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INTRODUCTION: To evaluate eye and orbital injuries in non-powered scooter, electric-scooter (e-scooter), and hoverboard riders in the United States (US) between 2014 and 2019. METHODS: The National Electronic Injury Surveillance System (NEISS) was queried for head and neck injuries by body part codes related to non-powered scooters and powered scooters/hoverboards from 2014 to 2019. The NEISS complex sampling design was used to obtain US population projections of injuries and hospital admissions. Keywords were queried in case narratives to analyze trends in location, type, and mechanism of eye and orbit injuries. RESULTS: Since their introduction, a 586% (p=0.01) increase in e-scooter injuries and 866% (p

ObjectiveTo investigate whether functional upper eyelid malposition is associated with unrecognized deficits in automated perimetry among glaucoma patients by examining patients undergoing eyelid surgery who had not been identified as requiring eyelid taping during glaucoma field testing.MethodsIn this retrospective pre-post study, an automated database search followed by manual chart review was used to identify eligible patients from January 2012 to March 2020. Included patients had reliable visual field testing within two years before and after functional upper blepharoplasty or ptosis repair and no comorbid ocular diagnoses. As part of routine practice, glaucoma visual field technicians taped patients with pupil-obstructing eyelid malposition; taped examinations were excluded from analysis. Clinical and demographic characteristics, mean deviation, and pattern standard deviation were evaluated within a two year period before and after eyelid surgery.ResultsThe final analysis included 60 eyes of 38 patients. Change in visual field parameters after eyelid surgery did not reach statistical significance in crude or adjusted analyses. Among patients with ptosis, the margin reflex distance-1 was not associated with change in mean deviation after surgery (Pearson R2 ​= ​0.0061; P ​= ​0.700). Five of 17 eyes excluded from analysis due to unreliable pre-operative visual fields demonstrated substantial improvement after surgery.ConclusionsFunctional upper eyelid malposition does not appear to cause spurious visual field abnormalities among glaucoma patients with reliable visual fields who were determined not to require eyelid taping at the time of their visual fields. Unreliable visual fields could be a sign of eyelid interference in this population.

The NASA human system risk board (HSRB) has long focused on trauma and acute medical illness as a key contributor to high level in-flight medical risk. However, ocular issues, trauma, and complaints during spaceflight are poorly characterized. In a retrospective case series, the NASA data from the life sciences data archieve (LSDA) and the lifetime surveillance of astronaught health (LSAH) was queried for eye related complaints and conditions in spaceflight across international space station (ISS) missions and space shuttle (STS) missions. The ISS dataset included missions from the year 2000 to 2020, and the STS dataset included missions from 1981 to 2011. Data were reviewed and segmented into categories of ocular complaints. 135 STS missions and 63 ISS missions were included in this analysis. Ocular events were only noted across 83 STS missions (61.5%) and 41 ISS missions (65.1%). Overall, the most common ocular complaints were eye irritation (n = 80, 33.1%), ocular foreign body or foreign body sensation (n = 55, 22.7%), dry eye syndromes (n = 38, 15.7%), epiphora or excessive tearing (n = 19, 7.85%). Of all ocular complaints or diagnoses, 9 (3.72%) were considered higher severity (keratitis, corneal ulcer, chemical exposure, and corneal abrasion). However, seemingly none required evacuation from mission. Improved depiction of ocular symptoms and diagnoses, and a more standard classification system and process to describe ocular symptoms, diagnoses, and treatments in space is crucial to provide more effective and comprehensive treatments.

Stramenopile algae contribute significantly to global primary productivity, and one class, Eustigmatophyceae, is increasingly studied for applications in high-value lipid production. Yet much about their basic biology remains unknown, including the nature of an enigmatic, pigmented globule found in vegetative cells. Here, we present an in-depth examination of this "red body," focusing on Nannochloropsis oceanica. During the cell cycle, the red body forms adjacent to the plastid, but unexpectedly it is secreted and released with the autosporangial wall following cell division. Shed red bodies contain antioxidant ketocarotenoids, and overexpression of a beta-carotene ketolase results in enlarged red bodies. Infrared spectroscopy indicates long-chain, aliphatic lipids in shed red bodies and cell walls, and UHPLC-HRMS detects a C32 alkyl diol, a potential precursor of algaenan, a recalcitrant cell wall polymer. We propose that the red body transports algaenan precursors from plastid to apoplast to be incorporated into daughter cell walls.

In prokaryotes, translation can occur on mRNA that is being transcribed in a process called coupling. How the ribosome affects the RNA polymerase (RNAP) during coupling is not well understood. Here, we reconstituted the E. coli coupling system and demonstrated that the ribosome can prevent pausing and termination of RNAP and double the overall transcription rate at the expense of fidelity. Moreover, we monitored single RNAPs coupled to ribosomes and show that coupling increases the pause-free velocity of the polymerase and that a mechanical assisting force is sufficient to explain the majority of the effects of coupling. Also, by cryo-EM, we observed that RNAPs with a terminal mismatch adopt a backtracked conformation, while a coupled ribosome allosterically induces these polymerases toward a catalytically active anti-swiveled state. Finally, we demonstrate that prolonged RNAP pausing is detrimental to cell viability, which could be prevented by polymerase reactivation through a coupled ribosome.

PURPOSE: This study aimed to identify baseline clinical features associated with the outcomes of patients enrolled in the COMBI-MB phase II study of dabrafenib and trametinib treatment in patients with V600 BRAF-mutant metastatic melanoma with melanoma brain metastases (MBM). Exploratory biomarker analysis was also conducted as part of the synergistic COMBI-BRV trial (BRV116521), to identify molecular and immunologic changes associated with dabrafenib in MBMs and extracranial metastases (ECM). PATIENTS AND METHODS: Post hoc analysis was performed for baseline features of patients (n = 125) enrolled in COMBI-MB. Analyses were performed to identify baseline clinical features associated with intracranial response rate (ICRR), progression-free survival (PFS), and overall survival (OS). UNLABELLED: Exploratory biomarker analysis was performed on biospecimen collected in the COMBI-BRV trial in which patients with BRAF-mutant, resectable MBM were treated with dabrafenib for 10 to 14 days prior to craniotomy. Accessible ECM were resected or biopsied at the time of craniotomy. Biospecimens underwent molecular and immunologic profiling for comparative analyses. RESULTS: In COMBI-MB baseline treatment with corticosteroids was independently associated with lower ICRR [39% vs. 63%; OR, 0.323; 95 % confidence interval (CI), 0.105-0.996; P = 0.049] and shorter PFS (HR, 1.93; 95% CI, 1.06-3.51; P = 0.031). Additional significant associations identified in the multivariate analysis were improved PFS in patients with a BRAFV600E genotype (HR, 0.565; 95% CI, 0.321-0.996; P = 0.048) and improved OS in patients with Eastern Cooperative Oncology Group 0 (HR, 0.44; 95% CI, 0.25-0.78; P = 0.005). CONCLUSIONS: Corticosteroid treatment was associated with reduced ICRR and PFS in COMBI-MB, similar to results with immunotherapy for MBMs. Baseline corticosteroid treatment is a key factor to consider in MBM patient management and clinical trial design/interpretation.

