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Recommendations from the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP) for interpreting sequence variants specify the use of computational predictors as "supporting" level of evidence for pathogenicity or benignity using criteria PP3 and BP4, respectively. However, score intervals defined by tool developers, and ACMG/AMP recommendations that require the consensus of multiple predictors, lack quantitative support. Previously, we described a probabilistic framework that quantified the strengths of evidence (supporting, moderate, strong, very strong) within ACMG/AMP recommendations. We have extended this framework to computational predictors and introduce a new standard that converts a tool's scores to PP3 and BP4 evidence strengths. Our approach is based on estimating the local positive predictive value and can calibrate any computational tool or other continuous-scale evidence on any variant type. We estimate thresholds (score intervals) corresponding to each strength of evidence for pathogenicity and benignity for thirteen missense variant interpretation tools, using carefully assembled independent data sets. Most tools achieved supporting evidence level for both pathogenic and benign classification using newly established thresholds. Multiple tools reached score thresholds justifying moderate and several reached strong evidence levels. One tool reached very strong evidence level for benign classification on some variants. Based on these findings, we provide recommendations for evidence-based revisions of the PP3 and BP4 ACMG/AMP criteria using individual tools and future assessment of computational methods for clinical interpretation.

Whole-genome sequencing (WGS) is the gold standard for fully characterizing genetic variation but is still prohibitively expensive for large samples. To reduce costs, many studies sequence only a subset of individuals or genomic regions, and genotype imputation is used to infer genotypes for the remaining individuals or regions without sequencing data. However, not all variants can be well imputed, and the current state-of-the-art imputation quality metric, denoted as standard Rsq, is poorly calibrated for lower-frequency variants. Here, we propose MagicalRsq, a machine-learning-based method that integrates variant-level imputation and population genetics statistics, to provide a better calibrated imputation quality metric. Leveraging WGS data from the Cystic Fibrosis Genome Project (CFGP), and whole-exome sequence data from UK BioBank (UKB), we performed comprehensive experiments to evaluate the performance of MagicalRsq compared to standard Rsq for partially sequenced studies. We found that MagicalRsq aligns better with true R

Background Lymphoedema is a chronic condition that is estimated to affect up to four people per 1000 of the UK population with this increasing with age. Men account for up to 20% of lymphoedema service caseloads with research focussing upon women affected. Aims To retrieve primary qualitative research on the experiences of men with chronic lymphoedema. Methods A qualitative review was undertaken using the Joanna Briggs Institute (JBI) meta-aggregation method. A search strategy was applied to 12 databases, from inception to February 2021, with 22 studies identified and appraised. The findings were extracted and synthesised via the JBI approach. Results Four synthesised findings were identified: (1) The ‘New Norm’, how diagnosis led to men being faced with a ‘new version’ of themselves; (2) ‘Journey into the Unknown’ relates to the unforeseen diagnosis of the condition; (3) ‘Access’ – challenge in receiving a diagnosis, and support; and (4) ‘Personhood’ – the impact of the condition upon external constructs and relationships. Conclusions Men are faced with similar challenges as women coupled with societal expectations with respect to gender identity and expression. This leads to those wishing to engage with men to adopt ‘gender-based tailoring’ within healthcare services, information and support.

