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Blake Oberfeld,1,2 Fatemeh Golsoorat Pahlaviani,1 Hani El Helwe,1 Henisk Falah,1 Nathan Hall,1 Jonathan Trzcinski,1 David Solá-Del Valle1,3,4 1Massachusetts Eye and Ear Infirmary and Harvard Medical School, Boston, MA, USA; 2University of Florida, Department of Ophthalmology, Gainesville, FL, USA; 3Chittick Eye Care, Champaign, IL, USA; 4Carle Illinois College of Medicine, Urbana, IL, USACorrespondence: David Solá-Del Valle, Fax +16175733707, Email david.sola@chittickeyecare.comPurpose: Despite holding promise, reports of using MIGS in severe glaucoma are scarce, and none has described combining multiple MIGS in this population. To the best of our knowledge, this is the largest study to report outcomes of phacoemulsification and MIGS (Phaco/MIGS) in patients with severe glaucoma.Methods: This retrospective review comprised 327 clinical visits of 71 patients with severe glaucoma who underwent Phaco/MIGS with iStent, endocyclodestruction, Kahook Dual Blade, Hydrus Microstent, or a combination of these MIGS (cMIGS) performed between 2016 and 2021. Primary outcomes included intraocular pressure (IOP) and medication burden evaluated by Generalized Estimating Equations, as well as Kaplan–Meier Estimates. Further analyses compared the efficacy of cMIGS and single Phaco/MIGS (sMIGS), procedure duration, visual acuity, and complications.Results: Mean preoperative IOP was 16.7 mmHg ± 5.8 (SD) on 2.3 ± 1.9 medications overall (N = 71), 16.9 ± 6.3 mmHg on 1.7 ± 1.9 medications in the sMIGS group (N = 37), and 16.4 ± 5.3 mmHg on 2.9 ± 1.6 medications in the cMIGS group (N = 34). Throughout 12 months, Phaco/MIGS led to significant reduction patterns in IOP (p < 0.001) and medications (p = 0.03). At 12 months, 47.5%, 87.5%, and 64.7% of the patients achieved IOP ≤ 12 mmHg, 17 mmHg, or predetermined goal IOP, respectively, without additional medication or procedure. Mean 12-month IOP was 13.5 ± 3.1 mmHg on 1.8 ± 1.7 medications. After adjusting for baseline medication burden, the reduction pattern in IOP (p < 0.05) was different between cMIGS and sMIGS, favoring cMIGS, and the groups had similar reduction patterns in medications (p = 0.75).Conclusion: The use of Phaco/MIGS in patients with cataract and severe glaucoma may significantly reduce IOP and medication burden throughout 12 months and, thus, may serve as a stepping stone in severe glaucoma patients with visually significant cataract before proceeding with more invasive glaucoma surgery. This effect may be potentiated by the combination effect of cMIGS.Plain Language Summary: Many patients with cataract and mild or moderate glaucoma who undergo cataract surgery also benefit from microinvasive glaucoma surgery (MIGS) performed at the same time, but the role of MIGS in patients with severe glaucoma and cataract is not clear. We report that combined cataract surgery and MIGS were associated with significant reductions in eye pressure in patients with severe glaucoma for more than 12 months.Keywords: microinvasive glaucoma surgery, MIGS, cMIGS, combined MIGS, generalized estimating equations, GEE, comparative effectiveness, combined efficacy, severe glaucoma