Ocular trauma or other ocular conditions can be significantly debilitating in space. A literature review of over 100 articles and NASA evidence books, queried for eye related trauma, conditions, and exposures was conducted. Ocular trauma and conditions during NASA space missions during the Space Shuttle Program and ISS through Expedition 13 in 2006 were reviewed. There were 70 corneal abrasions, 4 dry eyes, 4 eye debris, 5 complaints of ocular irritation, 6 chemical burns, and 5 ocular infections noted. Unique exposures on spaceflight, such as foreign bodies, including celestial dust, which may infiltrate the habitat and contact the ocular surface, as well as chemical and thermal injuries due to prolonged CO2 and heat exposure were reported. Diagnostic modalities used to evaluate the above conditions in space flight include vision questionnaires, visual acuity and Amsler grid testing, fundoscopy, orbital ultrasound, and ocular coherence tomography. Several types of ocular injuries and conditions, mostly affecting the anterior segment, are reported. Further research is necessary to understand the greatest ocular risks that astronauts face and how better we can prevent, but also diagnose and treat these conditions in space.

Purpose  Physician diversity is limited in ophthalmology and oculofacial plastic surgery. Determination of barriers within the application process for oculofacial plastic surgery may help target efforts to improve the recruitment of underrepresented groups. This study aimed to illuminate perceived barriers to increasing diversity in oculofacial plastic surgery trainees, according to the American Society of Ophthalmic Plastic and Reconstructive Surgery (ASOPRS) fellows and fellowship program directors (FPDs). Methods  During the month of February 2021, we sent surveys out to 54 current oculofacial plastic surgery fellows and 56 FPDs at 56 oculofacial plastic surgery programs recognized by the ASOPRS nationwide using a 15-question Qualtrics survey. Results  Sixty-three individuals (57%) responded to the survey: 34 fellows (63%) and 29 FPDs (52%). Eighty-eight percent of fellows and 68% of FPDs identified as non-underrepresented in medicine (UiM). Forty-four percent of fellows and 25% of FPDs identified as men. FPDs most commonly noted, "Not enough minorities applying to our program" and "The objective data (Ophthalmic Knowledge Assessment Program score, United States Medical Licensing Examination Step scores, clinical honors, Alpha Omega Alpha status, letter of recommendation) for minority applicants often do not meet the threshold required to offer an interview or to be ranked to match" as barriers. Among fellows, the lowest-rated considerations when applying to oculofacial plastic surgery were "Racially/ethnically diverse faculty" and "Perceptions of minority candidates by fellowship programs," whereas "Likelihood of matching in program of choice" was ranked highest in considerations. Fellows identifying as men indicated greater concern for "Financial factors related to fellowship (e.g., loans, salary, cost of living, or cost of interviewing)" compared to fellows identifying as women who noted greater concern for "Program or preceptor acceptance of starting or having a family during fellowship." Conclusion  Responses from FPDs suggest that efforts focused on recruiting and supporting diverse students to medicine and ophthalmology, mentoring applicants interested in oculofacial plastic surgery, and restructuring the application process to decrease bias, may improve diversity within the subspecialty. The lack of UiM representation in this study, 6% fellows and 7.4% FPDs identified as UiM, shows both the stark underrepresentation and the need for further research into this topic.

PurposePreclinical data suggest the combination of an anti-programmed death receptor 1 antibody plus dabrafenib and trametinib to have superior antitumor activity compared with dabrafenib plus trametinib alone. These observations are supported by translational evidence suggesting that immune checkpoint inhibitors plus targeted therapy may improve treatment outcomes in patients with BRAF V600-mutant metastatic melanoma. COMBI-i is a phase III trial evaluating spartalizumab, an anti-programmed death receptor 1 antibody, in combination with dabrafenib and trametinib (sparta-DabTram), versus placebo plus dabrafenib and trametinib (placebo-DabTram) in patients with BRAF V600-mutant unresectable or metastatic melanoma.MethodsPatients received spartalizumab 400 mg intravenously every 4 weeks plus dabrafenib 150 mg orally twice daily and trametinib 2 mg orally once daily or placebo-DabTram. Participants were age ≥ 18 years with unresectable or metastatic BRAF V600-mutant melanoma. The primary end point was investigator-assessed progression-free survival. Overall survival was a key secondary end point (ClinicalTrials.gov identifier: NCT02967692).ResultsAt data cutoff (July 1, 2020), the median progression-free survival was 16.2 months (95% CI, 12.7 to 23.9 months) in the sparta-DabTram arm versus 12.0 months (95% CI, 10.2 to 15.4 months) in the placebo-DabTram arm (hazard ratio, 0.82 [95% CI, 0.66 to 1.03]; P = .042 [one-sided; nonsignificant]). The objective response rates were 69% (183 of 267 patients) versus 64% (170 of 265 patients), respectively. Grade ≥ 3 treatment-related adverse events occurred in 55% (146 of 267) of patients in the sparta-DabTram arm and 33% (88 of 264) in the placebo-DabTram arm.ConclusionThe study did not meet its primary end point; broad first-line use of sparta-DabTram is not supported by these results. Further biomarker-driven investigation may identify patient subpopulations who could benefit from checkpoint inhibitor plus targeted therapy combinations.

ObjectivesTo evaluate whether use of an antibiotic improves the efficacy of care for a chalazion or hordeolum.MethodsA cross-sectional retrospective review was performed. All patients treated for a newly diagnosed chalazion or hordeolum at the University of California, San Francisco from 2012 to 2018 were identified. Patients were excluded when clinical notes were inaccessible or there was inadequate documentation of treatment modality or outcome. Patient demographics, setting of initial presentation, treatment modalities, antibiotic use, and outcomes were analyzed.ResultsA total of 2,712 patients met inclusion criteria. Management with an antibiotic was observed in 36.5% of patients. An antibiotic was 1.53 times (95% confidence interval [CI], 1.06-2.22, P=0.025) more likely to be prescribed in emergency or acute care setting for a chalazion. Older age was associated with a higher risk of receiving an antibiotic for a hordeolum (adjusted RR 1.07 per decade, 95% CI, 1.05-1.11, P

Background: With support from the Radcliffe Institute for Advanced Study at Harvard University, we convened researchers representing palliative care, psychosocial oncology, spiritual care, oncology, and psychedelic-assisted therapies. We aimed to define priorities and envision an agenda for future research on psychedelic-assisted therapies in patients with serious illness. Over two days in January 2020, participants engaged in an iterative series of reflective exercises that elicited their attitude and perspectives on scientific opportunities for this research. Objectives: The aim of the study is to identify themes that shape priorities and an agenda for research on psychedelic-assisted therapy for those affected by serious illness. Methods: We collected data through preconference interviews, audio recordings, flip charts, and sticky notes. We applied thematic qualitative analysis to elucidate key themes. Results: We identified seven key opportunities to advance the field of psychedelic-assisted therapies in serious illness care. Four opportunities were related to the science and design of psychedelic-assisted therapies: clarifying indications; developing and refining therapeutic protocols; investigating the impact of set and setting on therapeutic outcomes; and understanding the mechanisms of action. The other three pertained to institutional and societal drivers to support optimal and responsible research: education and certification for therapists; regulations and funding; and diversity and inclusion. Additionally, participants suggested epistemological limitations of the medical model to understand the potential value and therapeutic use of psychedelics. Conclusions: Medicine and society are witnessing a resurgence of interest in the effects and applications of psychedelic-assisted therapies in a wide range of settings. This article suggests key opportunities for research in psychedelic-assisted therapies for those affected by serious illness.

