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The means by which the physicochemical properties of different cellular components together determine bacterial cell shape remain poorly understood. Here, we investigate a programmed cell-shape change during Bacillus subtilis sporulation, when a rod-shaped vegetative cell is transformed to an ovoid spore. Asymmetric cell division generates a bigger mother cell and a smaller, hemispherical forespore. The septum traps the forespore chromosome, which is translocated to the forespore by SpoIIIE. Simultaneously, forespore size increases as it is reshaped into an ovoid. Using genetics, timelapse microscopy, cryo-electron tomography, and mathematical modeling, we demonstrate that forespore growth relies on membrane synthesis and SpoIIIE-mediated chromosome translocation, but not on peptidoglycan or protein synthesis. Our data suggest that the hydrated nucleoid swells and inflates the forespore, displacing ribosomes to the cell periphery, stretching septal peptidoglycan, and reshaping the forespore. Our results illustrate how simple biophysical interactions between core cellular components contribute to cellular morphology.
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División Celular Asimétrica/fisiología , Bacillus subtilis/fisiología , Cromosomas Bacterianos/metabolismo , Esporas Bacterianas/metabolismo , Translocación Genética , Bacillus subtilis/ultraestructura , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Cromosomas Bacterianos/genética , Peptidoglicano/biosíntesis , Peptidoglicano/genética , Biosíntesis de Proteínas/fisiología , Esporas Bacterianas/genética , Esporas Bacterianas/ultraestructuraRESUMEN
Transcription-coupled DNA repair removes bulky DNA lesions from the genome1,2 and protects cells against ultraviolet (UV) irradiation3. Transcription-coupled DNA repair begins when RNA polymerase II (Pol II) stalls at a DNA lesion and recruits the Cockayne syndrome protein CSB, the E3 ubiquitin ligase, CRL4CSA and UV-stimulated scaffold protein A (UVSSA)3. Here we provide five high-resolution structures of Pol II transcription complexes containing human transcription-coupled DNA repair factors and the elongation factors PAF1 complex (PAF) and SPT6. Together with biochemical and published3,4 data, the structures provide a model for transcription-repair coupling. Stalling of Pol II at a DNA lesion triggers replacement of the elongation factor DSIF by CSB, which binds to PAF and moves upstream DNA to SPT6. The resulting elongation complex, ECTCR, uses the CSA-stimulated translocase activity of CSB to pull on upstream DNA and push Pol II forward. If the lesion cannot be bypassed, CRL4CSA spans over the Pol II clamp and ubiquitylates the RPB1 residue K1268, enabling recruitment of TFIIH to UVSSA and DNA repair. Conformational changes in CRL4CSA lead to ubiquitylation of CSB and to release of transcription-coupled DNA repair factors before transcription may continue over repaired DNA.
