Non-therapeutic laparotomies in military trauma (2009-2014).

BACKGROUND: Combat casualties are frequently injured in austere settings where modern imaging modalities are unavailable. Exploratory laparotomies are often performed in these settings when there is suspicion for intra-abdominal injury. Prior studies of combat casualties reported non-therapeutic laparotomy (NTL) rates as high as 32%. Given improvements in combat casualty care over time, we evaluated NTLs performed during later years of the wars in Iraq and Afghanistan. METHODS: Military personnel with combat-related injuries (6/1/2009-12/31/2014) who underwent exploratory laparotomy based on concern for abdominal injury (i.e. not performed for proximal vascular control or fecal diversion) and were evacuated to Landstuhl Regional Medical Center (Germany) before being transferred to participating U.S. military hospitals were assessed. An NTL was defined as a negative laparotomy without substantial intra-abdominal injuries requiring repair. Characteristics, indications for laparotomy, operative findings, and outcomes were examined. RESULTS: Among 244 patients who underwent laparotomies, 41 (16.8%) had NTLs and 203 (83.2%) had therapeutic laparotomies (i.e. positive findings). Patients with NTLs had more computed tomography scans concerning for injury (48.8% vs 27.1%; p = 0.006), less penetrating injury mechanisms (43.9% vs 71.9%; p < 0.001), and lower Injury Severity Scores (26 vs 33; p = 0.003) compared to patients with therapeutic laparotomies. Patients with NTLs were also less likely to be admitted to the intensive care unit (70.7 vs 89.2% for patients with therapeutic laparotomies; p = 0.007). No patients with NTLs developed abdominal surgical site infections (SSI) compared to 16.7% of patients with therapeutic laparotomies (p = 0.002). There was no significant difference in mortality between the groups (p = 0.198). CONCLUSIONS: Our proportion of NTLs was lower than reported from earlier years during the wars in Iraq and Afghanistan. No infectious complications from NTLs (i.e. abdominal SSIs) were identified. Nevertheless, surgeons should continue to have a low threshold for exploratory laparotomy in military patients in austere settings with concern for intra-abdominal injury.

Contribution of intraflagellar transport to compartmentalization and maintenance of the photoreceptor cell.

The first steps of vision take place in the ciliary outer segment compartment of photoreceptor cells. The protein composition of outer segments is uniquely suited to perform this function. The most abundant among these proteins is the visual pigment, rhodopsin, whose outer segment trafficking involves intraflagellar transport (IFT). Here, we report three major findings from the analysis of mice in which ciliary transport was acutely impaired by conditional knockouts of IFT-B subunits. First, we demonstrate the existence of a sorting mechanism whereby mislocalized rhodopsin is recruited to and concentrated in extracellular vesicles prior to their release, presumably to protect the cell from adverse effects of protein mislocalization. Second, reducing rhodopsin expression significantly delays photoreceptor degeneration caused by IFT disruption, suggesting that controlling rhodopsin levels may be an effective therapy for some cases of retinal degenerative disease. Last, the loss of IFT-B subunits does not recapitulate a phenotype observed in mutants of the BBSome (another ciliary transport protein complex relying on IFT) in which non-ciliary proteins accumulate in the outer segment. Whereas it is widely thought that the role of the BBSome is to primarily participate in ciliary transport, our data suggest that the BBSome has another major function independent of IFT and possibly related to maintaining the diffusion barrier of the ciliary transition zone.

Downregulation of rhodopsin is an effective therapeutic strategy in ameliorating peripherin-2-associated inherited retinal disorders.