Hani El Helwe, Blake Oberfeld, Fatemeh Golsoorat Pahlaviani, Henisk Falah, Jonathan Trzcinski, David Solá-Del Valle Department of Ophthalmology, Harvard Medical School and Mass Eye and Ear, Boston, MA, USACorrespondence: David Solá-Del Valle, Massachusetts Eye and Ear, Harvard Medical School, 243 Charles Street, Boston, MA, 02114, USA, Email david_sola-delvalle@meei.harvard.eduPurpose: To compare outcomes of phacoemulsification and endocyclophotocoagulation with either dual blade goniotomy (PEcK) or two trabecular stents (ICE2).Setting: Retrospective, nonrandomized comparative study from a level 3 triage center.Methods: One hundred and seventy charts and a total of 1294 visits were reviewed following either PEcK or ICE2 from 2018 to 2022. One hundred and twenty-eight patients had PEcK and 42 underwent ICE2. Patients with less than 30 days of follow-up were excluded. The mean follow-up time was 505 ± 308 days. Two Kaplan–Meier curves (KM) assessed survival with ≤ baseline medications while maintaining (1) [GIC – Goal IOP Criteria] IOP ≤ goal IOP or (2) [PRC – Percent Reduction Criteria] IOP reduction ≥ 20% with 5 mmHg ≤ IOP ≤ 21 mmHg for at least two consecutive visits. IOP and medication burden reduction were compared using a paired t-test.Results: Most patients were Caucasian (65%) and had mild-stage glaucoma (43%). The most common glaucoma type was primary open-angle glaucoma (58%). Average age was 72.2 years at the time of surgery. Mean preoperative IOP was 17.58 ± 4.98 mmHg on 3.00 ± 1.41 medications in PEcK and 15.36 ± 3.58 mmHg on 1.81 ± 1.11 medications in ICE2 (p = 0.015 for IOP; p < 0.001 for medications). Under GIC, the success rate was significantly higher in PEcK at POM6 (69% vs 46%, p < 0.001) and POY1 (63% vs 36%, p < 0.001). Under PRC, the success rate was significantly higher in PEcK at POM6 (73% vs 61%, p = 0.031) and POY1 (67% vs 50%, p = 0.028). Mean reductions at POY1 were 5.00 ± 4.31 mmHg on 1.35 ± 1.08 less medications after PEcK and 3.14 ± 2.83 mmHg on 1.01 ± 0.94 less medications after ICE2 (p < 0.001 at POY1 for IOP; p < 0.05 after POW6 for medications).Conclusion: Both PEcK and ICE2 reduce medication and IOP from baseline, with PEcK having more favorable GIC and PRC success rates and greater IOP and medication reduction at 1 year.Keywords: microinvasive glaucoma surgery, phacoemulsification, endocyclophotocoagulation, Kahook Dual Blade, iStent inject, surgical outcomes

Blake Oberfeld,1,2 Fatemeh Golsoorat Pahlaviani,1 Nathan Hall,1 Henisk Falah-Trzcinski,1 Jonathan Trzcinski,1 Ta Chang,3 David Solá-Del Valle1 1Glaucoma Service, Massachusetts Eye and Ear Infirmary and Harvard Medical School, Boston, MA, USA; 2Department of Ophthalmology, University of Florida, Gainesville, FL, USA; 3Department of Ophthalmology, Bascom Palmer Eye Institute, Miami, FL, USACorrespondence: David Solá-Del Valle, Glaucoma Service, Massachusetts Eye and Ear Infirmary and Harvard Medical, 243 Charles St, Boston, MA, 02114, USA, Tel +1 617-573-3670, Fax +1 617-573-3707, Email David_Sola-DelValle@MEEI.HARVARD.EDUPurpose: Combining two or more MIGS (cMIGS) promises to be more efficacious than single MIGS (sMIGS). This study compared the efficacy of PEcK, which combines Phacoemulsification (Phaco), Endocyclophotocoagulation (ECP), and Kahook dual blade (KDB), relative to both of its constituent sMIGS, Phaco/ECP (Endo Optiks, NJ) and Phaco/KDB (New World Medical, CA) for the first time.Patients and methods: Data was collected retrospectively from 1833 visits of 271 patients who underwent PEcK, Phaco/ECP, or Phaco/KDB from 2016– 2021 at Massachusetts Eye and Ear. Primary outcomes included Generalized Estimating Equations (GEE) of intraocular pressure (IOP) and medication burden, as well as survival models.Results: Mean preoperative IOP was 17.6 ± 5.0 (SD) mmHg on 3.0 ± 1.4 medications in the PEcK group (n = 128), 17.9 ± 5.1 mmHg on 2.2 ± 1.5 medications in the Phaco/ECP group (n = 78), and 16.1 ± 4.3 mmHg on 0.4 ± 1.0 medications in the Phaco/KDB group (n = 65). For more than 36 months, all procedures resulted in significant patterns of IOP and medication reduction (all p < 0.001), before and after statistical adjustment. The reduction pattern in IOP was significantly different when comparing all groups over time and favored PEcK (p = 0.04), but the reduction pattern in medications was not significantly different (p = 0.11). Procedures did not differ in procedural time (p = 0.18) or in survival to maintain ≥ 20% IOP reduction (p = 0.43) without additional medication or procedure. There was a trend toward significant difference in maintaining IOP ≤ goal IOP that favored PEcK over Phaco/ECP after adjustment (p = 0.09).Conclusion: PEcK may confer greater IOP reduction without added procedural time compared to Phaco/ECP and Phaco/KDB in predominantly mild or moderate glaucoma. Further research on cMIGS may benefit from adopting this comparative analysis to constituent MIGS.Keywords: microinvasive glaucoma surgery, cMIGS, generalized estimating equations, GEE, comparative effectiveness, combined efficacy, glaucoma