Pharmacological ascorbate (P-AscH - , high-dose, intravenous vitamin C) has shown promise as an adjuvant therapy for pancreatic ductal adenocarcinoma (PDAC) treatment. The objective of this study was to determine the effects of P-AscH - when combined with PDAC chemotherapies.Clonogenic survival, combination indices, and DNA damage were determined in human PDAC cell lines treated with P-AscH - in combination with 5-fluorouracil, paclitaxel, or FOLFIRINOX (combination of leucovorin, 5-fluorouracil, irinotecan, oxaliplatin). Tumor volume changes, overall survival, blood analysis, and plasma ascorbate concentration were determined in vivo in mice treated with P-AscH - with or without FOLFIRINOX.P-AscH - combined with 5-fluorouracil, paclitaxel, or FOLFIRINOX significantly reduced clonogenic survival in vitro. The DNA damage, measured by γH2AX protein expression, was increased after treatment with P-AscH - , FOLFIRINOX, and their combination. In vivo, tumor growth rate was significantly reduced by P-AscH - , FOLFIRINOX, and their combination. Overall survival was significantly increased by the combination of P-AscH - and FOLFIRINOX. Treatment with P-AscH - increased red blood cell and hemoglobin values but had no effect on white blood cell counts. Plasma ascorbate concentrations were significantly elevated in mice treated with P-AscH - with or without FOLFIRINOX.The addition of P-AscH - to standard of care chemotherapy has the potential to be an effective adjuvant for PDAC treatment.

Background Cystic fibrosis (CF) is a recessive condition caused by variants in each CF transmembrane conductance regulator (CFTR) allele. Clinically affected individuals without two identified causal variants typically have no further interrogation of CFTR beyond examination of coding regions, but the development of variant-specific CFTR-targeted treatments necessitates complete understanding of CFTR genotype. Methods Whole genome sequences were analyzed on 5,058 individuals with CF. We focused on the full CFTR gene sequence and identified disease-causing variants in three phases: screening for known and structural variants; discovery of novel loss-of-function variants; and investigation of remaining variants. Results All variants identified in the first two phases and coding region variants found in the third phase were interpreted according to CFTR2 or ACMG criteria (n = 371; 16 [4.3%] previously unreported). Full gene sequencing enabled delineation of 18 structural variants (large insertions or deletions), of which two were novel. Additional CFTR variants of uncertain effect were found in 76 F508del homozygotes and in 21 individuals with other combinations of CF-causing variants. Both causative variants were identified in 98.1% (n = 4,960) of subjects, an increase of 2.3 percentage points from the 95.8% (n = 4,847) who had a registry- or chart-reported disease-causing CFTR genotype. Of the remaining 98 individuals, 78 carried one variant that has been associated with CF (CF-causing [n = 70] or resulting in varying clinical consequences n = 8]). Conclusions Complete CFTR gene sequencing in 5,058 individuals with CF identified at least one DNA variant in 99.6% of the cohort that is targetable by current molecular or emerging gene-based therapeutic technologies.

Ferumoxytol (FMX) is an iron oxide nanoparticle that is FDA approved for the treatment of iron deficiency anemia. FMX contains an Fe3O4 core. Currently, the redox chemistry of Fe3O4 nanoparticles remains relatively unexplored. FMX has recently gained interest as an anti-cancer agent. Ionizing radiation (IR) is a treatment modality employed to treat several types of cancer. Utilizing electron paramagnetic resonance (EPR) spectroscopy, we found that the products produced from the radiolysis of water can oxidize the Fe3O4 core of FMX. Because of the limited diffusion of the HO2• and HO• produced, these highly oxidizing species have little direct effect on FMX oxidation. We have determined that H2O2 is the primary oxidant of FMX. In the presence of labile Fe2+, we found that reducing species generated from the radiolysis of H2O are able to reduce the Fe3+ sites of the Fe3O4 core. Importantly, we also have shown that IR stimulates the release of ferric iron from FMX. Because of its release of iron, FMX may serve as an adjuvant to enhance radiotherapy.