Given the absence of approved treatments for pathogenic variants in Peripherin-2 (PRPH2), it is imperative to identify a universally effective therapeutic target for PRPH2 pathogenic variants. To test the hypothesis that formation of the elongated discs in presence of PRPH2 pathogenic variants is due to the presence of the full complement of rhodopsin in absence of the required amounts of functional PRPH2. Here we demonstrate the therapeutic potential of reducing rhodopsin levels in ameliorating disease phenotype in knockin models for p.Lys154del (c.458-460del) and p.Tyr141Cys (c.422 A > G) in PRPH2. Reducing rhodopsin levels improves physiological function, mitigates the severity of disc abnormalities, and decreases retinal gliosis. Additionally, intravitreal injections of a rhodopsin-specific antisense oligonucleotide successfully enhance the physiological function of photoreceptors and improves the ultrastructure of discs in mutant mice. Presented findings shows that reducing rhodopsin levels is an effective therapeutic strategy for the treatment of inherited retinal degeneration associated with PRPH2 pathogenic variants.

Sodium content in plant and insect food resources consumed by chimpanzees (Pan troglodytes schweinfurthii) in Gombe National Park, Tanzania.

OBJECTIVES: Many nonhuman primate diets are dominated by plant foods, yet plant tissues are often poor sources of sodium-a necessary mineral for metabolism and health. Among primates, chimpanzees (Pan troglodytes), which are ripe fruit specialists, consume diverse animal, and plant resources. Insects have been proposed as a source of dietary sodium for chimpanzees, yet published data on sodium values for specific foods are limited. We assayed plants and insects commonly eaten by chimpanzees to assess their relative value as sodium sources. MATERIALS AND METHODS: We used atomic absorption spectroscopy to determine sodium content of key plant foods and insects consumed by chimpanzees of Gombe National Park, Tanzania. Dietary contributions of plant and insect foods were calculated using feeding observational data. RESULTS: On a dry matter basis, mean sodium value of plant foods (n = 83 samples; mean = 86 ppm, SD = 92 ppm) was significantly lower than insects (n = 12; mean = 1549 ppm, SD = 807 ppm) (Wilcoxon rank sum test: W = 975, p < 0.001). All plant values were below the suggested sodium requirement (2000 ppm) for captive primates. While values of assayed insects were variable, sodium content of two commonly consumed insect prey for Gombe chimpanzees (Macrotermes soldiers and Dorylus ants) were four to five times greater than the highest plant values and likely meet requirements. DISCUSSION: We conclude that plant foods available to Gombe chimpanzees are generally poor sources of sodium while insects are important, perhaps critical, sources of sodium for this population.

A Seminested PCR Method for the Diagnosis of Invasive Fungal Infections in Combat Injured.

Background: Among combat injured, invasive fungal infections (IFIs) result in significant morbidity. Cultures and histopathology are the primary diagnostic methods for IFIs, but they have limitations. We previously evaluated a panfungal polymerase chain reaction assay, which was 83% sensitive and 99% specific for angioinvasive IFIs. Here, we evaluated 3 less resource-intensive seminested assays targeting clinically relevant fungi in the order Mucorales and genera Aspergillus and Fusarium. Methods: Formalin-fixed paraffin-embedded tissue specimens from a multicenter trauma IFI cohort (2009-2014) were used. Cases were US military personnel injured in Afghanistan with histopathologic IFI evidence. Controls were patients with similar injury patterns and no laboratory IFI evidence (negative culture and histopathology). Seminested assays specific to Mucorales (V4/V5 regions of 18S rDNA), Aspergillus (mitochondrial tRNA), and Fusarium (internal transcribed spacer [ITS]/28A regions of DNA) were compared with a panfungal assay amplifying the internal transcribed spacer 2 region of rDNA and to histopathology. Results: Specimens from 92 injury sites (62 subjects) were compared with control specimens from 117 injuries (101 subjects). We observed substantial agreement between the seminested and panfungal assays overall, especially for the order Mucorales. Moderate agreement was observed at the genus level for Aspergillus and Fusarium. When compared with histopathology, sensitivity and specificity of seminested assays were 67.4% and 96.6%, respectively (sensitivity increased to 91.7% when restricted to sites with angioinvasion). Conclusions: Prior studies of seminested molecular diagnostics have focused on culture-negative samples from immunocompromised patients. Our findings underscore the utility of the seminested approach in diagnosing soft-tissue IFIs using formalin-fixed paraffin-embedded tissue samples, especially with angioinvasion.