ObjectivePatients with melanoma and early stable disease (SD) with pembrolizumab have unclear prognosis. We present post hoc analyses of long-term outcomes for patients with early SD, partial response (PR) or complete response (CR) with pembrolizumab.Patients and methodsPatients who received pembrolizumab in the KEYNOTE-001 and KEYNOTE-006 studies and had SD, PR or CR at weeks 12 or 24 were included.ResultsOf 294 patients in the week 12 analysis, 107 (36.4%) had SD at week 12, of whom 7 (6.5%) had a best overall response of CR, 43 (40.2%) had PR and 57 (53.3%) had SD. Forty-eight-month overall survival (OS) rates were 95.2%, 73.0% and 47.7%, respectively, for patients with CR, PR and SD at week 12. Similar results were observed in the 241 patients in the week 24 analysis. Forty-eight-month OS rates were 72.1% for patients with SD at week 12 followed by subsequent response and 75.0% for patients with PR at week 12 followed by no change in response or progression. Thirty-six-month and 48-month OS rates were 11.6% and not reached, respectively, for patients with SD at week 12 followed by progression before week 24.ConclusionsA substantial proportion of patients (46.7%) with early (week 12) SD with pembrolizumab achieved subsequent PR or CR. Patients with SD at week 12 and subsequent CR/PR had similar survival to those who maintained PR. In contrast, patients with SD at week 12 and subsequent progression had poor survival outcomes. These findings may guide treatment decisions for patients achieving early SD.Trial registrationClinicaltrials.gov: NCT01295827 (KEYNOTE-001); NCT01866319 (KEYNOTE-006).

BackgroundMelanoma lacks validated blood-based biomarkers for monitoring and predicting treatment efficacy. Cell-free circulating tumour DNA (ctDNA) is a promising biomarker; however, various detection methods have been used, and, to date, no large studies have examined the association between serial changes in ctDNA and survival after BRAF, MEK, or BRAF plus MEK inhibitor therapy. We aimed to evaluate whether baseline ctDNA concentrations and kinetics could predict survival outcomes.MethodsIn this clinical validation study, we used analytically validated droplet digital PCR assays to measure BRAFV600-mutant ctDNA in pretreatment and on-treatment plasma samples from patients aged 18 years or older enrolled in two clinical trials. COMBI-d (NCT01584648) was a double-blind, randomised phase 3 study of dabrafenib plus trametinib versus dabrafenib plus placebo in previously untreated patients with BRAFV600 mutation-positive unresectable or metastatic melanoma. Patients had an Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1. COMBI-MB (NCT02039947) was an open-label, non-randomised, phase 2 study evaluating dabrafenib plus trametinib in patients with BRAFV600 mutation-positive metastatic melanoma and brain metastases. Patients in cohort A of COMBI-MB had asymptomatic brain metastases, no previous local brain-directed therapy, and an ECOG performance status of 0 or 1. Biomarker analysis was a prespecified exploratory endpoint in both trials and performed in the intention-to-treat populations in COMBI-d and COMBI-MB. We investigated the association between mutant copy number (baseline or week 4 or zero conversion status) and efficacy endpoints (progression-free survival, overall survival, and best overall response). We used Cox models, Kaplan-Meier plots, and log-rank tests to explore the association of pretreatment ctDNA concentrations with progression-free survival and overall survival. The effect of additional prognostic variables such as lactate dehydrogenase was also investigated in addition to the mutant copy number.FindingsIn COMBI-d, pretreatment plasma samples were available from 345 (82%) of 423 patients and on-treatment (week 4) plasma samples were available from 224 (53%) of 423 patients. In cohort A of COMBI-MB, pretreatment and on-treatment samples were available from 38 (50%) of 76 patients with intracranial and extracranial metastatic melanoma. ctDNA was detected in pretreatment samples from 320 (93%) of 345 patients (COMBI-d) and 34 (89%) of 38 patients (COMBI-MB). When assessed as a continuous variable, elevated baseline BRAFV600 mutation-positive ctDNA concentration was associated with worse overall survival outcome (hazard ratio [HR] 1·13 [95% CI 1·09-1·18], p

Acute invasive fungal rhinosinusitis is a rare, although highly morbid, infection primarily affecting immunosuppressed individuals. The same population is at particularly high risk of complications and mortality in the setting of SARS-CoV-2 infection and coronavirus disease 2019 (COVID-19)-related acute respiratory distress syndrome. The authors present a case of acute invasive fungal rhino-orbital mucormycosis in a patient with COVID-19 and discuss the prevalence, diagnosis, and treatment of fungal coinfections in COVID-19. Prompt recognition, initiation of therapy, and consideration of the challenges of rapidly evolving COVID-19 therapy guidelines are important for improving patient survival.

Monoclonal antibodies (mAbs) are the basis of treatments and diagnostics for pathogens and other biological phenomena. We conducted a structural characterization of mAbs against the N-terminal domain of nucleocapsid protein (NPNTD) from SARS-CoV-2 using small-angle X-ray scattering and transmission electron microscopy. Our solution-based results distinguished the mAbs' flexibility and how this flexibility affects the assembly of multiple mAbs on an antigen. By pairing two mAbs that bind different epitopes on the NPNTD, we show that flexible mAbs form a closed sandwich-like complex. With rigid mAbs, a juxtaposition of the antigen-binding fragments is prevented, enforcing a linear arrangement of the mAb pair, which facilitates further mAb polymerization. In a modified sandwich enzyme-linked immunosorbent assay, we show that rigid mAb-pairings with linear polymerization led to increased NPNTD detection sensitivity. These enhancements can expedite the development of more sensitive and selective antigen-detecting point-of-care lateral flow devices, which are critical for early diagnosis and epidemiological studies of SARS-CoV-2 and other pathogens.

Plastid isoprenoid-derived carotenoids serve essential roles in chloroplast development and photosynthesis. Although nearly all enzymes that participate in the biosynthesis of carotenoids in plants have been identified, the complement of auxiliary proteins that regulate synthesis, transport, sequestration, and degradation of these molecules and their isoprenoid precursors have not been fully described. To identify such proteins that are necessary for the optimal functioning of oxygenic photosynthesis, we screened a large collection of nonphotosynthetic (acetate-requiring) DNA insertional mutants of Chlamydomonas reinhardtii and isolated cpsfl1 The cpsfl1 mutant is extremely light-sensitive and susceptible to photoinhibition and photobleaching. The CPSFL1 gene encodes a CRAL-TRIO hydrophobic ligand-binding (Sec14) domain protein. Proteins containing this domain are limited to eukaryotes, but some may have been retargeted to function in organelles of endosymbiotic origin. The cpsfl1 mutant showed decreased accumulation of plastidial isoprenoid-derived pigments, especially carotenoids, and whole-cell focused ion-beam scanning-electron microscopy revealed a deficiency of carotenoid-rich chloroplast structures (e.g., eyespot and plastoglobules). The low carotenoid content resulted from impaired biosynthesis at a step prior to phytoene, the committed precursor to carotenoids. The CPSFL1 protein bound phytoene and β-carotene when expressed in Escherichia coli and phosphatidic acid in vitro. We suggest that CPSFL1 is involved in the regulation of phytoene synthesis and carotenoid transport and thereby modulates carotenoid accumulation in the chloroplast.

