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Purpose We herein compare topical interferon alpha 2b (IFN-α2b) to topical mitomycin C (MMC) in the adjuvant management after excision of primary acquired melanosis with atypia (PAM) and melanoma of the conjunctiva/cornea (CM). Methods We included 25 tumors from 25 patients (six with PAM and 19 with CM). After surgical excision, four patients started with adjuvant IFN-α2b (two in combination with radiotherapy), 19 with MMC, and two with radiotherapy alone. Five patients were switched from initial MMC/radiotherapy to IFN-α2b during follow-up. Efficacy was assessed via time to tumor recurrence and initial therapy response. Results With initial IFN-α2b, three patients (3/4, two with additional radiotherapy) showed complete remission (follow-up: 1478–1750 days) and one recurrence (1/4) was noted after 492 days. With initial MMC, no recurrence was recorded in 15 of the 19 patients (follow-up: 99–4732 days). Five patients were switched from MMC or radiotherapy to IFN-α2b: two patients showed complete remission (2/5), while another two (2/5) experienced recurrences and remained without recurrence after repeated courses of IFN-α2b (follow-up: 1798 and 1973 days). Only one patient showed incomplete response. Adverse effects were recorded in five patients, all received MMC. Conclusion Topical IFN-α2b (arguably together with radiotherapy) may be a viable alternative to MMC in PAM and CM. We observed fewer side effects at similar response rates. However, when response to MMC was poor, IFN-α2b may also be of limited utility.

Supplementary materials for the paper "Ribosomal protein RPL39L is an efficiency factor in the cotranslational folding of proteins with alpha helical domains". Analysis of public RNA-seq data The files named "RPL39L_public_rna_seq*" are related to the analysis of public RNA-seq data reflected in Figure 1 and S1 of the manuscript. To run the jupyter notebook with the analysis, create a local conda environment using a provided .yml file: conda env create -f RPL39L_public_rna_seq_env.yml Untar the archive with all the necessary data: tar -xvzf RPL39L_public_rna_seq.tar.gz In your juputer lab or hub, or standard notebooks instance, open the file "RPL39L_public_rna_seq.ipynb", select the environment "rpl39l", and run the analysis. The instructions forinstallation of the jupyter lab can be found here:https://jupyterlab.readthedocs.io/en/stable/getting_started/installation.html Analysis of Ribo-seq data from WT and RPL39L-KO mESC Extract the archive "RPL39L_Ribo_seq.tar.gz" with the following command: tar -xvzf RPL39L_Ribo_seq.tar.gz The analysis is executed in two steps, represented by independent Snakemake-based workflows. To run the workflows, pleaseinstall the Snakemake following the instructions from here:https://snakemake.readthedocs.io/en/stable/getting_started/installation.html Step 1: preparation of the enriched mouse genomeannotation The directory"prepare_annotation" contains the Snakemake workflow for preparation of custom mouse annotation. Navigate there and run the bash script "run_snakefile.sh" Step 2: analysis of the ribo-seq data The directory"process_data" contains the Snakemake workflow for running the analysis. Please go the subdirectory "./samples/Rpl39l_Samples/",put the riboseq .fastq files from the associated SRA study there, and run the bash script "rename.sh". After that, navigate back to the directory"process_data"and run the bash script "run_snakefile.sh".

Purpose Single center study to evaluate the incidence and long-term outcome of laser pointer maculopathy (LPM). Methods Medical records of 909,150 patients visiting our institution between 2007 and 2020 were screened in our electronic patient record system using the keywords "laserpointer," "laser pointer," and "solar." Results Eight patients (6/2 male/female, 11 eyes) with a history of LPM were identified by fundoscopy and optical coherence tomography (OCT), all of whom were children (6/2 male/female). Mean age at injury was 12.1 years (range 6–16). Five children (62.5%) were injured between 2019 and 2020, three (37.5%) between 2007 and 2018. Median best-corrected visual acuity (BCVA) of affected eyes at first presentation was 20/25 (range 20/50–20/16). Follow-up examination was performed in seven children (10 eyes) with a median follow-up period of 18 months (range 0.5–96). BCVA improved in 4 children (5 eyes; BCVA at follow-up 20/22.5, range 20/40–20/16). Three of these four children were treated with oral steroids. OCT revealed acute signs such as intraretinal fluid to resolve quickly, while outer retinal disruption persisted until the last follow-up in eight of eleven eyes. These lesions resembled lesions of patients with solar retinopathy of which seven cases (11 eyes) were identified between 2007 and 2020. Conclusion Readily available consumer laser pointers can damage the retina and the underlying retinal pigment epithelium, possibly leading to long-lasting visual impairments. The number of laser pointer injuries has increased over the last years. Therefore, access to laser pointers for children should be strictly controlled.