Functional annotation and meta-analysis of maize transcriptomes reveal genes involved in biotic and abiotic stress.

BACKGROUND: Environmental stress factors, such as biotic and abiotic stress, are becoming more common due to climate variability, significantly affecting global maize yield. Transcriptome profiling studies provide insights into the molecular mechanisms underlying stress response in maize, though the functions of many genes are still unknown. To enhance the functional annotation of maize-specific genes, MaizeGDB has outlined a data-driven approach with an emphasis on identifying genes and traits related to biotic and abiotic stress. RESULTS: We mapped high-quality RNA-Seq expression reads from 24 different publicly available datasets (17 abiotic and seven biotic studies) generated from the B73 cultivar to the recent version of the reference genome B73 (B73v5) and deduced stress-related functional annotation of maize gene models. We conducted a robust meta-analysis of the transcriptome profiles from the datasets to identify maize loci responsive to stress, identifying 3,230 differentially expressed genes (DEGs): 2,555 DEGs regulated in response to abiotic stress, 408 DEGs regulated during biotic stress, and 267 common DEGs (co-DEGs) that overlap between abiotic and biotic stress. We discovered hub genes from network analyses, and among the hub genes of the co-DEGs we identified a putative NAC domain transcription factor superfamily protein (Zm00001eb369060) IDP275, which previously responded to herbivory and drought stress. IDP275 was up-regulated in our analysis in response to eight different abiotic and four different biotic stresses. A gene set enrichment and pathway analysis of hub genes of the co-DEGs revealed hormone-mediated signaling processes and phenylpropanoid biosynthesis pathways, respectively. Using phylostratigraphic analysis, we also demonstrated how abiotic and biotic stress genes differentially evolve to adapt to changing environments. CONCLUSIONS: These results will help facilitate the functional annotation of multiple stress response gene models and annotation in maize. Data can be accessed and downloaded at the Maize Genetics and Genomics Database (MaizeGDB).

Enhanced pan-genomic resources at the maize genetics and genomics database.

Pan-genomes, encompassing the entirety of genetic sequences found in a collection of genomes within a clade, are more useful than single reference genomes for studying species diversity. This is especially true for a species like Zea mays, which has a particularly diverse and complex genome. Presenting pan-genome data, analyses, and visualization is challenging, especially for a diverse species, but more so when pan-genomic data is linked to extensive gene model and gene data, including classical gene information, markers, insertions, expression and proteomic data, and protein structures as is the case at MaizeGDB. Here, we describe MaizeGDB's expansion to include the genic subset of the Zea pan-genome in a pan-gene data center featuring the maize genomes hosted at MaizeGDB, and the outgroup teosinte Zea genomes from the Pan-Andropoganeae project. The new data center offers a variety of browsing and visualization tools, including sequence alignment visualization, gene trees and other tools, to explore pan-genes in Zea that were calculated by the pipeline Pandagma. Combined, these data will help maize researchers study the complexity and diversity of Zea, and to use the comparative functions to validate pan-gene relationships for a selected gene model.

Harnessing the predicted maize pan-interactome for putative gene function prediction and prioritization of candidate genes for important traits.