BackgroundPrevious analyses of BREAK-2 and BREAK-3 showed that durable outcomes lasting ≥3 years are achievable with dabrafenib in some patients with BRAF V600-mutant metastatic melanoma (MM); however, additional follow-up is needed to fully characterise the long-term impact of dabrafenib in these patients.MethodsBREAK-2 was a single-arm phase 2 study evaluating dabrafenib in treatment-naive or previously treated BRAF V600E/K-mutant MM. BREAK-3, a randomised (3:1) phase 3 study, assessed dabrafenib versus dacarbazine in previously untreated unresectable or metastatic BRAF V600E-mutant melanoma. Five-year analyses were performed.ResultsAll BREAK-2 patients (N = 92 [V600E, n = 76; V600K, n = 16]) discontinued treatment by the data cutoff. Median follow-up was 13.0 months. In BRAF V600E patients, 5-year progression-free survival (PFS) and overall survival (OS) were 11% and 20%, respectively. Subsequent immunotherapy was received by 22% of patients. In BREAK-3, median follow-up was 17.0 and 12.0 months in the dabrafenib (n = 187) and dacarbazine (n = 63) arms, respectively. Thirty-seven patients (59%) receiving dacarbazine crossed over to dabrafenib following disease progression as per protocol. Five-year PFS was 12% in the dabrafenib arm; all dacarbazine-arm patients progressed or were censored by 5 years. Dabrafenib improved PFS versus dacarbazine, regardless of baseline lactate dehydrogenase levels. Five-year OS rates were 24% and 22% in the dabrafenib and dacarbazine arms, respectively. Subsequent therapy in each arm included anti-CTLA-4 (dabrafenib [24%] and dacarbazine [24%]) and/or anti-PD-1 (8% and 2%) treatment. No new safety signals were observed.Conclusions and relevanceThese data, representing extended follow-up for dabrafenib monotherapy, demonstrate that durable benefit lasting ≥5 years is achievable in a subset of patients.Trial registrationClinicalTrials.gov (BREAK-2, NCT01153763; BREAK-3, NCT01227889).

This report describes the first documented case of Klebsiella pneumoniae endogenous endophthalmitis with concurrent orbital cellulitis in a patient without a liver abscess. A 34-year-old, caucasian male with a history of intravenous drug abuse and type 1 diabetes mellitus was transferred from an outside hospital for progressively worsening pain and swelling in the right eye. Careful history, physical examination, and imaging were consistent with a diagnosis of endogenous endophthalmitis with concurrent orbital cellulitis. Vitreous cultures were positive for Klebsiella pneumoniae. Despite aggressive and early intervention with antibiotics and vitrectomy, the patient's condition continued to worsen and evisceration was required to adequately control the infection. Klebsiella pneumoniae endogenous endophthalmitis with concurrent orbital cellulitis is a rare and often blinding infection, despite appropriately aggressive intervention.

The NAIP-NLRC4 family of inflammasomes are components of the innate immune system that sound a molecular alarm in the presence of intracellular pathogens. In this chapter, we provide an in-depth guide to using cryo-electron microscopy (cryo-EM) to investigate these inflammasomes, focusing especially on the techniques we used in our recent structural analysis of the NAIP5-NLRC4 inflammasome. We explain how to circumvent specific obstacles we encountered at each step, from sample preparation through data processing. The methods described here will be useful for further studies of the NAIP5-NLRC4 inflammasome and related supracomplexes involved in innate immune surveillance; they may also be useful for unrelated complexes that present similar issues, such as preferential orientations and compositional heterogeneity.

The photosynthesis machinery in chloroplast thylakoid membranes is comprised of multiple protein complexes and supercomplexes1,2. Here, we show a novel supramolecular organization of photosystem I (PSI) in the moss Physcomitrella patens by single-particle cryo-electron microscopy. The moss-specific light-harvesting complex (LHC) protein Lhcb9 is involved in this PSI supercomplex, which has been shown to have a molecular density similar to that of the green alga Chlamydomonas reinhardtii3. Our results show that the structural organization is unexpectedly different-two rows of the LHCI belt exist as in C. reinhardtii4, but the outer one is shifted toward the PsaK side. Furthermore, one trimeric LHC protein and one monomeric LHC protein position alongside PsaL/K, filling the gap between these subunits and the outer LHCI belt. We provide evidence showing that Lhcb9 is a key factor, acting as a linkage between the PSI core and the outer LHCI belt to form the unique supramolecular organization of the PSI supercomplex in P. patens.

BackgroundObesity has been linked to increased mortality in several cancer types; however, the relation between obesity and survival outcomes in metastatic melanoma is unknown. The aim of this study was to examine the association between body-mass index (BMI) and progression-free survival or overall survival in patients with metastatic melanoma who received targeted therapy, immunotherapy, or chemotherapy.MethodsThis retrospective study analysed independent cohorts of patients with metastatic melanoma assigned to treatment with targeted therapy, immunotherapy, or chemotherapy in randomised clinical trials and one retrospective study of patients treated with immunotherapy. Patients were classified according to BMI, following the WHO definitions, as underweight, normal, overweight, or obese. Patients without BMI and underweight patients were excluded. The primary outcomes were the associations between BMI and progression-free survival or overall survival, stratified by treatment type and sex. We did multivariable analyses in the independent cohorts, and combined adjusted hazard ratios in a mixed-effects meta-analysis to provide a precise estimate of the association between BMI and survival outcomes; heterogeneity was assessed with meta-regression analyses. Analyses were done on the predefined intention-to-treat population in the randomised controlled trials and on all patients included in the retrospective study.FindingsThe six cohorts consisted of a total of 2046 patients with metastatic melanoma treated with targeted therapy, immunotherapy, or chemotherapy between Aug 8, 2006, and Jan 15, 2016. 1918 patients were included in the analysis. Two cohorts containing patients from randomised controlled trials treated with targeted therapy (dabrafenib plus trametinib [n=599] and vemurafenib plus cobimetinib [n=240]), two cohorts containing patients treated with immunotherapy (one randomised controlled trial of ipilimumab plus dacarbazine [n=207] and a retrospective cohort treated with pembrolizumab, nivolumab, or atezolizumab [n=331]), and two cohorts containing patients treated with chemotherapy (two randomised controlled trials of dacarbazine [n=320 and n=221]) were classified according to BMI as normal (694 [36%] patients), overweight (711 [37%]), or obese (513 [27%]). In the pooled analysis, obesity, compared with normal BMI, was associated with improved survival in patients with metastatic melanoma (average adjusted hazard ratio [HR] 0·77 [95% CI 0·66-0·90] for progression-free survival and 0·74 [0·58-0·95] for overall survival). The survival benefit associated with obesity was restricted to patients treated with targeted therapy (HR 0·72 [0·57-0·91] for progression-free survival and 0·60 [0·45-0·79] for overall survival) and immunotherapy (HR 0·75 [0·56-1·00] and 0·64 [0·47-0·86]). No associations were observed with chemotherapy (HR 0·87 [0·65-1·17, pinteraction=0·61] for progression-free survival and 1·03 [0·80-1·34, pinteraction=0·01] for overall survival). The association of BMI with overall survival for patients treated with targeted and immune therapies differed by sex, with inverse associations in men (HR 0·53 [0·40-0·70]), but no associations observed in women (HR 0·85 [0·61-1·18, pinteraction=0·03]).InterpretationOur results suggest that in patients with metastatic melanoma, obesity is associated with improved progression-free survival and overall survival compared with those outcomes in patients with normal BMI, and that this association is mainly seen in male patients treated with targeted or immune therapy. These results have implications for the design of future clinical trials for patients with metastatic melanoma and the magnitude of the benefit found supports further investigation of the underlying mechanism of these associations.FundingASCO/CCF Young Investigator Award, ASCO/CCF Career Development Award, MD Anderson Cancer Center (MDACC) Melanoma Moonshot Program, MDACC Melanoma SPORE, and the Dr Miriam and Sheldon G Adelson Medical Research Foundation.