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Microscopía por Crioelectrón , Reparación del ADN , Complejos Multiproteicos/química , Complejos Multiproteicos/ultraestructura , ARN Polimerasa II/química , ARN Polimerasa II/ultraestructura , Transcripción Genética , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Proteínas Portadoras/ultraestructura , ADN Helicasas/química , ADN Helicasas/metabolismo , ADN Helicasas/ultraestructura , Enzimas Reparadoras del ADN/química , Enzimas Reparadoras del ADN/metabolismo , Enzimas Reparadoras del ADN/ultraestructura , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/ultraestructura , Humanos , Modelos Moleculares , Complejos Multiproteicos/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa/química , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa/ultraestructura , ARN Polimerasa II/metabolismo , Elongación de la Transcripción Genética , Factor de Transcripción TFIIH/química , Factor de Transcripción TFIIH/metabolismo , Factor de Transcripción TFIIH/ultraestructura , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Factores de Transcripción/ultraestructura , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/ultraestructura , UbiquitinaciónRESUMEN
Chromatin-remodelling complexes of the SWI/SNF family function in the formation of nucleosome-depleted, transcriptionally active promoter regions (NDRs)1,2. In the yeast Saccharomyces cerevisiae, the essential SWI/SNF complex RSC3 contains 16 subunits, including the ATP-dependent DNA translocase Sth14,5. RSC removes nucleosomes from promoter regions6,7 and positions the specialized +1 and -1 nucleosomes that flank NDRs8,9. Here we present the cryo-electron microscopy structure of RSC in complex with a nucleosome substrate. The structure reveals that RSC forms five protein modules and suggests key features of the remodelling mechanism. The body module serves as a scaffold for the four flexible modules that we call DNA-interacting, ATPase, arm and actin-related protein (ARP) modules. The DNA-interacting module binds extra-nucleosomal DNA and is involved in the recognition of promoter DNA elements8,10,11 that influence RSC functionality12. The ATPase and arm modules sandwich the nucleosome disc with the Snf2 ATP-coupling (SnAC) domain and the finger helix, respectively. The translocase motor of the ATPase module engages with the edge of the nucleosome at superhelical location +2. The mobile ARP module may modulate translocase-nucleosome interactions to regulate RSC activity5. The RSC-nucleosome structure provides a basis for understanding NDR formation and the structure and function of human SWI/SNF complexes that are frequently mutated in cancer13.
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Microscopía por Crioelectrón , Complejos Multiproteicos/química , Complejos Multiproteicos/ultraestructura , Nucleosomas/metabolismo , Nucleosomas/ultraestructura , Saccharomyces cerevisiae/química , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfatasas/ultraestructura , Secuencia de Aminoácidos , Animales , Transporte Biológico , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/ultraestructura , Drosophila melanogaster , Humanos , Ratones , Modelos Moleculares , Complejos Multiproteicos/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Proteínas Nucleares/ultraestructura , Nucleosomas/química , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Saccharomyces cerevisiae/ultraestructura , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/ultraestructura , Xenopus laevisRESUMEN
PURPOSE: The aim of this study is to investigate the distribution of spherical equivalent and axial length in the general population and to analyze the influence of education on spherical equivalent with a focus on ocular biometric parameters. METHODS: The Gutenberg Health Study is a population-based cohort study in Mainz, Germany. Participants underwent comprehensive ophthalmologic examinations as part of the 5-year follow-up examination in 2012-2017 including genotyping. The spherical equivalent and axial length distributions were modeled with gaussian mixture models. Regression analysis (on person-individual level) was performed to analyze associations between biometric parameters and educational factors. Mendelian randomization analysis explored the causal effect between spherical equivalent, axial length, and education. Additionally, effect mediation analysis examined the link between spherical equivalent and education. RESULTS: A total of 8532 study participants were included (median age: 57 years, 49% female). The distribution of spherical equivalent and axial length follows a bi-Gaussian function, partially explained by the length of education (i.e., < 11 years education vs. 11-20 years). Mendelian randomization indicated an effect of education on refractive error using a genetic risk score of education as an instrument variable (- 0.35 diopters per SD increase in the instrument, 95% CI, - 0.64-0.05, p = 0.02) and an effect of education on axial length (0.63 mm per SD increase in the instrument, 95% CI, 0.22-1.04, p = 0.003). Spherical equivalent, axial length and anterior chamber depth were associated with length of education in regression analyses. Mediation analysis revealed that the association between spherical equivalent and education is mainly driven (70%) by alteration in axial length. CONCLUSIONS: The distribution of axial length and spherical equivalent is represented by subgroups of the population (bi-Gaussian). This distribution can be partially explained by length of education. The impact of education on spherical equivalent is mainly driven by alteration in axial length.