OBJECTIVE The choice of surgical technique in sight-threatening Grave orbitopathy remains controversial. Available data are mostly derived from mixed cohorts with multiple surgical indications and techniques. The authors assessed predictors for visual outcome after standardized pterional orbital decompression for dysthyroid optic neuropathy. METHODS Retrospective analysis of 62 pterional orbital decompressions performed on 40 patients with dysthyroid optic neuropathy. RESULTS Visual acuity improved by an average of 3.8 lines in eyes with preoperative visual impairment (95% confidence interval [CI]: 1.8-5.8 lines, P < 0.001) and remained stable in eyes without prior visual impairment (95% CI -1.3 to 1 line, P = 0.81). Proptosis was reduced by an average of 3.1 mm (95% CI 1.8-4.3 mm, P < 0.001). Higher degrees of proptosis were predictive of worse visual outcomes (P = 0.017). New-onset diplopia developed in 2 patients, while previous diplopia resolved after surgery in 6 patients. CONCLUSIONS This cohort is the largest series of pterional orbit decompressions and the first to focus exclusively on dysthyroid neuropathy. Complication rates were low. Decompression surgery was highly effective at restoring and maintaining visual acuity in patients with dysthyroid optic neuropathy.

Zusammenfassung Hintergrund Die Symptome des akuten Winkelblocks, ein Notfallereignis, das ohne rechtzeitige Therapie zur irreversiblen Erblindung führen kann, sind vielfältig. Diese können initial als internistische oder neurologische Erkrankungen gedeutet werden, wenn Kopfschmerzen, Pupillenstarre oder Übelkeit im Vordergrund stehen. Ziel unserer Studie war es, die Rate belastender und invasiver Diagnostik durch primäre Vorstellung bei Nichtophthalmologen bei akutem primären Winkelblock zu erfassen. Methode Retrospektive Single-Center-Studie von Patienten mit akutem primärem Winkelblock. Zur Identifizierung dieser wurden alle Patienten erfasst, bei denen im Universitätsklinikum Freiburg, Klinik für Augenheilkunde im Zeitraum 2014–2018 eine chirurgische Iridektomie (5-133.0) oder Iridotomie durch Laser (5-136.1) durchgeführt wurde. Anschließend erfolgte durch Akteneinsicht die Datenanalyse zur Prüfung der Ein- und Ausschlusskriterien sowie des Krankheitsverlaufs. Ergebnisse Eingeschlossen wurden 91 Patienten mit akutem primären Winkelblock. Davon stellten sich 28 % (n = 25) initial bei nichtophthalmologischen Fachdisziplinen vor. In dieser Patientengruppe erhielten 56 % (n = 11) eine nichtzielführende Diagnostik, wobei bei 32 % (n = 8) eine kraniale Bildgebung und bei 8 % (n = 2) eine Lumbalpunktion durchgeführt wurde. Schlussfolgerung Bei akutem primären Winkelblock zeigt sich eine hohe Rate an nicht wegweisender Diagnostik durch Nichtophthalmologen, weshalb dieses Krankheitsbild fächerübergreifend präsent sein sollte. Bei unspezifischen Symptomen, wie Kopfschmerzen, Übelkeit und Erbrechen sowie Pupillenstarre, muss an die Möglichkeit eines akuten Augeninnendruckanstiegs durch einen akuten Winkelblock gedacht und das frühzeitige Hinzuziehen eines Ophthalmologen erwogen werden.

Purpose The aim of this study was to investigate HIF-1α, HIF-2α, and ProExC expression in conjunctival intraepithelial neoplasia (CIN), to differentiate between metaplasia and dysplasia, and to access their value as diagnostic and prognostic immunohistochemical markers. Recurrence and progression into SCC (squamous cell carcinoma) were defined as endpoints. Methods Forty-three specimens including CIN I (2), CIN II (9), CIN III (29), with and without metaplasia, and metaplasia alone (3), as well as 21 conjunctival control specimens, were stained with antibodies against HIF-1α, HIF-2α, and ProExC. The percentage of positively stained cells were calculated and used for further analysis. Results The mean percentages of HIF-1α and HIF-2α were not increased in CIN. In comparison, the expressions of these markers were even significantly elevated in control specimens (p < 0.001). Upper epithelial cells in CIN were more often ProExC-positive compared with normal conjunctiva or metaplasia (p = 0.06 and p = 0.07). Cox proportional-hazards analysis was performed for characterization of factors influencing the combined endpoint and showed a significant elevated hazard ratio for staining with ProExC (p = 0.04) compared with HIF-1α (p = 0.26) and HIF-2α (p = 0.49). Conclusion Our study shows that HIF-1α and HIF-2α do not serve as diagnostic or prognostic markers in CIN. ProExC seems to be a potential indicator for CIN, but not a reliable diagnostic marker. However, control specimens occasionally also display a high percentage of ProExC-positive cells and staining over the entire epithelial layer.