BackgroundSerous chorioretinopathy has been associated with MEK inhibitors, including cobimetinib. We describe the clinical features of serous retinopathy observed with cobimetinib in patients with BRAF V600-mutated melanoma treated in the Phase III coBRIM study.MethodsIn the coBRIM study, 493 patients were treated in two randomly assigned treatment groups: cobimetinib and vemurafenib (n = 247) or vemurafenib (n = 246). All patients underwent prospective ophthalmic examinations at screening, at regular intervals during the study, and whenever ocular symptoms developed. Patients with serous retinopathy were identified in the study database using a group of relevant and synonymous adverse event terms.ResultsEighty-six serous retinopathy events were reported in 70 patients (79 events in 63 cobimetinib and vemurafenib-treated patients vs seven events in seven vemurafenib-treated patients). Most patients with serous retinopathy identified by ophthalmic examination had no symptoms or had mild symptoms, among them reduced visual acuity, blurred vision, dyschromatopsia, and photophobia. Serous retinopathy usually occurred early during cobimetinib and vemurafenib treatment; median time to onset was 1.0 month. Most events were managed by observation and continuation of cobimetinib without dose modification and resolved or were resolving by the data cutoff date (19 Sept 2014).ConclusionsCobimetinib treatment was associated with serous retinopathy in patients with BRAF V600-mutated melanoma. Retinopathy was generally asymptomatic or mild. Periodic ophthalmologic evaluations at regular intervals and at the manifestation of any visual disturbance are recommended to facilitate early detection and resolution of serous retinopathy while patients are taking cobimetinib. Trial Registration Clinicaltrials.gov (NCT01689519). First received: September 18, 2012.

Robust innate immune detection of rapidly evolving pathogens is critical for host defense. Nucleotide-binding domain leucine-rich repeat (NLR) proteins function as cytosolic innate immune sensors in plants and animals. However, the structural basis for ligand-induced NLR activation has so far remained unknown. NAIP5 (NLR family, apoptosis inhibitory protein 5) binds the bacterial protein flagellin and assembles with NLRC4 to form a multiprotein complex called an inflammasome. Here we report the cryo-electron microscopy structure of the assembled ~1.4-megadalton flagellin-NAIP5-NLRC4 inflammasome, revealing how a ligand activates an NLR. Six distinct NAIP5 domains contact multiple conserved regions of flagellin, prying NAIP5 into an open and active conformation. We show that innate immune recognition of multiple ligand surfaces is a generalizable strategy that limits pathogen evolution and immune escape.

BackgroundPrevious analysis of COMBI-d (NCT01584648) demonstrated improved progression-free survival (PFS) and overall survival (OS) with combination dabrafenib and trametinib versus dabrafenib monotherapy in BRAF V600E/K-mutant metastatic melanoma. This study was continued to assess 3-year landmark efficacy and safety after ≥36-month follow-up for all living patients.Patients and methodsThis double-blind, phase 3 study enrolled previously untreated patients with BRAF V600E/K-mutant unresectable stage IIIC or stage IV melanoma. Patients were randomized to receive dabrafenib (150 mg twice daily) plus trametinib (2 mg once daily) or dabrafenib plus placebo. The primary endpoint was PFS; secondary endpoints were OS, overall response, duration of response, safety, and pharmacokinetics.ResultsBetween 4 May and 30 November 2012, a total of 423 of 947 screened patients were randomly assigned to receive dabrafenib plus trametinib (n = 211) or dabrafenib monotherapy (n = 212). At data cut-off (15 February 2016), outcomes remained superior with the combination: 3-year PFS was 22% with dabrafenib plus trametinib versus 12% with monotherapy, and 3-year OS was 44% versus 32%, respectively. Twenty-five patients receiving monotherapy crossed over to combination therapy, with continued follow-up under the monotherapy arm (per intent-to-treat principle). Of combination-arm patients alive at 3 years, 58% remained on dabrafenib plus trametinib. Three-year OS with the combination reached 62% in the most favourable subgroup (normal lactate dehydrogenase and

Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) can automatically generate 3D images with superior z-axis resolution, yielding data that needs minimal image registration and related post-processing. Obstacles blocking wider adoption of FIB-SEM include slow imaging speed and lack of long-term system stability, which caps the maximum possible acquisition volume. Here, we present techniques that accelerate image acquisition while greatly improving FIB-SEM reliability, allowing the system to operate for months and generating continuously imaged volumes > 106 µm3. These volumes are large enough for connectomics, where the excellent z resolution can help in tracing of small neuronal processes and accelerate the tedious and time-consuming human proofreading effort. Even higher resolution can be achieved on smaller volumes. We present example data sets from mammalian neural tissue, Drosophila brain, and Chlamydomonas reinhardtii to illustrate the power of this novel high-resolution technique to address questions in both connectomics and cell biology.

Orbital fractures are very common after facial trauma. The assessment of a patient with a suspected orbital wall injury includes a detailed oculofacial examination as well as radiologic imaging. Surgical repair with or without an implant may be indicated for diplopia, enophthalmos, or both. Cicatricial eyelid malposition is an iatrogenic complication commonly due to poor orbitotomy technique. Optimal repair involves direct exposure of the perimeter of the fractures' site through surgical planes that minimally scar the eyelids. A wide variety of implant options exist; however, thin, pliable, nonadherent materials such as nylon foil may offer several advantages. The authors describe the evaluation and management of orbital wall fractures.

The survival of advanced metastatic melanoma has been greatly improved within the past few years. New therapeutic strategies like kinase inhibitors for BRAF-mutant melanoma and immune checkpoint blockers proved to prolong survival times within clinical trials, and many of them have already entered routine clinical use. However, these different treatment modalities have not yet been tested against each other, which complicate therapy decisions. We performed an explorative analysis of survival data from recent clinical trials. Thirty-five Kaplan-Meier survival curves from 17 trials were digitised, re-grouped by matching inclusion criteria and treatment line, and averaged by therapy strategy. Notably, the survival curves grouped by therapy strategy revealed a very high concordance, even if different agents were used. The greatest survival improvement was observed with the combination of BRAF plus MEK inhibitors as well as with Programmed-death-1 (PD1) blockers with or without cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) blockers, respectively, with these two treatment strategies showing similar survival outcomes. For first-line therapy, averaged survival proportions of patients alive at 12 months were 74.5% with BRAF plus MEK inhibitor treatment versus 71.9% with PD-1 blockade. This explorative comparison shows the kinase inhibitors as similarly effective as immune checkpoint blockers with regard to survival. However, to confirm these first trends for implementation into an individualised treatment of melanoma patients, data from prospective clinical trials comparing the different treatment strategies head-to-head have to be awaited.