The recent assembly and annotation of the 26 maize nested association mapping population founder inbreds have enabled large-scale pan-genomic comparative studies. These studies have expanded our understanding of agronomically important traits by integrating pan-transcriptomic data with trait-specific gene candidates from previous association mapping results. In contrast to the availability of pan-transcriptomic data, obtaining reliable protein-protein interaction (PPI) data has remained a challenge due to its high cost and complexity. We generated predicted PPI networks for each of the 26 genomes using the established STRING database. The individual genome-interactomes were then integrated to generate core- and pan-interactomes. We deployed the PPI clustering algorithm ClusterONE to identify numerous PPI clusters that were functionally annotated using gene ontology (GO) functional enrichment, demonstrating a diverse range of enriched GO terms across different clusters. Additional cluster annotations were generated by integrating gene coexpression data and gene description annotations, providing additional useful information. We show that the functionally annotated PPI clusters establish a useful framework for protein function prediction and prioritization of candidate genes of interest. Our study not only provides a comprehensive resource of predicted PPI networks for 26 maize genomes but also offers annotated interactome clusters for predicting protein functions and prioritizing gene candidates. The source code for the Python implementation of the analysis workflow and a standalone web application for accessing the analysis results are available at https://github.com/eporetsky/PanPPI.

PanEffect: a pan-genome visualization tool for variant effects in maize.

SUMMARY: Understanding the effects of genetic variants is crucial for accurately predicting traits and functional outcomes. Recent approaches have utilized artificial intelligence and protein language models to score all possible missense variant effects at the proteome level for a single genome, but a reliable tool is needed to explore these effects at the pan-genome level. To address this gap, we introduce a new tool called PanEffect. We implemented PanEffect at MaizeGDB to enable a comprehensive examination of the potential effects of coding variants across 50 maize genomes. The tool allows users to visualize over 550 million possible amino acid substitutions in the B73 maize reference genome and to observe the effects of the 2.3 million natural variations in the maize pan-genome. Each variant effect score, calculated from the Evolutionary Scale Modeling (ESM) protein language model, shows the log-likelihood ratio difference between B73 and all variants in the pan-genome. These scores are shown using heatmaps spanning benign outcomes to potential functional consequences. In addition, PanEffect displays secondary structures and functional domains along with the variant effects, offering additional functional and structural context. Using PanEffect, researchers now have a platform to explore protein variants and identify genetic targets for crop enhancement. AVAILABILITY AND IMPLEMENTATION: The PanEffect code is freely available on GitHub (https://github.com/Maize-Genetics-and-Genomics-Database/PanEffect). A maize implementation of PanEffect and underlying datasets are available at MaizeGDB (https://www.maizegdb.org/effect/maize/).

PhosBoost: Improved phosphorylation prediction recall using gradient boosting and protein language models.

Protein phosphorylation is a dynamic and reversible post-translational modification that regulates a variety of essential biological processes. The regulatory role of phosphorylation in cellular signaling pathways, protein-protein interactions, and enzymatic activities has motivated extensive research efforts to understand its functional implications. Experimental protein phosphorylation data in plants remains limited to a few species, necessitating a scalable and accurate prediction method. Here, we present PhosBoost, a machine-learning approach that leverages protein language models and gradient-boosting trees to predict protein phosphorylation from experimentally derived data. Trained on data obtained from a comprehensive plant phosphorylation database, qPTMplants, we compared the performance of PhosBoost to existing protein phosphorylation prediction methods, PhosphoLingo and DeepPhos. For serine and threonine prediction, PhosBoost achieved higher recall than PhosphoLingo and DeepPhos (.78, .56, and .14, respectively) while maintaining a competitive area under the precision-recall curve (.54, .56, and .42, respectively). PhosphoLingo and DeepPhos failed to predict any tyrosine phosphorylation sites, while PhosBoost achieved a recall score of .6. Despite the precision-recall tradeoff, PhosBoost offers improved performance when recall is prioritized while consistently providing more confident probability scores. A sequence-based pairwise alignment step improved prediction results for all classifiers by effectively increasing the number of inferred positive phosphosites. We provide evidence to show that PhosBoost models are transferable across species and scalable for genome-wide protein phosphorylation predictions. PhosBoost is freely and publicly available on GitHub.