The use of microinvasive invasive glaucoma surgery (MIGS) in the treatment of glaucoma has increased exponentially over the last 10 years. However, practice patterns vary widely given the relative newness of these technologies. Some surgeons perform two or more MIGS simultaneously, such as those that target aqueous production and those that target aqueous outflow. These combined MIGS (cMIGS) may result in lower intraocular pressure (IOP) and reduced medication burden as compared to single MIGS (sMIGS). Current evidence suggests some cMIGS are more effective in reducing medication burden for at least 12 months versus sMIGS. This review focuses on the current evidence related to the efficacy of cMIGS as well as novel combinations of standalone MIGS, limitations of the current literature, and future directions for research.

Background: Chronic transportation noise exposure associates with cardiovascular events through a link involving heightened stress-associated neurobiological activity (as amygdalar metabolic activity, AmygA) on 18 F-fluorodeoxyglucose positron emission tomography/computed tomography ( 18 F-FDG-PET/CT). Increased AmygA also associates with greater visceral adipose tissue (VAT) and type 2 diabetes mellitus (DM). While relationships between noise exposure and VAT and DM have been reported, the underlying mechanisms remain incompletely understood. We tested whether: (1) transportation noise exposure associates with greater (a) baseline and gains in VAT and (b) DM risk, and (2) heightened AmygA partially mediates the link between noise exposure and these metabolic diseases., Methods: VAT was measured in a retrospective cohort (N = 403) who underwent clinical 18 F-FDG-PET/CT. AmygA was measured in those with brain imaging (N = 238). Follow-up VAT was remeasured on available imaging (N = 67). Among individuals (N = 224) without baseline DM, incident DM was adjudicated over 2 years from clinical records. Noise (24-h average) was modeled at each individual's home address. Linear regression, survival, and mediation analyses were employed., Results: Higher noise exposure (upper tertile vs. others) associated with greater: baseline VAT (standardized β [95% confidence interval (CI)]= 0.230 [0.021, 0.438], p = 0.031), gains in VAT (0.686 [0.185, 1.187], p = 0.008 adjusted for baseline VAT), and DM (hazard ratio [95% CI]=2.429 [1.031, 5.719], p = 0.042). The paths of: ↑noise exposure→↑AmygA→↑baseline VAT and ↑noise exposure→↑AmygA→↑subsequent DM were significant (p < 0.05)., Conclusions: Increased transportation noise exposure associates with greater VAT and DM. This relationship is partially mediated by stress-associated neurobiological activity. These findings suggest altered neurobiology contributes to noise exposure's link to metabolic diseases., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Aims: Air pollution [i.e. particulate matter with diameter <2.5 μm (PM2.5)] is a risk factor for major adverse cardiovascular events (MACE). While PM2.5 promotes leucopoiesis and atherosclerotic inflammation in experimental models, it is unknown whether this occurs in humans. We tested in humans (a) whether PM2.5 associates with higher leucopoietic tissue activity and arterial inflammation (ArtI), (ii) whether these associations persist after accounting for the effects of potential confounders including socioeconomics, traffic noise, and risk factors, and (iii) whether these tissue effects mediate the association between air pollution and MACE., Methods and Results: Individuals (N = 503) without cardiovascular disease (CVD) or active malignancy underwent 18 F-fluorodeoxyglucose positron emission tomography/computed tomography. Major adverse cardiovascular event was adjudicated over 5 years of follow-up. Leucopoietic tissue activity (in bone marrow and spleen) as well as ArtI were measured. Annual PM2.5 levels were assessed at each individual's home address. At baseline, higher PM2.5 associated with increased leucopoietic activity [standardized (95% CI): 0.129 (0.042, 0.215), P = 0.004] as well as ArtI [0.088 (0.006, 0.171), P = 0.036] after adjusting for CVD risk factors. Over a median 4.1 years, 40 individuals experienced MACE. PM2.5 exposure associated with MACE [Cox HR (95% CI): 1.404 (1.135, 1.737), P = 0.002], remaining significant after adjustment for CVD risk factors and other potential confounders. Mediation analysis demonstrated that increased leucopoietic activity and ArtI serially mediate the link between PM2.5 exposure and MACE., Conclusions: Higher air pollution exposure associates with heightened leucopoietic activity and ArtI and independently predicts MACE through a biological pathway that includes higher leucopoietic activity and ArtI in series., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2021. For permissions, please email: journals.permissions@oup.com.)