The Xeroderma pigmentosum complementation group C (XPC) complex is a versatile factor involved in both nucleotide excision repair and transcriptional coactivation as a critical component of the NANOG, OCT4, and SOX2 pluripotency gene regulatory network. Here we present the structure of the human holo-XPC complex determined by single-particle electron microscopy to reveal a flexible, ear-shaped structure that undergoes localized loss of order upon DNA binding. We also determined the structure of the complete yeast homolog Rad4 holo-complex to find a similar overall architecture to the human complex, consistent with their shared DNA repair functions. Localized differences between these structures reflect an intriguing phylogenetic divergence in transcriptional capabilities that we present here. Having positioned the constituent subunits by tagging and deletion, we propose a model of key interaction interfaces that reveals the structural basis for this difference in functional conservation. Together, our findings establish a framework for understanding the structure-function relationships of the XPC complex in the interplay between transcription and DNA repair.

The ∼230-residue C-terminal tail of the epidermal growth factor receptor (EGFR) is phosphorylated upon activation. We examined whether this phosphorylation is affected by deletions within the tail and whether the two tails in the asymmetric active EGFR dimer are phosphorylated differently. We monitored autophosphorylation in cells using flow cytometry and found that the first ∼80 residues of the tail are inhibitory, as demonstrated previously. The entire ∼80-residue span is important for autoinhibition and needs to be released from both kinases that form the dimer. These results are interpreted in terms of crystal structures of the inactive kinase domain, including two new ones presented here. Deletions in the remaining portion of the tail do not affect autophosphorylation, except for a six-residue segment spanning Tyr 1086 that is critical for activation loop phosphorylation. Phosphorylation of the two tails in the dimer is asymmetric, with the activator tail being phosphorylated somewhat more strongly. Unexpectedly, we found that reconstitution of the transmembrane and cytoplasmic domains of EGFR in vesicles leads to a peculiar phenomenon in which kinase domains appear to be trapped between stacks of lipid bilayers. This artifactual trapping of kinases between membranes enhances an intrinsic functional asymmetry in the two tails in a dimer.

The class III phosphatidylinositol 3-kinase complex I (PI3KC3-C1) that functions in early autophagy consists of the lipid kinase VPS34, the scaffolding protein VPS15, the tumor suppressor BECN1, and the autophagy-specific subunit ATG14. The structure of the ATG14-containing PI3KC3-C1 was determined by single-particle EM, revealing a V-shaped architecture. All of the ordered domains of VPS34, VPS15, and BECN1 were mapped by MBP tagging. The dynamics of the complex were defined using hydrogen-deuterium exchange, revealing a novel 20-residue ordered region C-terminal to the VPS34 C2 domain. VPS15 organizes the complex and serves as a bridge between VPS34 and the ATG14:BECN1 subcomplex. Dynamic transitions occur in which the lipid kinase domain is ejected from the complex and VPS15 pivots at the base of the V. The N-terminus of BECN1, the target for signaling inputs, resides near the pivot point. These observations provide a framework for understanding the allosteric regulation of lipid kinase activity.

Remodeling of thylakoid membranes in response to illumination is an important process for the regulation of photosynthesis. We investigated the thylakoid network from Arabidopsis thaliana using atomic force microscopy to capture dynamic changes in height, elasticity, and viscosity of isolated thylakoid membranes caused by changes in illumination. We also correlated the mechanical response of the thylakoid network with membrane ultrastructure using electron microscopy. We find that the elasticity of the thylakoid membranes increases immediately upon PSII-specific illumination, followed by a delayed height change. Direct visualization by electron microscopy confirms that there is a significant change in the packing repeat distance of the membrane stacks in response to illumination. Although experiments with Gramicidin show that the change in elasticity depends primarily on the transmembrane pH gradient, the height change requires both the pH gradient and STN7-kinase-dependent phosphorylation of LHCII. Our studies indicate that lumen expansion in response to illumination is not simply a result of the influx of water, and we propose a dynamic model in which protein interactions within the lumen drive these changes.

Adult-onset foveomacular vitelliform dystrophy (AOFVD) is a clinically heterogeneous maculopathy that may mimic other conditions and be difficult to diagnose. It is characterized by late onset, slow progression and high variability in morphologic and functional alterations. Diagnostic evaluation should include careful ophthalmoscopy and imaging studies. The typical ophthalmoscopic findings are bilateral, asymmetric, foveal or perifoveal, yellow, solitary, round to oval elevated subretinal lesions, often with central pigmentation. The lesions characteristically demonstrate increased autofluorescence and hypofluorescent lesions surrounded by irregular annular hyperfluorescence on fluorescein angiography. Optical coherence tomography studies demonstrate homogenous or heterogeneous hyperreflective material between the retinal pigment epithelium and the neurosensory retina. The visual prognosis is generally favorable, but visual loss can occur from chorioretinal atrophy and choroidal neovascularization.

The maintenance of mydriasis and the control of postoperative pain and inflammation are critical to the safety and success of cataract and intraocular lens replacement surgery. Appropriate mydriasis is usually achieved by topical and/or intracameral administration of anticholinergic agents, sympathomimetic agents, or both, with the most commonly used being cyclopentolate, tropicamide, and phenylephrine. Ocular inflammation is common after cataract surgery. Topical steroids and nonsteroidal anti-inflammatory drugs are widely used because they have been proved effective to control postsurgical inflammation and decrease pain. Topical nonsteroidal anti-inflammatory drugs have also been shown to help maintain dilation. However, use of multiple preoperative drops for pupil dilation, inflammation, and pain control have been shown to be time consuming, resulting in delays to the operating room, and they cause dissatisfaction among perioperative personnel; their use can also be associated with systemic side effects. Therefore, ophthalmologists have been in search of new options to streamline this process. This article will review the current medications commonly used for intraoperative mydriasis, as well as pain and inflammation control. In addition, a new combination of ketorolac, an anti-inflammatory agent, and phenylephrine, a mydriatic agent has recently been designed to maintain intraoperative mydriasis and to reduce postoperative pain and irritation from intraocular lens replacement surgery. Two Phase III clinical trials evaluating this combination have demonstrated statistically significant differences when compared to placebo in maintaining intraoperative mydriasis (P

We outline a strategy to enable non-directed Pd(II)-catalyzed C-H functionalization in the presence of Lewis basic heterocycles. In a high-throughput screen of two Pd-catalyzed C-H acetoxylation reactions, addition of a variety of N-containing heterocycles is found to cause low product conversion. A pyridine-containing test substrate is selected as representative of heterocyclic scaffolds that are hypothesized to cause catalyst arrest. We pursue two approaches in parallel that allow product conversion in this representative system: Lewis acids are found to be effective in situ blocking groups for the Lewis basic site, and a pre-formed pyridine N-oxide is shown to enable high yield of allylic C-H acetoxylation. Computational studies with density functional theory (M06) of binding affinities of selected heterocycles to Pd(OAc)2 provide an inverse correlation of the computed heterocycle-Pd(OAc)2 binding affinities with the experimental conversions to products. Additionally, 1H NMR binding studies provide experimental support for theoretical calculations.