Maize Feature Store: A centralized resource to manage and analyze curated maize multi-omics features for machine learning applications.

The big-data analysis of complex data associated with maize genomes accelerates genetic research and improves agronomic traits. As a result, efforts have increased to integrate diverse datasets and extract meaning from these measurements. Machine learning models are a powerful tool for gaining knowledge from large and complex datasets. However, these models must be trained on high-quality features to succeed. Currently, there are no solutions to host maize multi-omics datasets with end-to-end solutions for evaluating and linking features to target gene annotations. Our work presents the Maize Feature Store (MFS), a versatile application that combines features built on complex data to facilitate exploration, modeling and analysis. Feature stores allow researchers to rapidly deploy machine learning applications by managing and providing access to frequently used features. We populated the MFS for the maize reference genome with over 14 000 gene-based features based on published genomic, transcriptomic, epigenomic, variomic and proteomics datasets. Using the MFS, we created an accurate pan-genome classification model with an AUC-ROC score of 0.87. The MFS is publicly available through the maize genetics and genomics database. Database URL  https://mfs.maizegdb.org/.

A phenomenological, intersectional understanding of coping with ageism and racism among older adults.

The aim of this qualitative, phenomenological study was to understand how older adults cope with experiences of ageism and racism through an intersectional lens. Twenty adults 60+ residing in the U.S. Mountain West who identified as Black, Hispanic/Latino(a), Asian-American/Pacific Islander, Indigenous, or White participated individually in a one-hour, semi-structured interview. A team of five coders engaged in an inductive coding process through independent coding followed by critical discussion. Peer debriefing enhanced credibility. Nine themes were organized by three umbrella categories: Coping with ageism: 1) distancing via self-determination/defying stereotypes, 2) distancing by helping others; Coping with racism: 3) resistance, 4) exhaustion; Coping with both ageism and racism: 5) increased awareness through aging, 6) healthy lifestyle, 7) education, 8) acceptance/ 'let it go', and 9) avoidance. Novel findings include how older adults may cope with ageism and racism via increased awareness through aging and with ageism specifically by helping peer older adults, although instances of internalized ageism were noted and discussed. The themes exemplify problem-focused (e.g., helping others) and emotion-focused (acceptance), as well as individual (e.g., self-determination) and collective (e.g., resistance) coping strategies. This study can serve as a resource for practitioners in applying a more nuanced understanding of the ways older adults cope with ageism and racism in later life.

ROM1 is redundant to PRPH2 as a molecular building block of photoreceptor disc rims.

Visual signal transduction takes place within a stack of flattened membranous 'discs' enclosed within the light-sensitive photoreceptor outer segment. The highly curved rims of these discs, formed in the process of disc enclosure, are fortified by large hetero-oligomeric complexes of two homologous tetraspanin proteins, PRPH2 (a.k.a. peripherin-2 or rds) and ROM1. While mutations in PRPH2 affect the formation of disc rims, the role of ROM1 remains poorly understood. In this study, we found that the knockout of ROM1 causes a compensatory increase in the disc content of PRPH2. Despite this increase, discs of ROM1 knockout mice displayed a delay in disc enclosure associated with a large diameter and lack of incisures in mature discs. Strikingly, further increasing the level of PRPH2 rescued these morphological defects. We next showed that disc rims are still formed in a knockin mouse in which the tetraspanin body of PRPH2 was replaced with that of ROM1. Together, these results demonstrate that, despite its contribution to the formation of disc rims, ROM1 can be replaced by an excess of PRPH2 for timely enclosure of newly forming discs and establishing normal outer segment structure.

Seasonality of Microbiology of Combat-Related Wounds and Wound Infections in Afghanistan.