Mutational signatures discerned in cancer genomes, in aging tissues and in cells exposed to toxic agents, reflect complex processes underlying transformation of cells from normal to dysfunctional. Due to its ubiquitous and chronic nature, redox stress contributions to cellular makeover remain equivocal. The deciphering of a new mutational signature of an environmentally-relevant oxidizing agent, potassium bromate, in yeast single strand DNA uncovered a surprising heterogeneity in the mutational signatures of oxidizing agents. NMR-based analysis of molecular outcomes of redox stress revealed profound dissimilarities in metabolic landscapes following exposure to hydrogen peroxide versus potassium bromate. The predominance of G to T substitutions in the mutational spectra distinguished potassium bromate from hydrogen peroxide and paraquat and mirrored the observed metabolic changes. We attributed these changes to the generation of uncommon oxidizing species in a reaction with thiol-containing antioxidants; a nearly total depletion of intracellular glutathione and a paradoxical augmentation of potassium bromate mutagenicity and toxicity by antioxidants. Our study provides the framework for understanding multidimensional processes triggered by agents collectively known as oxidants. Detection of increased mutational loads associated with potassium bromate-related mutational motifs in human tumors may be clinically relevant as a biomarker of this distinct type of redox stress.

The clinical potential of pharmacologic ascorbate (P-AscH−; intravenous delivery achieving mmol/L concentrations in blood) as an adjuvant in cancer therapy is being reevaluated. At mmol/L concentrations, P-AscH− is thought to exhibit anticancer activity via generation of a flux of H2O2 in tumors, which leads to oxidative distress. Here, we use cell culture models of pancreatic cancer to examine the effects of P-AscH− on DNA damage, and downstream consequences, including changes in bioenergetics. We have found that the high flux of H2O2 produced by P-AscH− induces DNA damage. In response to this DNA damage, we observed that PARP1 is hyperactivated. Using our unique absolute quantitation, we found that P-AscH− mediated the overactivation of PARP1, which results in consumption of NAD+, and subsequently depletion of ATP leading to mitotic cell death. We have also found that Chk1 plays a major role in the maintenance of genomic integrity following treatment with P-AscH−. Hyperactivation of PARP1 and DNA repair are ATP-consuming processes. Using a Seahorse XF96 analyzer, we demonstrated that the severe decrease in ATP after challenging with P-AscH− is because of increased demand, not changes in the rate of production. Genetic deletion and pharmacologic inhibition of PARP1 preserved both NAD+ and ATP; however, the toxicity of P-AscH− remained. These data indicate that disruption of bioenergetics is a secondary factor in the toxicity of P-AscH−; damage to DNA appears to be the primary factor.Implications:Efforts to leverage P-AscH− in cancer therapy should first focus on DNA damage.

S1. There is differential susceptibility to P-AscH- across cell lines. S2. Bolus addition of hydrogen peroxide depletes intracellular ATP and NAD+ in MIA PaCa-2 and PANC-1 cells. S3. Pharmacological ascorbate depletes intracellular NADH in MIA PaCa-2 cells, but not in PANC-1, 339, and H6c7 cells. S4. Pharmacological inhibition of PARP1 by olaparib enhances the cytotoxicity of P-AscH- in MIA PaCa-2 cells. S5. A combination of P-AscH- and prexasertib synergistically inhibits cell proliferation of MIA PaCa-2 cells. Supplementary Table S1. Normal pancreatic cells are more resistant to P-AscH- than pancreatic cancer cells. Supplementary Table S2. The ED50's for depletion of ATP and NAD+ correlate with kcell for the removal of H2O2. Supplementary Table S3. The gene target and primer pairs used for QPCR.

Supplementary Table 1. Description of radiation treatment volumes (GTV, CTV, and PTV) Supplementary Table 2. Description of dose to organs at risk and treatment interruptions Supplemental Table 3. Subject count for key steps within the RT-phase of the clinical trial Supplemental Table 4. Subject count for key steps within the ADJ-phase of the clinical trial

Increased DUOX1 and DUOX2 expression is reversed by catalase.

Radiation induced intestinal damage histology and collagen deposition information.