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Longitud Axial del Ojo , Escolaridad , Humanos , Femenino , Masculino , Persona de Mediana Edad , Alemania/epidemiología , Longitud Axial del Ojo/patología , Distribución Normal , Biometría/métodos , Refracción Ocular/fisiología , Estudios de Seguimiento , Errores de Refracción/fisiopatología , Errores de Refracción/diagnóstico , Errores de Refracción/genética , Anciano , AdultoRESUMEN
Iatrogenic keratectasia is induced thinning and protrusion of the cornea after laser refractive surgery. Known risk factors include an excessively thin postoperative residual stromal bed, a thicker flap, or preoperatively undetected evidence of preexisting subclinical keratoconus. The rate of post-refractive ectasia in eyes without identifiable preoperative risk factors is 20 per 100â000 eyes for photorefractive keratectomy, 90 per 100â000 eyes for laser in situ keratomileusis, and 11 per 100â000 eyes for small incision lenticule extraction. Traditional screening tools for preoperative risk include the ectasia risk score system and percentage of tissue alteration. More recent methods include corneal elastography and epithelial mapping, in addition to Artificial Intelligence methods for data analysis. Therapy includes contact lenses, cross-linking, implantation of intracorneal ring segments, penetrating or lamellar keratoplasty, and, in early studies, implantation of corneal lenticules.
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Queratocono , Queratomileusis por Láser In Situ , Humanos , Dilatación Patológica/etiología , Inteligencia Artificial , Agudeza Visual , Topografía de la Córnea , Córnea/cirugía , Queratomileusis por Láser In Situ/efectos adversos , Queratocono/diagnóstico , Queratocono/cirugía , Enfermedad Iatrogénica/prevención & controlRESUMEN
PURPOSE: To evaluate the standard of care, in particular the use of topical or subconjunctival interferon-α2b, in treating ocular surface squamous neoplasia or melanocytic tumours in tertiary eye centres in Germany. METHODS: A survey containing 14 questions was sent to 43 tertiary eye centres in Germany. The questions addressed the surgical and medical management of ocular surface squamous neoplasia and melanocytic tumours (primary acquired melanosis and malignant melanoma), as well as the clinical experiences and difficulties in prescribing off-label interferon-α2b eye drops and subconjunctival injections. RESULTS: Twenty-four tertiary eye centres responded to the survey. Eighty-three percent of centres had used interferon-α2b in their clinical practice and 25% prescribed it as the first-line cytostatic agent following surgical excision of ocular surface squamous neoplasia, while 10% would do so for melanocytic tumours. Correspondingly, the majority of respondents selected mitomycin C as their first-line agent. Side effects were uncommon with topical interferon-α2b eye drops but were more frequently reported after subconjunctival interferon-α2b injections. In total, eight centres had experience with interferon-α2b injections. The most significant obstacles perceived by ophthalmologists when prescribing interferon-α2b were its high cost and the reimbursement thereof. CONCLUSION: Off-label mitomycin C was the preferred adjuvant therapy for epithelial and melanocytic tumours, with interferon-α2b being the standard second-line option. Interferon-α2b has predominantly been used to treat ocular surface squamous neoplasia and, to a lesser extent, melanocytic tumours at German tertiary eye centres. Following its market withdrawal, supply shortages of interferon-α2b are likely to have a profound impact on patient care and their quality of life.
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Carcinoma de Células Escamosas , Neoplasias de la Conjuntiva , Humanos , Mitomicina/uso terapéutico , Calidad de Vida , Carcinoma de Células Escamosas/tratamiento farmacológico , Carcinoma de Células Escamosas/patología , Carcinoma de Células Escamosas/cirugía , Interferón-alfa/uso terapéutico , Interferón-alfa/efectos adversos , Neoplasias de la Conjuntiva/tratamiento farmacológico , Encuestas y Cuestionarios , Soluciones Oftálmicas , Proteínas Recombinantes/uso terapéuticoRESUMEN
Photoreceptor cell transplantation into the mouse retina has been shown to result in the transfer of cytoplasmic material between donor and host photoreceptors. Recently it has been found that this inter-photoreceptor material transfer process is likely to be mediated by nanotube-like structures connecting donor and host photoreceptors. By leveraging cone-specific reporter mice and super-resolution microscopy we provide evidence for the transfer of cytoplasmic material also from endogenous cones to endogenous rod photoreceptors and the existence of nanotube-like cell-cell connections possibly mediating this process in the adult mouse retina, together with preliminary data indicating that horizontal material transfer may also occur in the human retina.