Purpose To evaluate the relationship between macular pigment optical density (MPOD) and glaucoma. Methods Forty-three patients with an established glaucoma diagnosis (25 females, 18 males, mean age 70 (range 34-84)) and 43 healthy controls (28 females, 15 males, mean age 62 (range 30-87)) were included in this prospective diagnostic case-control study. All subjects underwent detailed eye examination including ophthalmoscopy, best-corrected visual acuity, biomicroscopy, measurement of the axial length of the eye, objective refraction, lens status, central foveal thickness on spectral-domain optical coherence tomography (SD-OCT). In all glaucoma patients, a visual field assessment and a measurement of the retinal nerve fibre layer (RNFL) on SD-OCT were done. MPOD was determined using the macula pigment module of the Spectralis HRA+OCT (Heidelberg Engineering GmbH, Heidelberg, Germany) at 0.51°, 1.02° and 1.99° retinal eccentricity using two-wavelength autofluorescence imaging. Results In the glaucoma group, the median of the visual field mean defect was 5.1 db (quartiles 3.0 and 13.5) and the mean RNFL-thickness global was 65.9 µm (SD ± 16.1). Median MPOD measured at 0.51°, 1.02° and 1.99° retinal eccentricity in the glaucoma group was 0.42 DU, 0.34 DU and 0.13 DU, in the control group 0.40 DU, 0.35 DU and 0.12 DU respectively. There was no statistically significant difference of median MPOD between glaucomatous and control eyes (p=0.510, 0.735, 0.481). No significant relation between MPOD at 1.02 retinal eccentricity and the presence of glare symptoms was found (p=0.948). However, age seems to correlate with median MPOD measured at 1.02 retinal eccentricity (p=0.017). Conclusion There was no evidence for lower MPOD levels in our glaucoma patients; lower MPOD was not related to the presence of glare symptoms. However there seems to be a positive correlation between age and MPOD at 1.02° retinal eccentricity. To further investigate the relation between glare reported by glaucoma patients and glare disability linked to lower MPOD levels, additional studies are necessary that include both detailed inquiry of the quality of glare and a glare quantification and precise analysis of MPOD levels in glaucoma patients.

Purpose: To assess the additional value of 48-hour diurnal-nocturnal IOP profiles in comparison to only 24-hour diurnal-nocturnal IOP profiles in the management of glaucoma.Methods: All diurnal-nocturnal IOP profiles over 48 hours that were taken between 2017 and 2019 for diagnostic purposes in glaucoma patients at our hospital were reviewed. We counted elevated IOP values (>21 mmHg), higher short-term IOP fluctuations (> 6 mmHg) and nocturnal IOP peaks (measured at midnight and at 7 AM in supine position). In a second step, we repeated this analysis in the same profiles but censored the data to the first 24-hours. We compared the outcome rates by means of the Chi2-Test. Results: 661 IOP profiles were included. 59% of 48-hour IOP profiles revealed IOP values above 21 mmHg and 87% showed IOP fluctuation greater than 6 mmHg. Nocturnal peaks in supine position could be observed in 51% of the patients. In the profiles censored to the first 24 hours, the fractions were 50%, 71% and 48%, (p < 0,01, p < 0,01 and p = 0,12) respectively. Conclusion: Our data suggest that 48-hour diurnal-nocturnal IOP profiles are superior in identifying patients with clinically meaningful IOP events in comparison to observing the patients for only 24-hours. It is likely that the higher accuracy is worth the additional socioeconomic costs resulting from the prolonged inpatient stay.

Heterogenous nuclear ribonucleoproteins (hnRNPs) are abundant proteins implicated in various steps of RNA processing that assemble on nuclear RNA into larger complexes termed 40S hnRNP particles. Despite their initial discovery 55 years ago, our understanding of these intriguing macromolecular assemblies remains limited. Here, we report the biochemical purification of native 40S hnRNP particles and the determination of their complete protein composition by label-free quantitative mass spectrometry, identifying A-group and C-group hnRNPs as the major protein constituents. Isolated 40S hnRNP particles dissociate upon RNA digestion and can be reconstituted in vitro on defined RNAs in the presence of the individual protein components, demonstrating a scaffolding role for RNA in nucleating particle formation. Finally, we revealed their nanometer scale, condensate-like nature, promoted by intrinsically disordered regions of A-group hnRNPs. Collectively, we identify nuclear 40S hnRNP particles as novel dynamic biomolecular condensates., Nucleic Acids Research, 50 (11), ISSN:1362-4962, ISSN:0301-5610

Mitochondrial ribosomes (mitoribosomes) play a central role in synthesizing mitochondrial inner membrane proteins responsible for oxidative phosphorylation. Although mitoribosomes from different organisms exhibit considerable structural variations, recent insights into mitoribosome assembly suggest that mitoribosome maturation follows common principles and involves a number of conserved assembly factors. To investigate the steps involved in the assembly of the mitoribosomal small subunit (mt-SSU) we determined the cryoelectron microscopy structures of middle and late assembly intermediates of the Trypanosoma brucei mitochondrial small subunit (mt-SSU) at 3.6- and 3.7-Å resolution, respectively. We identified five additional assembly factors that together with the mitochondrial initiation factor 2 (mt-IF-2) specifically interact with functionally important regions of the rRNA, including the decoding center, thereby preventing premature mRNA or large subunit binding. Structural comparison of assembly intermediates with mature mt-SSU combined with RNAi experiments suggests a noncanonical role of mt-IF-2 and a stepwise assembly process, where modular exchange of ribosomal proteins and assembly factors together with mt-IF-2 ensure proper 9S rRNA folding and protein maturation during the final steps of assembly., Proceedings of the National Academy of Sciences of the United States of America, 119 (3), ISSN:0027-8424, ISSN:1091-6490