INTRODUCTION: Battlefield-related wound infections are a significant source of morbidity among combat casualties. Seasonality of these infections was demonstrated in previous conflicts (e.g., Korea) but has not been described with trauma-related health care-associated infections from the war in Afghanistan. METHODS: The study population included military personnel wounded in Afghanistan (2009-2014) medevac'd to Landstuhl Regional Medical Center and transitioned to participating military hospitals in the United States with clinical suspicion of wound infections and wound cultures collected ≤7 days post-injury. Analysis was limited to the first wound culture from individuals. Infecting isolates were collected from skin and soft-tissue infections, osteomyelitis, and burn soft-tissue infections. Data were analyzed by season (winter [ December 1-February 28/29], spring [March 1-May 31], summer [June 1-August 31], and fall [September 1-November 30]). RESULTS: Among 316 patients, 297 (94.0%) sustained blast injuries with a median injury severity score and days from injury to initial culture of 33 and 3.5, respectively. Although all patients had a clinical suspicion of a wound infection, a diagnosis was confirmed in 198 (63%) patients. Gram-negative bacilli (59.5% of 316) were more commonly isolated from wound cultures in summer (68.1%) and fall (67.1%) versus winter (43.9%) and spring (45.1%; P < .001). Multidrug-resistant (MDR) Gram-negative bacilli (21.8%) were more common in summer (21.8%) and fall (30.6%) versus winter (7.3%) and spring (19.7%; P = .028). Findings were similar for infecting Gram-negative bacilli (72.7% of 198)-summer (79.5%) and fall (83.6%; P = .001)-and infecting MDR Gram-negative bacilli (27.3% of 198)-summer (25.6%) and fall (41.8%; P = .015). Infecting anaerobes were more common in winter (40%) compared to fall (11%; P = .036). Gram-positive organisms were not significantly different by season. CONCLUSION: Gram-negative bacilli, including infecting MDR Gram-negative bacilli, were more commonly recovered in summer/fall months from service members injured in Afghanistan. This may have implications for empiric antibiotic coverage during these months.

ROM1 is redundant to PRPH2 as a molecular building block of photoreceptor disc rims.

Visual signal transduction takes place within a stack of flattened membranous "discs" enclosed within the light-sensitive photoreceptor outer segment. The highly curved rims of these discs, formed in the process of disc enclosure, are fortified by large hetero-oligomeric complexes of two homologous tetraspanin proteins, PRPH2 (a.k.a. peripherin-2 or rds) and ROM1. While mutations in PRPH2 affect the formation of disc rims, the role of ROM1 remains poorly understood. In this study, we found that the knockout of ROM1 causes a compensatory increase in the disc content of PRPH2. Despite this increase, discs of ROM1 knockout mice displayed a delay in disc enclosure associated with a large diameter and lack of incisures in mature discs. Strikingly, further increasing the level of PRPH2 rescued these morphological defects. We next showed that disc rims are still formed in a knockin mouse in which the tetraspanin body of PRPH2 was replaced with that of ROM1. Together, these results demonstrate that, despite its contribution to the formation of disc rims, ROM1 can be replaced by an excess of PRPH2 for timely enclosure of newly forming discs and establishing normal outer segment structure.

Absolute Quantification of Photoreceptor Outer Segment Proteins.

Photoreceptor cells generate neuronal signals in response to capturing light. This process, called phototransduction, takes place in a highly specialized outer segment organelle. There are significant discrepancies in the reported amounts of many proteins supporting this process, particularly those of low abundance, which limits our understanding of their molecular organization and function. In this study, we used quantitative mass spectrometry to simultaneously determine the abundances of 20 key structural and functional proteins residing in mouse rod outer segments. We computed the absolute number of molecules of each protein residing within an individual outer segment and the molar ratio among all 20 proteins. The molar ratios of proteins comprising three well-characterized constitutive complexes in outer segments differed from the established subunit stoichiometries of these complexes by less than 7%, highlighting the exceptional precision of our quantification. Overall, this study resolves multiple existing discrepancies regarding the outer segment abundances of these proteins, thereby advancing our understanding of how the phototransduction pathway functions as a single, well-coordinated molecular ensemble.