Photoautotrophic organisms efficiently regulate absorption of light energy to sustain photochemistry while promoting photoprotection. Photoprotection is achieved in part by triggering a series of dissipative processes termed non-photochemical quenching (NPQ), which depend on the re-organization of photosystem (PS) II supercomplexes in thylakoid membranes. Using atomic force microscopy, we characterized the structural attributes of grana thylakoids from Arabidopsis thaliana to correlate differences in PSII organization with the role of SOQ1, a recently discovered thylakoid protein that prevents formation of a slowly reversible NPQ state. We developed a statistical image analysis suite to discriminate disordered from crystalline particles and classify crystalline arrays according to their unit cell properties. Through detailed analysis of the local organization of PSII supercomplexes in ordered and disordered phases, we found evidence that interactions among light-harvesting antenna complexes are weakened in the absence of SOQ1, inducing protein rearrangements that favor larger separations between PSII complexes in the majority (disordered) phase and reshaping the PSII crystallization landscape. The features we observe are distinct from known protein rearrangements associated with NPQ, providing further support for a role of SOQ1 in a novel NPQ pathway. The particle clustering and unit cell methodology developed here is generalizable to multiple types of microscopy and will enable unbiased analysis and comparison of large data sets.

Gene transcription is regulated in response to environmental changes and developmental cues. In mammalian cells subjected to stress conditions such as heat shock, transcription of most protein-coding genes decreases, while the transcription of heat shock protein genes increases. Repression involves direct binding to RNA polymerase II (Pol II) of certain noncoding RNAs (ncRNAs) that are upregulated upon heat shock. Another class of ncRNAs is also upregulated and binds to Pol II but does not inhibit transcription. Incorporation of repressive ncRNAs into pre-initiation complexes prevents transcription initiation, while non-repressive ncRNAs are displaced from Pol II by TFIIF. Here, we present cryo-electron microscopy reconstructions of human Pol II in complex with six different ncRNAs from mouse and human. Our structures show that both repressive and non-repressive ncRNAs bind to a conserved binding site within the cleft of Pol II. The site, which is also shared with a previously characterized yeast aptamer, is close to the active center and, thus, in an ideal position to regulate transcription. Importantly, additional RNA elements extend flexibly beyond the docking site. We propose that the differences concerning the repressive activity of the ncRNAs analyzed must be due to the distinct character of these more unstructured, flexible segments of the RNA that emanate from the cleft.

We demonstrate two ways in which the Fourier transforms of images that consist solely of randomly distributed electrons (shot noise) can be used to compare the relative performance of different electronic cameras. The principle is to determine how closely the Fourier transform of a given image does, or does not, approach that of an image produced by an ideal camera, i.e. one for which single-electron events are modeled as Kronecker delta functions located at the same pixels where the electrons were incident on the camera. Experimentally, the average width of the single-electron response is characterized by fitting a single Lorentzian function to the azimuthally averaged amplitude of the Fourier transform. The reciprocal of the spatial frequency at which the Lorentzian function falls to a value of 0.5 provides an estimate of the number of pixels at which the corresponding line-spread function falls to a value of 1/e. In addition, the excess noise due to stochastic variations in the magnitude of the response of the camera (for single-electron events) is characterized by the amount to which the appropriately normalized power spectrum does, or does not, exceed the total number of electrons in the image. These simple measurements provide an easy way to evaluate the relative performance of different cameras. To illustrate this point we present data for three different types of scintillator-coupled camera plus a silicon-pixel (direct detection) camera.

Age-related macular degeneration (AMD) is a leading cause of visual impairment in aging populations in industrialized countries. Here we investigated whether the genotype of vascular endothelial growth factor A (VEGFA) gene is associated with response to anti-VEGF therapy. 223 eyes with neovascular AMD were treated with intravitreal anti-VEGF therapy. Responders were defined as patients who had an improvement in best corrected visual acuity (BCVA) of at least 5 letters or one line on the EDTRS visual acuity chart along with resolution of intraretinal or subretinal fluid over 12 months. Patients who did not meet the definition of responders were classified as poor-responders. The vision of responders (n = 148) improved while the vision of poor-responders (n = 75) worsened (P

PurposeTo determine whether there is an association between hepatic lipase (LIPC) and age-related macular degeneration (AMD) in two independent Caucasian cohorts.MethodsA discovery cohort of 1626 patients with advanced AMD and 859 normal controls and a replication cohort of 2159 cases and 1150 controls were genotyped for two single-nucleotide polymorphisms (SNPs) in the promoter region of LIPC. The associations between the SNPs and AMD were examined by χ(2) tests.ResultsIn the discovery cohort, rs493258 and rs10468017 were both associated with advanced AMD (P=9.63E-3 and P=0.048, respectively). The association was corroborated in the replication cohort (P=4.48E-03 for rs493258 and P=0.015 for rs10468017). Combined analysis resulted in even more significant associations (P=1.21E-04 for rs493258 and P=1.67E-03 for rs10468017).ConclusionThe LIPC promoter variants rs493258 and rs10468017 were associated with advanced AMD in two independent Caucasian populations, confirming that LIPC polymorphisms may be a genetic risk factor for AMD in the Caucasian population.

The molecular underpinnings of synaptic vesicle fusion for fast neurotransmitter release are still unclear. Here, we used a single vesicle-vesicle system with reconstituted SNARE and synaptotagmin-1 proteoliposomes to decipher the temporal sequence of membrane states upon Ca(2+)-injection at 250-500 μM on a 100-ms timescale. Furthermore, detailed membrane morphologies were imaged with cryo-electron microscopy before and after Ca(2+)-injection. We discovered a heterogeneous network of immediate and delayed fusion pathways. Remarkably, all instances of Ca(2+)-triggered immediate fusion started from a membrane-membrane point-contact and proceeded to complete fusion without discernible hemifusion intermediates. In contrast, pathways that involved a stable hemifusion diaphragm only resulted in fusion after many seconds, if at all. When complexin was included, the Ca(2+)-triggered fusion network shifted towards the immediate pathway, effectively synchronizing fusion, especially at lower Ca(2+)-concentration. Synaptic proteins may have evolved to select this immediate pathway out of a heterogeneous network of possible membrane fusion pathways.DOI:http://dx.doi.org/10.7554/eLife.00109.001.

The rs1061170T/C variant encoding the Y402H change in complement factor H (CFH) has been identified by genome-wide association studies as being significantly associated with age-related macular degeneration (AMD). However, the precise mechanism by which this CFH variant impacts the risk of AMD remains largely unknown. Oxidative stress plays an important role in many aging diseases, including cardiovascular disease and AMD. A large amount of oxidized phospholipids (oxPLs) are generated in the eye because of sunlight exposure and high oxygen content. OxPLs bind to the retinal pigment epithelium and macrophages and strongly activate downstream inflammatory cascades. We hypothesize that CFH may impact the risk of AMD by modulating oxidative stress. Here we demonstrate that CFH binds to oxPLs. The CFH 402Y variant of the protective rs1061170 genotype binds oxPLs with a higher affinity and exhibits a stronger inhibitory effect on the binding of oxPLs to retinal pigment epithelium and macrophages. In addition, plasma from non-AMD subjects with the protective genotype has a lower level of systemic oxidative stress measured by oxPLs per apolipoprotein B (oxPLs/apoB). We also show that oxPL stimulation increases expression of genes involved in macrophage infiltration, inflammation, and neovascularization in the eye. OxPLs colocalize with CFH in drusen in the human AMD eye. Subretinal injection of oxPLs induces choroidal neovascularization in mice. In addition, we show that the CFH risk allele confers higher complement activation and cell lysis activity. Together, these findings suggest that CFH influences AMD risk by modulating oxidative stress, inflammation, and abnormal angiogenesis.