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Células Fotorreceptoras Retinianas Conos , Células Fotorreceptoras Retinianas Bastones , Animales , Mamíferos , Ratones , RetinaRESUMEN
The bacterial transpeptidase sortase A is a well-established tool in protein chemistry and catalyzes the chemoselective ligation of peptides and proteins. During catalysis sortase A cleaves the conserved Leu-Pro-X-Thr-Gly sorting motif at the Thr residue under concomitant thioester formation at active site Cys184. We have used expressed protein ligation (EPL) to generate sortase mutants with Cys184 replaced by selenocysteine (Sec) and homocysteine (Hcy). Sec-sortase showed a moderate 2-3-fold reduction in catalytic activity in contrast to Hcy-sortase which is a poor catalyst with less than 1% of wild-type activity. The sensitivity of the active site nucleophiles towards an alkylation reagent correlated with the pKa values of the mutated residues. Furthermore, the pH-profile of Sec-sortase was shifted to more acidic conditions when compared to the wild-type enzyme. These observations provide information on sortase catalysis and the semisynthetic enzymes might represent useful tools for further biochemical investigations and engineering approaches of sortases A.
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Aminoaciltransferasas/química , Aminoaciltransferasas/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Cisteína Endopeptidasas/química , Cisteína Endopeptidasas/genética , Homocisteína/genética , Ingeniería de Proteínas , Selenocisteína/genética , Alquilación , Secuencia de Aminoácidos , Aminoaciltransferasas/metabolismo , Proteínas Bacterianas/metabolismo , Catálisis , Dominio Catalítico , Cisteína Endopeptidasas/metabolismo , Escherichia coli/genética , Homocisteína/química , Homocisteína/metabolismo , Mutación , Pliegue de Proteína , Selenocisteína/química , Selenocisteína/metabolismo , Técnicas de Síntesis en Fase Sólida , Transformación GenéticaAsunto(s)
Enfermedades de la Córnea/cirugía , Lámina Limitante Posterior/cirugía , Hemorragia del Ojo/cirugía , Cirugía Filtrante/efectos adversos , Glaucoma/cirugía , Anciano , Conjuntiva/cirugía , Enfermedades de la Córnea/diagnóstico por imagen , Enfermedades de la Córnea/etiología , Enfermedades de la Córnea/fisiopatología , Lámina Limitante Posterior/diagnóstico por imagen , Lámina Limitante Posterior/patología , Hemorragia del Ojo/diagnóstico por imagen , Hemorragia del Ojo/etiología , Hemorragia del Ojo/fisiopatología , Femenino , Humanos , Presión Intraocular , Microscopía con Lámpara de Hendidura , Colgajos Quirúrgicos , Tomografía de Coherencia Óptica , Agudeza Visual/fisiologíaRESUMEN
PURPOSE: To evaluate the interchangeability of different tomography devices used for ray tracing-based intraocular lens (IOL) calculation. SETTING: Eye clinic, Castrop-Rauxel, Germany. DESIGN: Retrospective analysis. METHOD: Measurements from 3 Placido-Scheimpflug devices and 3 optical coherence tomography (OCT) devices were compared in 83 and 161 other eyes after cataract surgery, respectively. 2-dimensional matrices of anterior local corneal curvature and local corneal thickness are transferred to the ray-tracing software OKULIX. Calculations are performed with the same IOL in the same position of an eye with the same axial length. Differences in spherical equivalent (SE), astigmatism, and spherical aberration are evaluated. Furthermore, the influence of the size of the matrices (optical zone) on the accuracy is quantified. RESULTS: For the Placido-Scheimpflug devices, the deviations from the average of three measurements taken for each eye in SE (mean ± SD) were 0.17 ± 0.24 diopters (D), -0.26 ± 0.29 D, and 0.08 ± 0.39 D ( P < .001, analysis of variance [ANOVA]), for the centroids of the astigmatic differences 0.04 D/173 degrees, 0.14 D/93 degrees, and 0.10 D/7 degrees, and for the median of the absolute values of the vector differences 0.31 D, 0.33 D, and 0.29 D. For OCT devices, the corresponding results were 0.01 ± 0.21 D, -0.03 ± 0.21 D, and 0.02 ± 0.20 D ( P = .005, ANOVA); 0.18 D/120 degrees, 0.07 D/70 degrees, and 0.22 D/4 degrees; and 0.26 D, 0.30 D, and 0.33 D. The accuracy of the calculated spherical aberrations allows for an individual selection of the best fitting IOL model in most cases. CONCLUSIONS: The differences are small enough to make the devices interchangeable regarding astigmatism and spherical aberration. Although there are significant differences in SE between Scheimpflug and OCT devices, the differences between OCT devices are also small enough to make them interchangeable, but the differences between Placido-Scheimpflug devices are too large to make these devices interchangeable.