Current understanding of the impact of coronavirus disease-2019 (COVID-19) on arrhythmias continues to evolve as new data emerge. Cardiac arrhythmias are more common in critically ill COVID-19 patients. The potential mechanisms that could result in arrhythmogenesis among COVID-19 patients include hypoxia caused by direct viral tissue involvement of lungs, myocarditis, abnormal host immune response, myocardial ischemia, myocardial strain, electrolyte derangements, intravascular volume imbalances, and drug sides effects. To manage these arrhythmias, it is imperative to increase the awareness of potential drug-drug interactions, to monitor QTc prolongation while receiving COVID therapy and provide special considerations for patients with inherited arrhythmia syndromes. It is also crucial to minimize exposure to COVID-19 infection by stratifying the need for intervention and using telemedicine. As COVID-19 infection continues to prevail with a potential for future surges, more data are required to better understand pathophysiology and to validate management strategies., (Copyright © 2020. Published by Elsevier Inc.)

Objectives: This study sought to identify midmyocardial arrhythmogenic substrates by examining the frequency content of unipolar endocardial surface electrograms, comparing sites with transmural scar versus sites with intramural excitable substrate (IES) as identified during needle catheter ablation for ventricular tachycardia (VT)., Background: Midmyocardial arrhythmogenic substrates are a common reason catheter ablation for VT may fail., Methods: A total of 659 intramural needle sites were studied in 26 patients (age 61 ± 9 years, 85% male, 69% nonischemic cardiomyopathy) who underwent intramural needle catheter ablation for VT. Among 136 sites where endocardial pacing did not capture (threshold >10 mA), needle pacing captured at 29 indicating IES, and did not capture at 107 indicating transmural scar. Intramural needle ablation was performed at 21 of 29 IES sites. Analysis of voltage amplitude, duration, and power spectra of endocardial and intramural needle electrograms was performed., Results: IES sites compared with transmural scar had higher endocardial unipolar electrogram voltage, 0.99 (interquartile range [IQR]: 0.69 to 1.62) mV versus 0.78 (IQR: 0.61 to 1.09) mV; p = 0.038; higher unipolar intramural needle electrogram voltage, 1.16 (0.80 to 1.69) mV versus 0.76 (0.6 to 1.12) mV; p = 0.003; higher endocardial unipolar frequency power particularly in the 5- to 20-Hz band, 1.97 (IQR: 0.93 to 3.89) mV 2 /s versus 1.03 (IQR: 0.63 to 2.22) mV 2 /s; p = 0.002; and higher unipolar intramural electrogram frequency particularly in the 0 to 10 Hz range, 3.02 (IQR: 0.98 to 6.95) mV 2 /s versus 1.33 (IQR: 0.70 to 3.13) mV 2 /s; p = 0.018. Endocardial unipolar frequency in the 5- to 20-Hz band identified sites with IES, area under the curve of 0.676; p = 0.002; power frequency integral of >0.77 mV 2 /s provided a 90% sensitivity and 41% specificity., Conclusions: The frequency content of unipolar electrograms may complement voltage in the detection of deep intramural substrates to facilitate VT catheter ablation. (Intramural Needle Ablation for Ablation of Recurrent Ventricular Tachycardia; NCT01791543)., (Copyright © 2020 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.)