Purpose: Pancreatic ductal adenocarcinoma (PDA) cells are known to produce excessive amounts of reactive oxygen species (ROS), particularly superoxide, which may contribute to the aggressive and refractory nature of this disease. Extracellular superoxide dismutase (EcSOD) is an antioxidant enzyme that catalyzes the dismutation of superoxide in the extracellular environment. This study tests the hypothesis that EcSOD modulates PDA growth and invasion by modifying the redox balance in PDA.Experimental Design: We evaluated the prognostic significance of EcSOD in a human tissue microarray (TMA) of patients with PDA. EcSOD overexpression was performed in PDA cell lines and animal models of disease. The impact of EcSOD on PDA cell lines was evaluated with Matrigel invasion in combination with a superoxide-specific SOD mimic and a nitric oxide synthase (NOS) inhibitor to determine the mechanism of action of EcSOD in PDA.Results: Loss of EcSOD expression is a common event in PDA, which correlated with worse disease biology. Overexpression of EcSOD in PDA cell lines resulted in decreased invasiveness that appeared to be related to reactions of superoxide with nitric oxide. Pancreatic cancer xenografts overexpressing EcSOD also demonstrated slower growth and peritoneal metastasis. Overexpression of EcSOD or treatment with a superoxide-specific SOD mimic caused significant decreases in PDA cell invasive capacity.Conclusions: These results support the hypothesis that loss of EcSOD leads to increased reactions of superoxide with nitric oxide, which contributes to the invasive phenotype. These results allow for the speculation that superoxide dismutase mimetics might inhibit PDA progression in human clinical disease. Clin Cancer Res; 21(7); 1741–51. ©2015 AACR.

Supplementary Legend

The effect of ascorbate and catalase on clonogenic cell survival in PDAC.

Purpose: Pharmacologic concentrations of ascorbate may be effective in cancer therapeutics. We hypothesized that ascorbate concentrations achievable with i.v. dosing would be cytotoxic in pancreatic cancer for which the 5-year survival is Experimental Design: Pancreatic cancer cell lines were treated with ascorbate (0, 5, or 10 mmol/L) for 1 hour, then viability and clonogenic survival were determined. Pancreatic tumor cells were delivered s.c. into the flank region of nude mice and allowed to grow at which time they were randomized to receive either ascorbate (4 g/kg) or osmotically equivalent saline (1 mol/L) i.p. for 2 weeks.Results: There was a time- and dose-dependent increase in measured H2O2 production with increased concentrations of ascorbate. Ascorbate decreased viability in all pancreatic cancer cell lines but had no effect on an immortalized pancreatic ductal epithelial cell line. Ascorbate decreased clonogenic survival of the pancreatic cancer cell lines, which was reversed by treatment of cells with scavengers of H2O2. Treatment with ascorbate induced a caspase-independent cell death that was associated with autophagy. In vivo, treatment with ascorbate inhibited tumor growth and prolonged survival.Conclusions: These results show that pharmacologic doses of ascorbate, easily achievable in humans, may have potential for therapy in pancreatic cancer. Clin Cancer Res; 16(2); 509–20

Plasma ascorbate concentrations.

H1299T-CAT cells overexpress catalase in vivo.

Hemoglobin concentrations.

Supplementary Figures 1-3. S1: Creation and characterization of EcSOD stably expressing PDA cell lines.; S2: Activity of EcSOD activity from stably expression PDA cell lines; S3: L-NNA decreases NOS activity in PDA cells.

Supplemental Figure 1: P-AscH- treatment in combination with RT and TMZ decreases the systemic the systemic oxidaitive stress marker 4-hydroxy-2-nonenal modified proteins. S1A-SAB.

Additional Clinical Data for Comparator Patients.

Ascorbate-2-phosphate treatment does not induce sustained DUOX expression.

DUOX1 expression is increased in normal pancreas compared to PDAC samples.