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Astigmatismo , Lentes Intraoculares , Facoemulsificación , Humanos , Córnea , Astigmatismo/cirugía , Topografía de la Córnea/métodos , Estudios Retrospectivos , Tomografía de Coherencia Óptica/métodos , Biometría/métodosRESUMEN
Background: The aim of this study was to evaluate the short-term efficacy and safety of the Paul Glaucoma Implant (PGI) in pediatric eyes diagnosed with glaucoma following congenital cataract surgery (GFCS). Methods: A retrospective, single-center, descriptive study was conducted on consecutive children diagnosed with GFCS who underwent PGI implantation between July 2022 and November 2023 at the University Medical Center Mainz. The primary outcome measure was the reduction in IOP at the last follow-up visit. Results: Ten eyes of nine children were included in the study. The mean follow-up time was 7.70 ± 4.22 months (4.68-10.72 months). At the end of the study follow-up, the mean (95% CI) reduction in IOP was -14.8 ± 8.73 mmHg (-8.56 to -21.04 mmHg, p < 0.001). At the last follow-up, 30.0% (3/10) of patients achieved an IOP (intraocular pressure) of ≥6 and ≤21 mmHg with a reduction in IOP of ≥25% without treatment, while 90.0% (9/10) achieved this target IOP regardless of glaucoma medication treatment. The mean number of antiglaucoma medications was significantly reduced from 3.50 (IQR = 1) to 2.0 (IQR = 2, p = 0.01), and the visual acuity logMAR improved from 1.26 ± 0.62 to 1.03 ± 0.48 (p = 0.04). Only one eye experienced numerical hypotony (4 mmHg) without choroidal detachment or anterior chamber shallowing within the first 24 h. No other adverse events were observed during the follow-up period. Conclusions: PGI implantation significantly lowered IOP and the number of antiglaucoma eye drops with a favorable safety profile in children diagnosed with GFCS, thereby achieving a high rate of qualified surgical success in the short term.
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OBJECTIVE: To evaluate the long-term outcomes of trabeculectomy (TE) surgery in a large cohort with a minimum follow-up of 3 years. DESIGN: Retrospective cohort study. SETTING: University Eye Hospital, Germany. PARTICIPANTS: Three hundred and seventy-nine patients with open-angle glaucoma underwent TE with mitomycin C (MMC) between January 2013 and February 2017 with a minimal follow-up of 3 years. Eligible patients were identified via an electronic surgical case register. INTERVENTIONS: All patients had undergone TE with MMC following a set surgical protocol. To assess the influence of cataract surgery following TE, eyes which underwent cataract surgery at least 6 months after TE were matched 1:3 by sex and age to eyes who did not undergo cataract surgery during the follow-up period. MAIN OUTCOME MEASURES: Primary outcome was the proportion of surgical success based on intraocular pressure (IOP), surgical complications, the need for revision surgery, loss of light perception and the need for additional pressure-lowering medication. RESULTS: The mean follow-up time was 6 (±0.8, IQR: 5.4-6.5) years. Seventy-three per cent of eyes achieved qualified surgical success at the last follow-up (IOP≥5 mm Hg and ≤18 mm Hg, without surgical complications or complete loss of vision) but necessitated additional medical therapy, complete surgical success with no additional medical therapy was achieved in 69% of eyes. There was no significant difference in the success probability between eyes that had undergone cataract surgery after TE and those that had not (p=0.45). CONCLUSIONS: The results demonstrate a high and stable success rate of TE after a mean follow-up time of approximately 6 years, that is, not affected by later cataract surgery.