To compare a new no-touch alignment technique for toric intraocular lenses (IOL) with the conventional technique that uses a manual pendulum. In this retrospective case–control study, patients who underwent toric IOL implantation using two different alignment techniques (digital Callisto® system vs. manual-pendulum-based marking) were compared in a vector analysis using the Alpins method and an analysis of variance regarding corrected and uncorrected visual acuity and the deviation of the achieved IOL axis from the targeted axis. Sixty-one eyes were included into analysis. Thirty-six of these surgeries were performed via the Callisto® system and 25 eyes via pendulum-based corneal markings. Median IOL axis misalignment was 3° in both groups. Median uncorrected distance visual acuity was 0.097 logMAR versus 0.200. Median best-corrected visual acuity was 0.000 logMAR versus 0.097. All these data were below the range of statistical significance (p > 0.05). Vector analysis showed no significant difference for TIA [median of 3.14 diopters (D) vs. 2.73 D], SIA (median of 3.82 D vs. 3.79 D), DV (1.18 D vs. 1.08 D), and CI (1.23 vs. 1.29). Median angle of error was 1.96° versus − 0.44° (p > 0.05). We found no significant difference in the refractive results, the IOL positioning, and the best-corrected and uncorrected distance visual acuity between the two compared methods. Nevertheless, the Callisto® IOL alignment system delivers a standardized and easy-to-use technology. In particular, less-experienced surgeons might benefit from this marking technique.

Assembly pathway for mitoribosome The biogenesis of ribosomes is a multistep process facilitated by assembly factors. Saurer et al. provided structural information on the maturation process of the mitochondrial ribosome, or mitoribosome, in the parasitic protozoan Trypanosoma brucei (see the Perspective by Karbstein). Cells evolved a dedicated machinery for maturation of the small subunit of the mitoribosome, including the formation of three distinct and well-structured assembly intermediates. Comparison of intermediates and the mature mitoribosome reveals how assembly factors and ribosomal proteins work together to fold and stabilize ribosomal RNA. Science , this issue p. 1144 ; see also p. 1077

Intraocular pressure (IOP) measurement can be performed with different methods. Newer methods have to be compared to the standard method, the Goldmann applanation tonometry (GAT). We herein compare two air-puff tonometers, the non-contact tonometer (Tomey NCT) and the Corvis ST (CST) with GAT in eyes with a broad spectrum of IOP. Two hundred and forty-nine eyes of 249 patients (with diagnosis of either glaucoma or ocular hypertension) were included in this monocenter prospective cohort study. Each eye underwent IOP measurements via GAT, NCT and CST. Bland–Altman plots were calculated to compare the different methods in the three groups. Paired t tests were used for statistical comparison between the three measurement methods. The difference between the different methods was tested on correlation against central corneal thickness (CCT). Mean IOP in GAT was 17.6 mmHg (standard deviation (SD) 5.9), 16.3 mmHg (SD 5.6) in NCT and 18.0 mmHg (SD 5.5) in CST. Comparisons between GAT and CST vs. NCT showed significant differences (p

The conserved signal recognition particle (SRP) cotranslationally delivers ~30% of the proteome to the eukaryotic endoplasmic reticulum (ER). The molecular mechanism by which eukaryotic SRP transitions from cargo recognition in the cytosol to protein translocation at the ER is not understood. Here, structural, biochemical, and single-molecule studies show that this transition requires multiple sequential conformational rearrangements in the targeting complex initiated by guanosine triphosphatase (GTPase)–driven compaction of the SRP receptor (SR). Disruption of these rearrangements, particularly in mutant SRP54G226E linked to severe congenital neutropenia, uncouples the SRP/SR GTPase cycle from protein translocation. Structures of targeting intermediates reveal the molecular basis of early SRP-SR recognition and emphasize the role of eukaryote-specific elements in regulating targeting. Our results provide a molecular model for the structural and functional transitions of SRP throughout the targeting cycle and show that these transitions provide important points for biological regulation that can be perturbed in genetic diseases., Science Advances, 7 (21), ISSN:2375-2548

BACKGROUND/AIMS Retinal haemangioblastomas (RH) remain a major cause of visual impairment in patients with von Hippel-Lindau (VHL) disease. Identification of genotype-phenotype correlation is an important prerequisite for better management, treatment and prognosis. METHODS Retrospective, single-centre cohort study of 200 VHL patients. Genetic data and date of onset of RH, central nervous system haemangioblastomas (CNSH), pheochromocytoma/paraganglioma (PPGL), clear cell renal cell carcinoma (ccRCC) and pancreatic neuroendocrine neoplasm (PNEN) were collected. The number and locations of RH were recorded. RESULTS The first clinical finding occurred at an age of 26 ± 14 years (y) [mean ± SD]. In 91 ± 3% (95% CI 88-94) of the patients, at least one RH occur until the age of 60y. A total of 42 different rare VHL gene variants in 166 patients were detected. A higher age-related incidence of RH, CNSH, ccRCC and PNEN was detected in patients with a truncating variant (TV) compared to patients with a single amino-acid substitution/deletion (AASD) (all p