Supplementary Figure Legends

Chemoradiation therapy is the mainstay for treatment of locally advanced, borderline resectable pancreatic cancer. Pharmacologic ascorbate (P-AscH−, i.e., intravenous infusions of ascorbic acid, vitamin C), but not oral ascorbate, produces high plasma concentrations capable of selective cytotoxicity to tumor cells. In doses achievable in humans, P-AscH− decreases the viability and proliferative capacity of pancreatic cancer via a hydrogen peroxide (H2O2)-mediated mechanism. In this study, we demonstrate that P-AscH− radiosensitizes pancreatic cancer cells but inhibits radiation-induced damage to normal cells. Specifically, radiation-induced decreases in clonogenic survival and double-stranded DNA breaks in tumor cells, but not in normal cells, were enhanced by P-AscH−, while radiation-induced intestinal damage, collagen deposition, and oxidative stress were also reduced with P-AscH− in normal tissue. We also report on our first-in-human phase I trial that infused P-AscH− during the radiotherapy “beam on.” Specifically, treatment with P-AscH− increased median overall survival compared with our institutional average (21.7 vs. 12.7 months, P = 0.08) and the E4201 trial (21.7 vs. 11.1 months). Progression-free survival in P-AscH−–treated subjects was also greater than our institutional average (13.7 vs. 4.6 months, P < 0.05) and the E4201 trial (6.0 months). Results indicated that P-AscH− in combination with gemcitabine and radiotherapy for locally advanced pancreatic adenocarcinoma is safe and well tolerated with suggestions of efficacy. Because of the potential effect size and minimal toxicity, our findings suggest that investigation of P-AscH− efficacy is warranted in a phase II clinical trial.Significance:These findings demonstrate that pharmacologic ascorbate enhances pancreatic tumor cell radiation cytotoxicity in addition to offering potential protection from radiation damage in normal surrounding tissue, making it an optimal agent for improving treatment of locally advanced pancreatic adenocarcinoma.

Purpose:Standard treatment for glioblastoma (GBM) includes surgery, radiation therapy (RT), and temozolomide (TMZ), yielding a median overall survival (OS) of approximately 14 months. Preclinical models suggest that pharmacologic ascorbate (P-AscH−) enhances RT/TMZ antitumor effect in GBM. We evaluated the safety of adding P-AscH− to standard RT/TMZ therapy.Patients and Methods:This first-in-human trial was divided into an RT phase (concurrent RT/TMZ/P-AscH−) and an adjuvant (ADJ) phase (post RT/TMZ/P-AscH− phase). Eight P-AscH− dose cohorts were evaluated in the RT phase until targeted plasma ascorbate levels were achieved (≥20 mmol/L). In the ADJ phase, P-AscH− doses were escalated in each subject at each cycle until plasma concentrations were ≥20 mmol/L. P-AscH− was infused 3 times weekly during the RT phase and 2 times weekly during the ADJ phase continuing for six cycles or until disease progression. Adverse events were quantified by CTCAE (v4.03).Results:Eleven subjects were evaluable. No dose-limiting toxicities occurred. Observed toxicities were consistent with historical controls. Adverse events related to study drug were dry mouth and chills. Targeted ascorbate plasma levels of 20 mmol/L were achieved in the 87.5 g cohort; diminishing returns were realized in higher dose cohorts. Median progression-free survival (PFS) was 9.4 months and median OS was 18 months. In subjects with undetectable MGMT promoter methylation (n = 8), median PFS was 10 months and median OS was 23 months.Conclusions:P-AscH−/RT/TMZ is safe with promising clinical outcomes warranting further investigation.

Basal oxygen consumption rate is sustained after exposure to P-AscH- in MIA PaCa-2 cells.

No changes in NOX enzymes are observed following P-AscH-.

