base equilibrium between 3 and 2 was achieved through spectral titrations using a derivatized phosphazene base. The pKa of 3 was thus determined to be 24 ± 0.6 in MeTHF at -125 °C. Using a thermodynamic square scheme and the Bordwell relationship, the hydroperoxo complex (3) O-H bond dissociation free energy (BDFE) was calculated as 81.8 ± 1.5 (BDE = 86.8) kcal/mol. The observed oxidizing capability of [CuII2(XYLO)(O2•-)]2+ (1), as demonstrated in H atom abstraction reactions with certain phenolic ArO-H and hydrocarbon C-H substrates, provides direct support for this experimentally determined O-H BDFE. A kinetic study reveals a very fast reaction of TEMPO-H with 1 in MeTHF, with k (-100 °C) = 5.6 M-1 s-1. Density functional theory (DFT) calculations reveal how the structure of 1 may minimize stabilization of the superoxide moiety, resulting in its enhanced reactivity. The thermodynamic insights obtained herein highlight the importance of the interplay between ligand design and the generation and properties of copper (or other metal ion) bound O2-derived reduced species, such as pKa, reduction potential, and BDFE; these may be relevant to the capabilities (i.e., oxidizing power) of reactive oxygen intermediates in metalloenzyme chemical system mediated oxidative processes.

Spin Interconversion of Heme-Peroxo-Copper Complexes Facilitated by Intramolecular Hydrogen-Bonding Interactions.

Synthetic peroxo-bridged high-spin (HS) heme-(µ-η2:η1-O22-)-Cu(L) complexes incorporating (as part of the copper ligand) intramolecular hydrogen-bond (H-bond) capabilities and/or steric effects are herein demonstrated to affect the complex's electronic and geometric structure, notably impacting the spin state. An H-bonding interaction with the peroxo core favors a low-spin (LS) heme-(µ-η1:η1-O22-)-Cu(L) structure, resulting in a reversible temperature-dependent interconversion of spin state (5 coordinate HS to 6 coordinate LS). The LS state dominates at low temperatures, even in the absence of a strong trans-axial heme ligand. Lewis base addition inhibits the H-bond facilitated spin interconversion by competition for the H-bond donor, illustrating the precise H-bonding interaction required to induce spin-crossover (SCO). Resonance Raman spectroscopy (rR) shows that the H-bonding pendant interacts with the bridging peroxide ligand to stabilize the LS but not the HS state. The H-bond (to the Cu-bound O atom) acts to weaken the O-O bond and strengthen the Fe-O bond, exhibiting ν(M-O) and ν(O-O) values comparable to analogous known LS complexes with a strong donating trans-axial ligand, 1,5-dicyclohexylimidazole, (DCHIm)heme-(µ-η1:η1-O22-)-Cu(L). Variable-temperature (-90 to -130 °C) UV-vis and 2H NMR spectroscopies confirm the SCO process and implicate the involvement of solvent binding. Examining a case of solvent binding without SCO, thermodynamic parameters were obtained from a van't Hoff analysis, accounting for its contribution in SCO. Taken together, these data provide evidence for the H-bond group facilitating a core geometry change and allowing solvent to bind, stabilizing a LS state. The rR data, complemented by DFT analysis, reveal a stronger H-bonding interaction with the peroxo core in the LS compared to the HS complexes, which enthalpically favors the LS state. These insights enhance our fundamental understanding of secondary coordination sphere influences in metalloenzymes.

Usefulness of surveillance imaging in patients with head and neck cancer who are treated with definitive radiotherapy.

BACKGROUND: The current study was performed to assess the efficacy of surveillance imaging in patients with head and neck cancer (HNC) who are treated definitively with radiotherapy. METHODS: Eligible patients included those with a demonstrable disease-free interval (≥1 follow-up imaging procedure without evidence of disease and a subsequent visit/imaging procedure) who underwent treatment of HNC from 2000 through 2010. RESULTS: A total of 1508 patients were included. The median overall survival was 99 months, with a median imaging follow-up period of 59 months. Of the 1508 patients, 190 patients (12.6%) experienced disease recurrence (107 patients had locoregional and 83 had distant disease recurrence). A total of 119 patients (62.6%) in the group with disease recurrence were symptomatic and/or had an adverse clinical finding associated with the recurrence. Approximately 80% of patients with locoregional disease recurrences presented with a clinical finding, whereas 60% of distant disease recurrences were detected by imaging in asymptomatic patients. Despite the earlier detection of disease recurrence via imaging, those patients in the group of patients with clinically detected disease recurrence were significantly more likely to undergo salvage therapy compared with those whose recurrence was detected on imaging (odds ratio, 0.35). There was no difference in overall survival noted between those patients with disease recurrences that were detected clinically or with imaging alone. Approximately 70% of disease recurrences occurred within the first 2 years. In those patients who developed disease recurrence after 2 years, the median time to recurrence was 51 months. After 2 years, the average number of imaging procedures per patient for the detection of a salvageable recurrence for the imaging-detected group was 1539. CONCLUSIONS: Surveillance imaging in asymptomatic patients with HNC who are treated definitively with radiotherapy without clinically suspicious findings beyond 2 years has a low yield and a high cost. Physicians ordering these studies must use judicious consideration and discretion.