Photoreceptor disc incisures form as an adaptive mechanism ensuring the completion of disc enclosure.

The first steps of vision take place within a stack of tightly packed disc-shaped membranes, or 'discs', located in the outer segment compartment of photoreceptor cells. In rod photoreceptors, discs are enclosed inside the outer segment and contain deep indentations in their rims called 'incisures'. The presence of incisures has been documented in a variety of species, yet their role remains elusive. In this study, we combined traditional electron microscopy with three-dimensional electron tomography to demonstrate that incisures are formed only after discs become completely enclosed. We also observed that, at the earliest stage of their formation, discs are not round as typically depicted but rather are highly irregular in shape and resemble expanding lamellipodia. Using genetically manipulated mice and frogs and measuring outer segment protein abundances by quantitative mass spectrometry, we further found that incisure size is determined by the molar ratio between peripherin-2, a disc rim protein critical for the process of disc enclosure, and rhodopsin, the major structural component of disc membranes. While a high perpherin-2 to rhodopsin ratio causes an increase in incisure size and structural complexity, a low ratio precludes incisure formation. Based on these data, we propose a model whereby normal rods express a modest excess of peripherin-2 over the amount required for complete disc enclosure in order to ensure that this important step of disc formation is accomplished. Once the disc is enclosed, the excess peripherin-2 incorporates into the rim to form an incisure.

Wildfires and Older Adults: A Scoping Review of Impacts, Risks, and Interventions.

Climate change is leading to worsening disasters that disproportionately impact older adults. While research has begun to measure disparities, there is a gap in examining wildfire-specific disasters. To address this gap, this scoping review analyzed literature to explore the nexus of wildfires and older adults. We searched peer-reviewed literature using the following inclusion criteria: (1) published in a peer-reviewed journal; (2) available in English; (3) examines at least one topic related to wildfires; and (4) examines how criterion three relates to older adults in at least one way. Authors screened 261 titles and abstracts and 138 were reviewed in full, with 75 articles meeting inclusion criteria. Findings heavily focused on health impacts of wildfires on older adults, particularly of smoke exposure and air quality. While many articles mentioned a need for community-engaged responses that incorporate the needs of older adults, few addressed firsthand experiences of older adults. Other common topics included problems with evacuation, general health impacts, and Indigenous elders' fire knowledge. Further research is needed at the nexus of wildfires and older adults to highlight both vulnerabilities and needs as well as the unique experience and knowledge of older adults to inform wildfire response strategies and tactics.

Photoreceptor disc incisures form as an adaptive mechanism ensuring the completion of disc enclosure.

The first steps of vision take place within a stack of tightly packed disc-shaped membranes, or "discs", located in the outer segment compartment of photoreceptor cells. In rod photoreceptors, discs are enclosed inside the outer segment and contain deep indentations in their rims called "incisures". The presence of incisures has been documented in a variety of species, yet their role remains elusive. In this study, we combined traditional electron microscopy with three-dimensional electron tomography to demonstrate that incisures are formed only after discs become completely enclosed. We also observed that, at the earliest stage of their formation, discs are not round as typically depicted but rather are highly irregular in shape and resemble expanding lamellipodia. Using genetically manipulated mice and frogs and measuring outer segment protein abundances by quantitative mass spectrometry, we further found that incisure size is determined by the molar ratio between peripherin-2, a disc rim protein critical for the process of disc enclosure, and rhodopsin, the major structural component of disc membranes. While a high perpherin-2 to rhodopsin ratio causes an increase in incisure size and structural complexity, a low ratio precludes incisure formation. Based on these data, we propose a model whereby normal rods express a modest excess of peripherin-2 over the amount required for complete disc enclosure in order to ensure that this important step of disc formation is accomplished. Once the disc is enclosed, the excess peripherin-2 incorporates into the rim to form an incisure.