Background/Aims Surgical procedures in ophthalmology underlie variations over different time periods and are strongly dependent on the technical process and the invention of innovative surgical techniques. Especially, in glaucoma surgery a lot of surgical devices and techniques have been introduced during the last years. Until now, the use of these newer techniques has not been shown on a robust data basis. We herein present the numbers of different types of glaucoma surgeries performed at German hospitals between 2006 and 2018. Methods and Design The quality reports of hospitals in Germany from 2006 to 2018 were analyzed concerning all procedural codes for glaucoma surgery and intervention. Especially, the change in usage of “classical” and “modern” surgical techniques (MIGS: “minimally invasive glaucoma surgery”) or devices was compared. Results The number of glaucoma procedures performed increased by 75% from 27,811 in 2006 to 48,794 in 2018. Absolute numbers of trabeculectomies, goniotomies, ab externo trabeculotomies and to a certain level cyclodestructive procedures decreased during the examined years while use of MIGS has increased in absolute and relative numbers since 2012. From 2015 a strong increase in the usage of XEN implants could be seen. Drainage implants (such as Baerveldt/Ahmed) showed stable absolute numbers over the time period covered. Absolute numbers of laser trabeculoplasty peaked in 2014 and decreased afterwards. Iridotomies and iridectomies increased by 120%/248% over the whole period. Conclusion Our data show a trend towards the modern surgical options and especially MIGS during the recent years. Classical procedures showed a decrease in total numbers emphasized from 2013. These numbers confirm the assumption that modern glaucoma surgery is becoming more and more popular and established in German hospitals.

PURPOSE To quantify retinal vasculature changes in Stargardt disease1 (STGD1) with volume-rendered optical coherence tomography angiography. METHODS Optical coherence tomography angiography volumes from healthy subjects and two subgroups of patients with STGD1 with the presence/absence of definitely decreased autofluorescence areas were compared. Optical coherence tomography angiography vessel surface area and vessel volume were measured in central zones (Z) of 1-, 2-, and 3-mm diameter. RESULTS Twenty nine eyes of 15 patients with STGD1 (20/9 eyes with/without definitely decreased autofluorescence) and 30 eyes of 15 controls contributed data. An enlarged foveal avascular zone was found in patients with STGD1 without and even more with definitely decreased autofluorescence associated with a vessel rarefication in central and also paracentral zones with unnoticeable autofluorescence. Vessel surface area and vessel volume were reduced in both STGD1 subgroups for all zones (P < 0.0001). Stargardt disease 1 eyes when compared to without definitely decreased autofluorescence showed reduced vessel surface area and vessel volume in Z2+3 (both P < 0.05). CONCLUSION Volume rendering of optical coherence tomography angiography in STGD1 shows a reduced retinal flow in the central macula. This is most likely secondary to loss of neurosensory tissue with disease progression and therefore not likely be favorably influenced by gene transfer and retinal pigment epithelial transplantation. Retinal blood flow assessed by 3D volume-rendered optical coherence tomography angiography could serve as surrogate marker for vascular changes of the central retina.

Support films are commonly used during cryo-EM specimen preparation to both immobilise the sample and minimise the exposure of particles at the air-water interface. Here we report preparation protocols for carbon and graphene supported single particle electron microscopy samples using a novel 3D-printed sample transfer block to facilitate the direct, wetted, movement of both carbon and graphene supports from the substrate on which they were generated to small volumes (10 μL) of sample. These approaches are simple and inexpensive to implement, minimise hydrophobic contamination of the support films, and are widely applicable to single particle studies. Our approach also allows the direct exchange of the sample buffer on the support film in cases in which it is unsuitable for vitrification, e.g. for samples from centrifugal density gradients that help to preserve sample integrity. © 2020 The Authors, Journal of Structural Biology, 213 (1), ISSN:1047-8477, ISSN:1095-8657

The human GID (hGID) complex is a conserved E3 ubiquitin ligase regulating diverse biological processes, including glucose metabolism and cell cycle progression. However, the biochemical function and substrate recognition of the multi-subunit complex remain poorly understood. Using biochemical assays, cross-linking mass spectrometry, and cryo-electron microscopy, we show that hGID engages two distinct modules for substrate recruitment, dependent on either WDR26 or GID4. WDR26 and RanBP9 cooperate to ubiquitinate HBP1 in vitro, while GID4 is dispensable for this reaction. In contrast, GID4 functions as an adaptor for the substrate ZMYND19, which surprisingly lacks a Pro/N-end degron. GID4 substrate binding and ligase activity is regulated by ARMC8α, while the shorter ARMC8β isoform assembles into a stable hGID complex that is unable to recruit GID4. Cryo-EM reconstructions of these hGID complexes reveal the localization of WDR26 within a ring-like, tetrameric architecture and suggest that GID4 and WDR26/Gid7 utilize different, non-overlapping binding sites. Together, these data advance our mechanistic understanding of how the hGID complex recruits cognate substrates and provides insights into the regulation of its E3 ligase activity., EMBO Reports, 22 (11), ISSN:1469-221X, ISSN:1469-3178