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Catarata , Glaucoma de Ángulo Abierto , Trabeculectomía , Humanos , Trabeculectomía/métodos , Glaucoma de Ángulo Abierto/tratamiento farmacológico , Estudios Retrospectivos , Ojo , Presión Intraocular , Mitomicina/uso terapéutico , Catarata/complicaciones , Resultado del Tratamiento , Estudios de SeguimientoRESUMEN
BACKGROUND: To evaluate the long-term efficacy and safety of modified canaloplasty versus trabeculectomy in open-angle glaucoma. METHODS: In total, 210 subjects with open-angle glaucoma were included. 70 were treated with Mitomycin C-augmented modified canaloplasty with enhanced subconjunctival filtration and 140 with Mitomycin C-augmented trabeculectomy. Cases were matched 1:2 by sex and age. RESULTS: In canaloplasty and trabeculectomy groups, 61.4% and 57.9% of participants were female. Mean age was 60.0 ± 13.9 and 63.0 ± 12.2 years, median follow-up time was 4.6 [IQR 4.3, 5.05] years and 5.8 [IQR 5.4, 6.3]. Strict success was achieved in 20.0% and 56.4%, complete success in 24.3% and 66.4%, and qualified success in 34.3% and 73.6% (each p < 0.001). Kaplan-Meier survival analysis showed a better survival probability for trabeculectomy than for canaloplasty (p < 0.001) and Cox regression analysis revealed an HR of 6.03 (95%-CI 3.66, 9.93, p < 0.001) after canaloplasty. Trabeculectomy showed superiority in terms of IOP decrease (9.2 ± 7.9 mmHg vs. 13.7 ± 10.4 mmHg, p = 0.002), use of AGM (50.0% vs. 10.7%, p < 0.001), and the number of revision surgeries (41.4% vs. 21.4%, p = 0.004). Occurrence of complications was similar in both groups (14.5% vs. 7.5%, p = 0.19). CONCLUSIONS: Trabeculectomy showed superiority in efficacy and equality in safety compared to modified canaloplasty.
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Neurodegenerative diseases remain incompletely understood and therapies are needed. Stem cell-derived organoid models facilitate fundamental and translational medicine research. However, to which extent differential neuronal and glial pathologic processes can be reproduced in current systems is still unclear. Here, we tested 16 different chemical, physical, and cell functional manipulations in mouse retina organoids to further explore this. Some of the treatments induce differential phenotypes, indicating that organoids are competent to reproduce distinct pathologic processes. Notably, mouse retina organoids even reproduce a complex pathology phenotype with combined photoreceptor neurodegeneration and glial pathologies upon combined (not single) application of HBEGF and TNF, two factors previously associated with neurodegenerative diseases. Pharmacological inhibitors for MAPK signaling completely prevent photoreceptor and glial pathologies, while inhibitors for Rho/ROCK, NFkB, and CDK4 differentially affect them. In conclusion, mouse retina organoids facilitate reproduction of distinct and complex pathologies, mechanistic access, insights for further organoid optimization, and modeling of differential phenotypes for future applications in fundamental and translational medicine research.