PDF file - 964K, Results presented in this section show: (1) A minimal difference in cell cycle phase distributions in MnSOD (+/+), and (-/-) MEFs following re-entry into the proliferative cycle (Table I) (2) An increase in MnSOD protein levels and activity in MnSOD overexpressing MB231 human mammary epithelial cancer cells (Figure 1) (3) An inhibition of MnSOD activity in quiescent normal human fibroblasts was associated with an increase in glucose consumption and percent S-phase (Figure 2) (4) Cell cycle phase-associated increase in cellular ROS levels was absent in MnSOD (-/-) MEFs (Figure 3) (5) Cell cycle phase specific increase in DHE-fluorescence is primarily due to an increase in cellular steady-state levels of superoxide (Figure 4) (6) Mass spectrometry results identifying lysine and arginine methylation pattern of MnSOD during quiescence and proliferation; species conservation of lysine and arginine methylation sites in MnSOD (Figure 5) (7) Site directed mutagenesis approach to mutate lysine 89 and 202 of MnSOD, and engineer expression vectors of wild-type and K-to-A mutant carrying human MnSOD cDNAs (Figure 6). (8) Computer modeling of MnSOD-methylation pattern (Figure 7)

PDF file - 102K, Table 1. AscH- and MnT4MPyP are synergistic as seen by Combination Index (CI) and Dose Reduction Index (DRI). Table 2. Summary statistics for tumor volume measurements in the generalized estimating equations analysis.

Supplemental Figure S5. Bioluminescence imaging microscopy.

Supplemental Figure S1. Timing for the administration of pharmacological ascorbate enhances decreases in clonogenic survival.

Supplemental Figure S3. Ascorbate radiosensitization induces formation of γ-H2AX.

Supplemental Figure S4. Weight changes of mice during treatment periods.

Supplemental Figure S2. IR + ascorbate induces G0/G1 cell cycle arrest. Sub-G1 population in MIA PaCa-2 cells treated with 2 Gy IR {plus minus} 2 mM ascorbate.

Supplemental Table S2. Summary statistics for the survival analysis.

The toxicity of pharmacologic ascorbate is mediated by the generation of H2O2 via the oxidation of ascorbate. Because pancreatic cancer cells are sensitive to H2O2 generated by ascorbate, they would also be expected to become sensitized to agents that increase oxidative damage such as ionizing radiation. The current study demonstrates that pharmacologic ascorbate enhances the cytotoxic effects of ionizing radiation as seen by decreased cell viability and clonogenic survival in all pancreatic cancer cell lines examined, but not in nontumorigenic pancreatic ductal epithelial cells. Ascorbate radiosensitization was associated with an increase in oxidative stress–induced DNA damage, which was reversed by catalase. In mice with established heterotopic and orthotopic pancreatic tumor xenografts, pharmacologic ascorbate combined with ionizing radiation decreased tumor growth and increased survival, without damaging the gastrointestinal tract or increasing systemic changes in parameters indicative of oxidative stress. Our results demonstrate the potential clinical utility of pharmacologic ascorbate as a radiosensitizer in the treatment of pancreatic cancer. Cancer Res; 75(16); 3314–26. ©2015 AACR.

Supplemental Table S1. Summary statistics for tumor volume measurements in the mixed linear regression analysis in MIA PaCa-2 tumor xenografts.

Supplemental Table S3. Summary statistics for tumor volume measurements in the mixed linear regression analysis in orthotopic tumor xenografts.

Supplemental Table S4. Summary statistics for tumor volume measurements in the mixed linear regression analysis in PANC-1 tumor xenografts.

PDF file - 123K, Supplementary Figure 1. AsPC-1 and Panc-1 cells treated with MnT2EPyP or MnTBAP in combination with AscH-.