Ribosomes have been suggested to directly control gene regulation, but regulatory roles for ribosomal RNA (rRNA) remain largely unexplored. Expansion segments (ESs) consist of multitudes of tentacle-like rRNA structures extending from the core ribosome in eukaryotes. ESs are remarkably variable in sequence and size across eukaryotic evolution with largely unknown functions. In characterizing ribosome binding to a regulatory element within a Homeobox (Hox) 5′ UTR, we identify a modular stem-loop within this element that binds to a single ES, ES9S. Engineering chimeric, “humanized” yeast ribosomes for ES9S reveals that an evolutionary change in the sequence of ES9S endows species-specific binding of Hoxa9 mRNA to the ribosome. Genome editing to site-specifically disrupt the Hoxa9-ES9S interaction demonstrates the functional importance for such selective mRNA-rRNA binding in translation control. Together, these studies unravel unexpected gene regulation directly mediated by rRNA and how ribosome evolution drives translation of critical developmental regulators. © 2020 Elsevier Inc. ISSN:1097-2765 ISSN:1097-4164

Cellular life requires a high degree of molecular complexity and self-organization, some of which must have originated in a prebiotic context. Here, we demonstrate how both of these features can emerge in a plausibly prebiotic system. We found that chemical gradients in simple mixtures of activated amino acids and fatty acids can lead to the formation of amyloid-like peptide fibrils that are localized inside of a proto-cellular compartment. In this process, the fatty acid or lipid vesicles act both as a filter, allowing the selective passage of activated amino acids, and as a barrier, blocking the diffusion of the amyloidogenic peptides that form spontaneously inside the vesicles. This synergy between two distinct building blocks of life induces a significant increase in molecular complexity and spatial order thereby providing a route for the early molecular evolution that could give rise to a living cell.

The protein kinase mammalian target of rapamycin (mTOR) is the central regulator of cell growth. Aberrant mTOR signaling is linked to cancer, diabetes, and neurological disorders. mTOR exerts its functions in two distinct multiprotein complexes, mTORC1 and mTORC2. Here, we report a 3.2-Å resolution cryo-EM reconstruction of mTORC2. It reveals entangled folds of the defining Rictor and the substrate-binding SIN1 subunits, identifies the carboxyl-terminal domain of Rictor as the source of the rapamycin insensitivity of mTORC2, and resolves mechanisms for mTORC2 regulation by complex destabilization. Two previously uncharacterized small-molecule binding sites are visualized, an inositol hexakisphosphate (InsP6) pocket in mTOR and an mTORC2-specific nucleotide binding site in Rictor, which also forms a zinc finger. Structural and biochemical analyses suggest that InsP6 and nucleotide binding do not control mTORC2 activity directly but rather have roles in folding or ternary interactions. These insights provide a firm basis for studying mTORC2 signaling and for developing mTORC2-specific inhibitors., Science Advances, 6 (45), ISSN:2375-2548

Recent advances in the field of biomedical research allow for elucidation of the transcriptional signature of rare tumors such as conjunctival squamous cell carcinoma (SCC). In this study we compare its expression profile to conjunctival papilloma (Pap) and healthy conjunctival tissue (Ctrl) and develop a classification tool to differentiate these entities. Seven conjunctival SCC, seven Pap and ten Ctrl were formalin-fixed and paraffin-embedded (FFPE) and analyzed using Massive Analysis of cDNA Ends (MACE) RNA sequencing. Differentially expressed genes (DEG) and gene ontology (GO) clusters were explored and the abundance of involved cell types was quantified by xCell. Finally, a classification model was developed to distinguish SCC from Pap and Ctrl. Among the most prominent DEG in SCC a plethora of keratins were upregulated when compared to Pap and Ctrl. xCell analysis revealed an enrichment of immune cells, including activated dendritic cells and T-helper type 1 cells (Th1), in SCC when compared to Ctrl. The generated classification model could reliably discriminate between the three entities according to the expression pattern of 30 factors. This study provides a transcriptome-wide gene expression profile of rare conjunctival SCC. The analysis identifies distinct keratins, as well as dendritic and Th1 cells as important mediators in SCC. Finally, the provided gene expression classifier may become an aid to the conventional histological classification of conjunctival tumors in uncertain cases.

Royal jelly (RJ) is produced by honeybees (Apis mellifera) as nutrition during larval development. The high viscosity of RJ originates from high concentrations of long lipoprotein filaments that include the glycosylated major royal jelly protein 1 (MRJP1), the small protein apisimin and insect lipids. Using cryo-electron microscopy we reveal the architecture and the composition of RJ filaments, in which the MRJP1 forms the outer shell of the assembly, surrounding stacked apisimin tetramers harbouring tightly packed lipids in the centre. The structural data rationalize the pH-dependent disassembly of RJ filaments in the gut of the larvae., Nature Communications, 11 (1), ISSN:2041-1723

In contrast to the bacterial translation machinery, mitoribosomes and mitochondrial translation factors are highly divergent in terms of composition and architecture. There is increasing evidence that the biogenesis of mitoribosomes is an intricate pathway, involving many assembly factors. To better understand this process, we investigated native assembly intermediates of the mitoribosomal large subunit from the human parasite Trypanosoma brucei using cryo-electron microscopy. We identify 28 assembly factors, 6 of which are homologous to bacterial and eukaryotic ribosome assembly factors. They interact with the partially folded rRNA by specifically recognizing functionally important regions such as the peptidyltransferase center. The architectural and compositional comparison of the assembly intermediates indicates a stepwise modular assembly process, during which the rRNA folds toward its mature state. During the process, several conserved GTPases and a helicase form highly intertwined interaction networks that stabilize distinct assembly intermediates. The presented structures provide general insights into mitoribosomal maturation. © 2020 Elsevier Inc. The structures of two assembly intermediates of the Trypanosoma brucei mitoribosomal large subunit in combination with biochemical analysis provide insights into the stepwise mitoribosomal biogenesis process that involves numerous assembly factors functioning as enzymes or scaffold components. © 2020 Elsevier Inc. ISSN:1097-2765 ISSN:1097-4164