Background/aimsTo determine the genetic basis of myotonia congenita (MC) and strabismus in a large Caucasian family.MethodsSeven patients making up four generations of a family with MC and strabismus were recruited. All patients had at least one standard ophthalmic examination, including best-corrected visual acuity, refraction, and ocular motility measurements. CLCN1 and SCN4A genes were sequenced and analysed for mutations.ResultsFive out of the seven family members were diagnosed with MC by clinical history and electromyography. Ophthalmic history and exam revealed eyelid myotonia and strabismus. All patients with MC were diagnosed with strabismus between the ages of 3 and 6 and required surgical restoration of ocular alignment. Sequencing results revealed a c. 1333G>A; p. Val445Met mutation in the SCN4A gene.ConclusionThere are few reports describing eyelid myotonia and strabismus in patients diagnosed with MC. We found significant ocular involvement in a family with a mutation in SCN4A. Future studies may confirm that MC with significant ocular involvement can be used to direct genetic analysis.

AimTo investigate clinical presentation and genotypes in patients with simultaneous geographic atrophy (GA) and choroidal neovascularization (CNV) and to compare with patients with GA or CNV only.Patients and methodsTwenty patients with combined CNV-GA and 154 CNV only and 154 GA only were chosen based on clinical exam and imaging. Six single-nucleotide polymorphisms (SNPs)-rs2274700 and rs1061170 (complement factor H), rs10490924 and rs11200638 (HTRA1/LOC387715), rs2230199 (C3), rs9332739 (C2)-were genotyped using the SNaPshot method. Chi-squared tests were used for genetic analysis.ResultsIn patients with CNV-GA, GA progressed slowly and often preceded CNV. CNV presented as subretinal haemorrhage or fluid, with a sudden drop in visual acuity (VA). Comparing combined CNV-GA to GA and CNV only, patients with both had a higher frequency of at-risk alleles at both SNPs within the HTRA1 gene-rs10490924 (52.5%), rs11200638 (52.6%). Statistical significance was not achieved. CNV-GA patients had no protective alleles at SNP rs9332739 (C2), compared with GA (27%) and CNV only (10%).ConclusionThere is a paucity of reports describing simultaneous CNV-GA. Clinical and genetic results may support the fact that GA and CNV fit on an age-related macular degeneration (AMD)-disease continuum and may clarify the disease processes in AMD.

The macromolecular assembly required to initiate transcription of protein-coding genes, known as the Pre-Initiation Complex (PIC), consists of multiple protein complexes and is approximately 3.5 MDa in size. At the heart of this assembly is the Mediator complex, which helps regulate PIC activity and interacts with the RNA polymerase II (pol II) enzyme. The structure of the human Mediator-pol II interface is not well-characterized, whereas attempts to structurally define the Mediator-pol II interaction in yeast have relied on incomplete assemblies of Mediator and/or pol II and have yielded inconsistent interpretations. We have assembled the complete, 1.9 MDa human Mediator-pol II-TFIIF complex from purified components and have characterized its structural organization using cryo-electron microscopy and single-particle reconstruction techniques. The orientation of pol II within this assembly was determined by crystal structure docking and further validated with projection matching experiments, allowing the structural organization of the entire human PIC to be envisioned. Significantly, pol II orientation within the Mediator-pol II-TFIIF assembly can be reconciled with past studies that determined the location of other PIC components relative to pol II itself. Pol II surfaces required for interacting with TFIIB, TFIIE, and promoter DNA (i.e., the pol II cleft) are exposed within the Mediator-pol II-TFIIF structure; RNA exit is unhindered along the RPB4/7 subunits; upstream and downstream DNA is accessible for binding additional factors; and no major structural re-organization is necessary to accommodate the large, multi-subunit TFIIH or TFIID complexes. The data also reveal how pol II binding excludes Mediator-CDK8 subcomplex interactions and provide a structural basis for Mediator-dependent control of PIC assembly and function. Finally, parallel structural analysis of Mediator-pol II complexes lacking TFIIF reveal that TFIIF plays a key role in stabilizing pol II orientation within the assembly.

BackgroundThe mitotic spindle is a complex mechanical apparatus required for accurate segregation of sister chromosomes during mitosis. We designed a genetic screen using automated microscopy to discover factors essential for mitotic progression. Using a RNA interference library of 49,164 double-stranded RNAs targeting 23,835 human genes, we performed a loss of function screen to look for small interfering RNAs that arrest cells in metaphase.ResultsHere we report the identification of genes that, when suppressed, result in structural defects in the mitotic spindle leading to bent, twisted, monopolar, or multipolar spindles, and cause cell cycle arrest. We further describe a novel analysis methodology for large-scale RNA interference datasets that relies on supervised clustering of these genes based on Gene Ontology, protein families, tissue expression, and protein-protein interactions.ConclusionThis approach was utilized to classify functionally the identified genes in discrete mitotic processes. We confirmed the identity for a subset of these genes and examined more closely their mechanical role in spindle architecture.

In the brief period before it was legally terminated, considerable human subjects research was conducted on psychedelics. More than 1,000 clinical reports documented a wide array of psychological effects and therapeutic possibilities. The findings held major implications for disciplines as diverse as psychology, psychiatry, neuro-science, anthropology, sociology, and religious studies. Psychological and psychiatric findings included insights into states of consciousness, the unconscious, and the relationship of different schools of psychology, motivation, self-actualization, spirituality, psychotherapy, and adjunctive therapies. With further human subjects studies virtually impossible, the original investigators clearly constitute an irreplaceable resource of information. An interdisciplinary group of surviving investigators was therefore convened and interviewed to obtain an oral history of the findings and implications of their research. This article provides a summary of their conclusions, of the psychological and social implications of their research, and of the impact their research had on diverse academic disciplines. © 2006 Sage Publications.

3,4-Methylenedioxymethamphetamine (MDMA) has been at the center of a debate over its potential benefits as an adjunct to psychotherapy versus its capability for neurotoxic effects and is currently classified as a Schedule 1 drug by the Drug Enforcement Administration (DEA). However, as yet, there is very little methodological data on the subjective experience of the MDMA-induced state or its psychological and behavioral sequelae. The present study was, therefore, designed to obtain this kind of information. Twenty psychiatrists who had taken MDMA previously were evaluated using a semistructured interview. Subjective experience of the actual MDMA-induced state, as well as both short-term (less than 1 week) and relatively longer term (greater than 1 week) sequelae, were examined retrospectively. Side effects, insight gained, pleasure, and intensity of the MDMA experience were evaluated as were the influence of set and setting at the time the MDMA was taken and the dosage utilized. Finally, the authors discuss methodological problems and limitations of a study of this type.