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Building stock management is becoming a global societal and political issue, inter alia because of growing sustainability concerns. Comprehensive and openly accessible building stock data can enable impactful research exploring the most effective policy options. In Europe, efforts from citizen and governments generated numerous relevant datasets but these are fragmented and heterogeneous, thus hindering their usability. Here, we present EUBUCCO v0.1, a database of individual building footprints for ~202 million buildings across the 27 European Union countries and Switzerland. Three main attributes - building height, construction year and type - are included for respectively 73%, 24% and 46% of the buildings. We identify, collect and harmonize 50 open government datasets and OpenStreetMap, and perform extensive validation analyses to assess the quality, consistency and completeness of the data in every country. EUBUCCO v0.1 provides the basis for high-resolution urban sustainability studies across scales - continental, comparative or local studies - using a centralized source and is relevant for a variety of use cases, e.g., for energy system analysis or natural hazard risk assessments.
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The possible applications for human retinal organoids (HROs) derived from human induced pluripotent stem cells (hiPSC) rely on the robustness and transferability of the methodology for their generation. Standardized strategies and parameters to effectively assess, compare, and optimize organoid protocols are starting to be established, but are not yet complete. To advance this, we explored the efficiency and reliability of a differentiation method, called CYST protocol, that facilitates retina generation by forming neuroepithelial cysts from hiPSC clusters. Here, we tested seven different hiPSC lines which reproducibly generated HROs. Histological and ultrastructural analyses indicate that HRO differentiation and maturation are regulated. The different hiPSC lines appeared to be a larger source of variance than experimental rounds. Although previous reports have shown that HROs in several other protocols contain a rather low number of cones, HROs from the CYST protocol are consistently richer in cones and with a comparable ratio of cones, rods, and Müller glia. To provide further insight into HRO cell composition, we studied single cell RNA sequencing data and applied CaSTLe, a transfer learning approach. Additionally, we devised a potential strategy to systematically evaluate different organoid protocols side-by-side through parallel differentiation from the same hiPSC batches: In an explorative study, the CYST protocol was compared to a conceptually different protocol based on the formation of cell aggregates from single hiPSCs. Comparing four hiPSC lines showed that both protocols reproduced key characteristics of retinal epithelial structure and cell composition, but the CYST protocol provided a higher HRO yield. So far, our data suggest that CYST-derived HROs remained stable up to at least day 200, while single hiPSC-derived HROs showed spontaneous pathologic changes by day 200. Overall, our data provide insights into the efficiency, reproducibility, and stability of the CYST protocol for generating HROs, which will be useful for further optimizing organoid systems, as well as for basic and translational research applications.
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Disturbances of retinal perfusion are involved in the onset and maintenance of several ocular diseases, including diabetic retinopathy, glaucoma, and retinal vascular occlusion. Hence, knowledge on ocular vascular anatomy and function is highly relevant for basic research studies and for clinical judgment and treatment. The retinal vasculature is composed of the superficial, intermediate, and deep vascular layer. Detection of changes in blood flow and vascular diameter especially in smaller vessels is essential to understand and to analyze vascular diseases. Several methods to evaluate blood flow regulation in the retina have been described so far, but no gold standard has been established. For highly reliable assessment of retinal blood flow, exact determination of vessel diameter is necessary. Several measurement methods have already been reported in humans. But for further analysis of retinal vascular diseases, studies in laboratory animals, including genetically modified mice, are important. As for mice, the small vessel size is challenging requiring devices with high optic resolution. In this review, we recapitulate different methods for retinal blood flow and vessel diameter measurement. Moreover, studies in humans and in experimental animals are described.