Purpose To characterize the choriocapillaris (CC) structure in relation to subretinal fluid (SRF) as a possible systematic error source using spectral domain (SD-OCTA) compared to swept-source optical coherence tomography angiography (SS-OCTA). Methods This is a prospective case-control study of 23 eyes. Ten patients with acute central serous chorioretinopathy (CSC), three patients with partial macular-off retinal detachment (RD) and ten healthy, age-matched controls were included. Abnormal CC decorrelation signals were quantitatively compared in CSC and controls by means of custom image processing. To investigate the influence of SRF on CC OCTA signal, the extent of SRF was quantified with a macular heatmap and compared with the corresponding OCTA signal of the CC. Results SS-OCTA yielded a more homogeneous OCTA signal from the CC than SD-OCTA, offering less signal dispersion and variability in healthy and diseased eyes. Both devices demonstrated CC signal voids in CSC and RD, respectively. In CCS, the voids were predominantly located in the area with SRF. Compared to SD-OCTA, SS-OCTA delivered a more homogenous OCTA signal and reduced signal voids in the CC underneath SRF in both RD and CSC (CSC, 7.6% ± 6.3% vs, 19.7% ± 9.6%, p p Conclusion Understanding OCTA artifacts is critical to ensure accurate clinical evaluations. In this study, we describe the presence of SRF as an important shadow-causing artifact source for CC OCTA analysis which can be mitigated but not completely eliminated by employing SS-OCTA.

Ribosomes have been suggested to directly control gene regulation, however regulatory roles for ribosomal RNA (rRNA) remain largely unexplored. Expansion segments (ESs) consist of a multitude of tentacle-like rRNA structures that extend from the core ribosome in eukaryotes. ESs are remarkably variable in sequence and size across eukaryotic evolution with a largely unknown function. In characterizing ribosome binding to a regulatory element within a Homeobox (Hox) 5’ UTR, we unexpectedly identify a modular stem-loop within this element that binds to a single ES, ES9S. Engineering chimeric, “humanized” yeast ribosomes for ES9S reveals that an evolutionary change in the sequence of ES9S endows species-specific binding of Hoxa9 mRNA to the ribosome. Genome editing to site-specifically disrupt the Hoxa9-ES9S interaction in neural stem cells demonstrates the functional importance for such selective mRNA-rRNA binding in translation control. Together, these studies unravel unexpected gene regulation directly mediated by rRNA.

Oxazolidinones are synthetic antibiotics used for treatment of infections caused by Gram-positive bacteria. They target the bacterial protein synthesis machinery by binding to the peptidyl transferase centre (PTC) of the ribosome and interfering with the peptidyl transferase reaction. Cadazolid is the first member of quinoxolidinone antibiotics, which are characterized by combining the pharmacophores of oxazolidinones and fluoroquinolones, and it is evaluated for treatment of Clostridium difficile gastrointestinal infections that frequently occur in hospitalized patients. In vitro protein synthesis inhibition by cadazolid was shown in Escherichia coli and Staphylococcus aureus, including an isolate resistant against linezolid, the prototypical oxazolidinone antibiotic. To better understand the mechanism of inhibition, we determined a 3.0 Å cryo-electron microscopy structure of cadazolid bound to the E. coli ribosome in complex with mRNA and initiator tRNA. Here we show that cadazolid binds with its oxazolidinone moiety in a binding pocket in close vicinity of the PTC as observed previously for linezolid, and that it extends its unique fluoroquinolone moiety towards the A-site of the PTC. In this position, the drug inhibits protein synthesis by interfering with the binding of tRNA to the A-site, suggesting that its chemical features also can enable the inhibition of linezolid-resistant strains., Scientific Reports, 9, ISSN:2045-2322

U1 small nuclear ribonucleoparticle (U1 snRNP) plays a central role during RNA processing. Previous structures of U1 snRNP revealed how the ribonucleoparticle is organized and recognizes the pre-mRNA substrate at the exon–intron junction. As with many other ribonucleoparticles involved in RNA metabolism, U1 snRNP contains extensions made of low complexity sequences. Here, we developed a protocol to reconstitute U1 snRNP in vitro using mostly full-length components in order to perform liquid-state NMR spectroscopy. The accuracy of the reconstitution was validated by probing the shape and structure of the particle by SANS and cryo-EM. Using an NMR spectroscopy-based approach, we probed, for the first time, the U1 snRNP tails at atomic detail and our results confirm their high degree of flexibility. We also monitored the labile interaction between the splicing factor PTBP1 and U1 snRNP and validated the U1 snRNA stem loop 4 as a binding site for the splicing regulator on the ribonucleoparticle. Altogether, we developed a method to probe the intrinsically disordered regions of U1 snRNP and map the interactions controlling splicing regulation. This approach could be used to get insights into the molecular mechanisms of alternative splicing and screen for potential RNA therapeutics.