Inflammatory agents, microbial products, or stromal factors pre-activate or prime neutrophils to respond to activating stimuli in a rapid and aggressive manner. Primed neutrophils exhibit enhanced chemotaxis, phagocytosis, and respiratory burst when stimulated by secondary activating stimuli. We previously reported that Triggering Receptor Expressed on Myeloid cells-1 (TREM-1) mediates neutrophil effector functions such as increased superoxide generation, transepithelial migration, and chemotaxis. However, it is unclear whether TREM-1 is required for the process of priming itself or for primed responses to subsequent stimulation. To investigate this, we utilized in vitro and in vivo differentiated neutrophils that were primed with TNF-α and then stimulated with the particulate agonist, opsonized zymosan (OpZ). Bone marrow progenitors isolated from WT and Trem-1–/– mice were transduced with estrogen regulated Homeobox8 (ER-Hoxb8) fusion transcription factor and differentiated in vitro into neutrophils following estrogen depletion. The resulting neutrophils expressed high levels of TREM-1 and resembled mature in vivo differentiated neutrophils. The effects of priming on phagocytosis and oxidative burst were determined. Phagocytosis did not require TREM-1 and was not altered by priming. In contrast, priming significantly enhanced OpZ-induced oxygen consumption and superoxide production in WT but not Trem-1–/– neutrophils indicating that TREM-1 is required for primed oxidative burst. TREM-1-dependent effects were not mediated during the process of priming itself as priming enhanced degranulation, ICAM-1 shedding, and IL-1ß release to the same extent in WT and Trem-1–/- neutrophils. Thus, TREM-1 plays a critical role in primed phagocytic respiratory burst and mediates its effects following priming.

Chronic Pseudomonas aeruginosa (Pa) infection is associated with increased morbidity and mortality in people with cystic fibrosis (CF). There is no gold standard definition of chronic Pa infection in CF. We compared chronic Pa definitions using encounter-based versus annualized data in the Early Pseudomonas Infection Control (EPIC) Observational study cohort, and subsequently compared annualized chronic Pa definitions across a range of U.S. cohorts spanning decades of CF care. We found that an annualized chronic Pa definition requiring at least 1 Pa+ culture in 3 of 4 consecutive years ("Green 3/4") resulted in chronic Pa metrics similar to established encounter-based modified Leeds criteria definitions, including a similar age at and proportion who fulfilled chronic Pa criteria, and a similar proportion with sustained Pa infection after meeting the chronic Pa definition. The Green 3/4 chronic Pa definition will be valuable for longitudinal analyses in cohorts with limited culture frequency.

At pharmacological levels, ascorbate (P-AscH-) acts as a pro-oxidant by generating H2O2, depleting ATP in sensitive cells leading to cell death. The aim of this study was to determine the role of ATP production by oxidative phosphorylation or glycolysis in mechanisms of resistance to P-AscH–induced cell death. Pancreatic cancer cells were used to generate ρ0 cells by mitochondrial overexpression of the Y147A mutant uracil-N-glycosylase or Herpes Simplex Virus protein. The ρ0 phenotype was confirmed by probing for mitochondrial DNA, mitochondrial DNA-encoded cytochrome c oxidase subunit 2, and monitoring the rate of oxygen consumption. In ρ0 cells, glycolysis accounted for 100% of ATP production as there was no mitochondrial oxygen consumption. Even though the activities of H2O2-removing antioxidant enzymes were similar in both the parental and ρ0 clones, P-AscH- -induced clonogenic cell death in ρ0 cells showed more resistance than the parental cell line. In addition, P-AscH- induced more DNA damage and more consumption of NAD+ and greater decreases in the production of ATP in the parental cell line compared to the ρ0 cells. Thus, cancer cells that largely use oxidative phosphorylation to generate ATP may be more sensitive to P-AscH- compared with cells that are glycolysis-dependent.

Cystic fibrosis (CF) is a multiorgan disease caused by a wide variety of mutations in the cystic fibrosis transmembrane conductance regulator gene. As treatment has progressed from symptom mitigation to targeting of specific molecular defects, genetics has played an important role in identifying the proper precision therapies for each individual. Novel therapeutic approaches are focused on expanding treatment to a greater number of individuals as well as working toward a cure. This review discusses the role of genetics in our understanding of CF with a particular emphasis on how genetics informs the exciting landscape of current and novel CF therapies.