Coronavirus disease 2019 (COVID-19) is a novel respiratory illness caused by SARS-CoV-2. Viral entry is mediated through viral spike protein and host ACE2 enzyme interaction. Most cases are mild; severe disease often involves cytokine storm and organ failure. Therapeutics including antivirals, immunomodulators, and vaccines are in development. To view this SnapShot, open or download the PDF., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Aims: Chronic noise exposure associates with increased cardiovascular disease (CVD) risk; however, the role of confounders and the underlying mechanism remain incompletely defined. The amygdala, a limbic centre involved in stress perception, participates in the response to noise. Higher amygdalar metabolic activity (AmygA) associates with increased CVD risk through a mechanism involving heightened arterial inflammation (ArtI). Accordingly, in this retrospective study, we tested whether greater noise exposure associates with higher: (i) AmygA, (ii) ArtI, and (iii) risk for major adverse cardiovascular disease events (MACE)., Methods and Results: Adults (N = 498) without CVD or active cancer underwent clinical 18F-fluorodeoxyglucose positron emission tomography/computed tomography imaging. Amygdalar metabolic activity and ArtI were measured, and MACE within 5 years was adjudicated. Average 24-h transportation noise and potential confounders were estimated at each individual's home address. Over a median 4.06 years, 40 individuals experienced MACE. Higher noise exposure (per 5 dBA increase) predicted MACE [hazard ratio (95% confidence interval, CI) 1.341 (1.147-1.567), P < 0.001] and remained robust to multivariable adjustments. Higher noise exposure associated with increased AmygA [standardized β (95% CI) 0.112 (0.051-0.174), P < 0.001] and ArtI [0.045 (0.001-0.090), P = 0.047]. Mediation analysis suggested that higher noise exposure associates with MACE via a serial mechanism involving heightened AmygA and ArtI that accounts for 12-26% of this relationship., Conclusion: Our findings suggest that noise exposure associates with MACE via a mechanism that begins with increased stress-associated limbic (amygdalar) activity and includes heightened arterial inflammation. This potential neurobiological mechanism linking noise to CVD merits further evaluation in a prospective population., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2019. For permissions, please email: journals.permissions@oup.com.)

Background: Lower socioeconomic status (SES) associates with a higher risk of major adverse cardiac events (MACE) via mechanisms that are not well understood., Objectives: Because psychosocial stress is more prevalent among those with low SES, this study tested the hypothesis that stress-associated neurobiological pathways involving up-regulated inflammation in part mediate the link between lower SES and MACE., Methods: A total of 509 individuals, median age 55 years (interquartile range: 45 to 66 years), underwent clinically indicated whole-body 18 F-fluorodeoxyglucose positron emission tomography/computed tomography imaging and met pre-defined inclusion criteria, including absence of known cardiovascular disease or active cancer. Baseline hematopoietic tissue activity, arterial inflammation, and in a subset of 289, resting amygdalar metabolism (a measure of stress-associated neural activity) were quantified using validated 18 F-fluorodeoxyglucose positron emission tomography/computed tomography methods. SES was captured by neighborhood SES factors (e.g., median household income and crime). MACE within 5 years of imaging was adjudicated., Results: Over a median 4.0 years, 40 individuals experienced MACE. Baseline income inversely associated with amygdalar activity (standardized β: -0.157 [95% confidence interval (CI): -0.266 to -0.041]; p = 0.007) and arterial inflammation (β: -0.10 [95% CI: -0.18 to -0.14]; p = 0.022). Further, income associated with subsequent MACE (standardized hazard ratio: 0.67 [95% CI: 0.47 to 0.96]; p = 0.029) after multivariable adjustments. Mediation analysis demonstrated that the path of: ↓ neighborhood income to ↑ amygdalar activity to ↑ bone marrow activity to ↑ arterial inflammation to ↑ MACE was significant (β: -0.01 [95% CI: -0.06 to -0.001]; p < 0.05)., Conclusions: Lower SES: 1) associates with higher amygdalar activity; and 2) independently predicts MACE via a serial pathway that includes higher amygdalar activity, bone marrow activity, and arterial inflammation. These findings illuminate a stress-associated neurobiological mechanism by which SES disparities may potentiate adverse health outcomes., (Copyright © 2019 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.)