Mycobacterium tuberculosis harbors proteasomes that recruit substrates for degradation through an ubiquitin-like modification pathway. Recently, a non-ATPase activator termed Bpa (bacterial proteasome activator) was shown to support an alternate proteasomal degradation pathway. Here, we present the cryo-electron microscopy (cryo-EM) structure of Bpa in complex with the 20S core particle (CP). For docking into the cryo-EM density, we solved the X-ray structure of Bpa, showing that it forms tight four-helix bundles arranged into a 12-membered ring with a 40 Å wide central pore and the C-terminal helix of each protomer protruding from the ring. The Bpa model was fitted into the cryo-EM map of the Bpa-CP complex, revealing its architecture and striking symmetry mismatch. The Bpa-CP interface was resolved to 3.5 Å, showing the interactions between the C-terminal GQYL motif of Bpa and the proteasome α-rings. This docking mode is related to the one observed for eukaryotic activators with features specific to the bacterial complex.

Purpose To compare spectral-domain (SD) and swept-source (SS) optical coherence tomography angiography (OCTA) for imaging retinal capillary hemangioblastomas (RCHs) in von Hippel-Lindau disease (VHLD). Methods Prospective single-center cross-sectional study. Tumor size (TS) of perfused RCHs was assessed clinically in relation to the optic disc size. For both technologies, SD-OCTA and SS-OCTA, corresponding images with a scan size of 3 × 3 mm2 and 6 × 6 mm2, respectively, were overlaid according to the set of marker positions to determine the TS. Results From 200 patients with VHLD, 48 patients showed 84 RCHs. SD-OCTA images of 39 RCHs (46.4%) and SS-OCTA images of 48 RCHs (57.2%) were suitable for analysis. The average in OCTA-measured TS of 1.60 ± 2.58 mm2 (range, 0.01–10.43) was congruent to the clinically assessed TS in 81.3% of cases (r = 0.86, P < 0.0001). TS measured in SD-OCTA compared to SS-OCTA showed similar values and a high correlation (all P < 0.0001). Nevertheless, despite the similarities, a slight trend in SS-OCTA was observed whereby with increasing TS, an elevated TS was detected compared to SD-OCTA (3 × 3-mm2 scans: mean difference of 0.03 ± 0.04 mm2, 6 × 6-mm2 scans: 0.08 ± 0.19 mm2). However, within the same imaging technology method, TS values almost did not differ (SD-OCTA: mean difference of 0.01 ± 0.02 mm2, SS-OCTA: 0.001 ± 0.01 mm2). Conclusions OCTA may serve as an additional tool for diagnosis and monitoring of RCHs. Nevertheless, due to the differences between the technologies, the values cannot be used interchangeably. Translational Relevance SD-OCTA and SS-OCTA are suitable to detect and monitor RCHs and provide a more detailed assessment about the TS than this is clinically possible.

Structure of the largest, most complex ribosome Ribosomes are two-subunit ribonucleoprotein assemblies that catalyze the translation of messenger RNA into protein. Ribosomal RNAs (rRNAs) play key structural and functional roles. Ramrath et al. report the high-resolution structure of mitochondrial ribosomes from the unicellular parasite Trypanosoma brucei that contain the smallest known rRNAs. The trypanosomal mitoribosome is the most complex ribosomal assembly characterized, with two rRNAs and 126 proteins. The increased protein subunits have substituted for rRNA as an architectural scaffold. The structure also reveals the minimal core needed for ribosome function. Science , this issue p. eaau7735

First steps of translocation elucidated Ribosomes synthesizing membrane or secretory proteins are targeted to the endoplasmic reticulum (ER) in eukaryotic cells by the signal recognition particle (SRP). Upon reaching the ER, the SRP interacts with its receptor to promote transfer of the signal sequence to the protein-conducting channel or translocon. Kobayashi et al. studied the ribosomal complex that forms on the ER, in which the SRP and its receptor interact to transfer the newly synthesized protein to the translocon. The observed organization of the assembly reveals the roles of multiple eukaryotic-specific protein components present in the SRP and its receptor in stabilizing the conformation that facilitates signal sequence handover. Science , this issue p. 323

Final maturation of eukaryotic ribosomes occurs in the cytoplasm and requires the sequential removal of associated assembly factors and processing of the immature 20S pre‐RNA. Using cryo‐electron microscopy (cryo‐EM), we have determined the structure of a yeast cytoplasmic pre‐40S particle in complex with Enp1, Ltv1, Rio2, Tsr1, and Pno1 assembly factors poised to initiate final maturation. The structure reveals that the pre‐rRNA adopts a highly distorted conformation of its 3′ major and 3′ minor domains stabilized by the binding of the assembly factors. This observation is consistent with a mechanism that involves concerted release of the assembly factors orchestrated by the folding of the rRNA in the head of the pre‐40S subunit during the final stages of maturation. Our results provide a structural framework for the coordination of the final maturation events that drive a pre‐40S particle toward the mature form capable of engaging in translation.