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BACKGROUND: Autonomous diagnosis and assessment of medical emergencies are important skills to acquire for medical students. Ophthalmology features certain specialty-specific "red flag" signs and symptoms, which pose a challenge for educators in ophthalmology. To support medical students in identifying those "red flags" we developed and implemented interactive cases for our elearning platform. MATERIAL AND METHODS: A total of seven interactive cases with key feature problems regarding potentially dangerous signs and symptoms, such as painless loss of vision or red eye were developed. Medical students were guided through a case and performed formative assessments. The interactive cases were created with elearning authoring software and were available on the learning management system presence of the department of ophthalmology. They were mandatory for medical students in the ophthalmology course. Students evaluated the cases after the course. RESULTS: The interactive cases were rated on average at 1.51⯱ 0.68 (mean⯱ standard deviation; nâ¯= 163) on a grade scale (1â¯= best, 6â¯= worst). On a Likert scale they were perceived as helpful for individual learning at 1.60⯱ 0.81 (1â¯= very helpful, 7â¯= not helpful at all; nâ¯= 164). The information provided on the cases and selection of scenarios was positively evaluated. CONCLUSION: To support students in identifying and managing ophthalmic emergencies in the context of limited time in tightly packed curricula, interactive key feature cases can be part of corresponding elearning resources. An integration of such cases was evaluated as desirable.
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Educación de Pregrado en Medicina , Oftalmología , Estudiantes de Medicina , Curriculum , Urgencias Médicas , Humanos , Oftalmología/educaciónRESUMEN
Purpose: Prenatal growth restriction is associated with impaired neurodevelopment in childhood. This study investigated the effects of being born small for gestational age (SGA) on peripapillary retinal nerve fiber layer (pRNFL) thickness in adults born at term. Methods: A retrospective cohort study was conducted with a prospective ophthalmologic examination of participants born at full-term (gestational age ≥37 weeks) between 1969 and 2002. All participants were examined with spectral-domain optical coherence tomography and grouped according to their birth weight in correlation to gestational age as former moderate (birth weight (BW) percentile 3rd to <10th) and severe SGA (<3rd percentile), normal (10th-90th percentile, AGA), and moderately (>90th to 97th percentile) and severely (>97th percentile) large for gestational age (LGA) adults (18 to 52 years). Results: Overall, 547 eyes of 285 individuals (age 29.9±9.4 years, 151 females) born at term were included. Multivariable regression analyses revealed a strong association between a lower global pRNFL thickness in the severe SGA (B=-8.99 [95%-CI: -12.68; -5.30] µm; p<0.001) and in the moderate SGA groups (B=-6.40 [95%-CI: -10.29; -2.50] µm; p=0.001) compared to the reference AGA group. Conclusion: Our results indicate that restricted fetal growth affects neurologic tissue development of the optic nerve head, particularly in individuals born severely SGA at term. This indicates that fetal growth restriction may exert disturbances in the development of neurologic tissue, which persists in adulthood.
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PURPOSE: To evaluate long-term results of glaucoma surgery in newborn and infants with glaucoma. METHODS: Seventy-nine eyes of 52 children (age: 3 weeks-15.3 years) with primary congenital or secondary glaucoma treated between 2015 and 2017 were included. The median follow-up time was 3.9 years. Conventional probe trabeculotomy, 360° catheter-assisted trabeculotomy, filtering and cyclodestructive surgery were compared. Strict criteria for surgical success were applied: Complete surgical success (IOP below target IOP, no further surgery) and incomplete surgical success (additional surgery allowed) were analyzed, and IOP at baseline and last follow-up was compared. RESULTS: Intraocular pressure (IOP) was significantly reduced in primary congenital (preoperative IOP: 27.8 ± 7.5 mmHg vs. postoperative IOP: 14.2 ± 4.5 mmHg) and secondary glaucoma (preoperative IOP: 29.2 ± 9.1 mmHg vs. postoperative IOP: 16.6 ± 4.7 mmHg). 90% of all eyes reached target IOP with or without medication allowing for additional surgeries. As first surgery, 360° catheter-assisted trabeculotomy had a tendency to higher surgical success than other surgical approaches, while cyclodestructive procedures had lowest. CONCLUSIONS: We found very promising surgical results in our childhood glaucoma patient group. Surgical success in both congenital and secondary glaucoma was high.