Context: Epidemiologic data link psychological stress to adiposity. The underlying mechanisms remain uncertain., Objectives: To test whether (i) higher activity of the amygdala, a neural center involved in the response to stress, associates with greater visceral adipose tissue (VAT) volumes and (ii) this association is mediated by increased bone marrow activity., Setting: Massachusetts General Hospital, Boston, Massachusetts., Patients: Two hundred forty-six patients without active oncologic, cardiovascular, or inflammatory disease who underwent clinical 18F-fluorodeoxyglucose positron emission tomography/computed tomography imaging were studied. VAT imaging was repeated ∼1 year later in 68 subjects., Design: Metabolic activity of the amygdala (AmygA), hematopoietic tissue activity, and adiposity volumes were measured with validated methods., Main Outcome Measure: The relationship between AmygA and baseline and follow-up VAT., Results: AmygA associated with baseline body mass index (standardized β = 0.15; P = 0.01), VAT (0.19; P = 0.002), and VAT/subcutaneous adipose tissue ratio (0.20; P = 0.002), all remaining significant after adjustment for age and sex. AmygA also associated with bone marrow activity (0.15; P = 0.01), which in turn associated with VAT (0.34; P < 0.001). Furthermore, path analysis showed that 48% of the relationship between AmygA and baseline VAT was mediated by increased bone marrow activity (P = 0.007). Moreover, AmygA associated with achieved VAT after 1 year (P = 0.02) after adjusting for age, sex, and baseline VAT., Conclusions: These results suggest a neurobiological pathway involving the amygdala and bone marrow linking psychosocial stress to adiposity in humans. Future studies should test whether targeting this mechanism attenuates adiposity and its complications., (Copyright © 2019 Endocrine Society.)

Regulation of the epithelial Na(+) channel (ENaC) by ubiquitylation is controlled by the activity of two counteracting enzymes, the E3 ubiquitin-protein ligase Nedd4-2 (mouse ortholog of human Nedd4L) and the ubiquitin-specific protease Usp2-45. Previously, Usp2-45 was shown to decrease ubiquitylation and to increase surface function of ENaC in Xenopus laevis oocytes, whereas the splice variant Usp2-69, which has a different N-terminal domain, was inactive toward ENaC. It is shown here that the catalytic core of Usp2 lacking the N-terminal domain has a reduced ability relative to Usp2-45 to enhance ENaC activity in Xenopus oocytes. In contrast, its catalytic activity toward the artificial substrate ubiquitin-AMC is fully maintained. The interaction of Usp2-45 with ENaC exogenously expressed in HEK293 cells was tested by coimmunoprecipitation. The data indicate that different combinations of ENaC subunits, as well as the α-ENaC cytoplasmic N-terminal but not C-terminal domain, coprecipitate with Usp2-45. This interaction is decreased but not abolished when the cytoplasmic ubiquitylation sites of ENaC are mutated. Importantly, coimmunoprecipitation in HEK293 cells and GST pull-down of purified recombinant proteins show that both the catalytic domain and the N-terminal tail of Usp2-45 physically interact with the HECT domain of Nedd4-2. Taken together, the data support the conclusion that Usp2-45 action on ENaC is promoted by various interactions, including through binding to Nedd4-2 that is suggested to position Usp2-45 favorably for ENaC deubiquitylation.

The occupancy of the central cavity of the membrane-embedded c ring of the ATP synthase of Escherichia coli was investigated with a photo-cross-linking approach. Single cysteine mutants were created at c subunit positions 4, 8, and 11, which are oriented to the inside of the ring. These cysteines were alkylated with reagents carrying a photoactivatable substituent and illuminated. Subunit c and derivatives were then isolated and subjected to mass spectrometric analyses. The most noticeable product, which was found exclusively in irradiated samples, had a mass increase of 719 Da, consistent with a cross-link product between the substituted c subunit and phosphatidylethanolamine. Digestion with phospholipase C converted this product into one with a mass diminished by 126 Da, indicating that the phosphoethanolamine moiety was cleaved off. Hence, the cross-link forms to the diacylglycerol moiety of phosphatidylethanolamine. Control experiments showed that the subunit c-phospholipid adducts were formed in the ATP synthase complex in its natural membrane environment and were not artifacts arising from monomeric c subunits. We conclude therefore that the inner lumen of the c ring is occupied with phospholipids. No evidence was found for an extension of subunit a into this space.