Pathological mechanisms underlying single large-scale mitochondrial DNA deletions.

OBJECTIVE: Single, large-scale deletions in mitochondrial DNA (mtDNA) are a common cause of mitochondrial disease. This study aimed to investigate the relationship between the genetic defect and molecular phenotype to improve understanding of pathogenic mechanisms associated with single, large-scale mtDNA deletions in skeletal muscle. METHODS: We investigated 23 muscle biopsies taken from adult patients (6 males/17 females with a mean age of 43 years) with characterized single, large-scale mtDNA deletions. Mitochondrial respiratory chain deficiency in skeletal muscle biopsies was quantified by immunoreactivity levels for complex I and complex IV proteins. Single muscle fibers with varying degrees of deficiency were selected from 6 patient biopsies for determination of mtDNA deletion level and copy number by quantitative polymerase chain reaction. RESULTS: We have defined 3 "classes" of single, large-scale deletion with distinct patterns of mitochondrial deficiency, determined by the size and location of the deletion. Single fiber analyses showed that fibers with greater respiratory chain deficiency harbored higher levels of mtDNA deletion with an increase in total mtDNA copy number. For the first time, we have demonstrated that threshold levels for complex I and complex IV deficiency differ based on deletion class. INTERPRETATION: Combining genetic and immunofluorescent assays, we conclude that thresholds for complex I and complex IV deficiency are modulated by the deletion of complex-specific protein-encoding genes. Furthermore, removal of mt-tRNA genes impacts specific complexes only at high deletion levels, when complex-specific protein-encoding genes remain. These novel findings provide valuable insight into the pathogenic mechanisms associated with these mutations. Ann Neurol 2018;83:115-130.

Heme-Cu Binucleating Ligand Supports Heme/O2 and FeII-CuI/O2 Reactivity Providing High- and Low-Spin FeIII-Peroxo-CuII Complexes.

The focus of this study is in the description of synthetic heme/copper/O2 chemistry employing a heme-containing binucleating ligand which provides a tridentate chelate for copper ion binding. The addition of O2 (-80 °C, tetrahydrofuran (THF) solvent) to the reduced heme compound (PImH)FeII (1), gives the oxy-heme adduct, formally a heme-superoxide complex FeIII-(O2•-) (2) (resonance Raman spectroscopy (rR): νO-O, 1171 cm-1 (Δ18O2, -61 cm-1); νFe-O, 575 cm-1 (Δ18O2, -24 cm-1)). Simple warming of 2 to room temperature regenerates reduced complex 1; this reaction is reversible, as followed by UV-vis spectroscopy. Complex 2 is electron paramagnetic resonance (EPR)-silent and exhibits upfield-shifted pyrrole resonances (δ 9.12 ppm) in 2H NMR spectroscopy, indicative of a six-coordinate low-spin heme. The coordination of the tethered imidazolyl arm to the heme-superoxide complex as an axial base ligand is suggested. We also report the new fully reduced heme-copper complex [(PImH)FeIICuI]+ (3), where the copper ion is bound to the tethered tridentate portion of PImH. This reacts with O2 to give a distinctive low-temperature-stable, high-spin (S = 2, overall) peroxo-bridged complex [(PImH)FeIII-(O22-)-CuII]+ (3a): λmax, 420 (Soret), 545, 565 nm; δpyrr, 93 ppm; νO-O, 799 cm-1 (Δ18O2, -48 cm-1); νFe-O, 524 cm-1 (Δ18O2, -23 cm-1). To 3a, the addition of dicyclohexylimidazole (DCHIm), which serves as a heme axial base, leads to low-spin (S = 0 overall) species complex [(DCHIm)(PImH)FeIII-(O22-)-CuII]+ (3b): λmax, 425 (Soret), 538 nm; δpyrr, 10.2 ppm; νO-O, 817 cm-1 (Δ18O2, -55 cm-1); νFe-O, 610 cm-1 (Δ18O2, -26 cm-1). These investigations into the characterization of the O2-adducts from (PImH)FeII (1) with/without additional copper chelation advance our understanding of the dioxygen reactivity of heme-only and heme/Cu-ligand heterobinuclear system, thus potentially relevant to O2 reduction in heme-copper oxidases or fuel-cell chemistry.

Impact of Intramolecular Hydrogen Bonding on the Reactivity of Cupric Superoxide Complexes with O-H and C-H Substrates.

The dioxygen reactivity of a series of TMPA-based copper(I) complexes (TMPA=tris(2-pyridylmethyl)amine), with and without secondary-coordination-sphere hydrogen-bonding moieties, was studied at -135 °C in 2-methyltetrahydrofuran (MeTHF). Kinetic stabilization of the H-bonded [( (X1)(X2) TMPA)CuII (O2.- )]+ cupric superoxide species was achieved, and they were characterized by resonance Raman (rR) spectroscopy. The structures and physical properties of [( (X1)(X2) TMPA)CuII (N3- )]+ azido analogues were compared, and the O2.- reactivity of ligand-CuI complexes when an H-bonding moiety is replaced by a methyl group was contrasted. A drastic enhancement in the reactivity of the cupric superoxide towards phenolic substrates as well as oxidation of substrates possessing moderate C-H bond-dissociation energies is observed, correlating with the number and strength of the H-bonding groups.