In this study, X-ray Photoelectron Spectroscopy (XPS) is used as a fast technique to characterize the composition of nanosized small extracellular vesicles (EVs), with the aim of evaluating its possible application in cancer screening and diagnosis. This spectroscopic technique is an ideal approach to characterize membranes due to its sensitivity and its penetration depth, in the nanometric range, meaning that the results will mainly correspond to the membrane composition, with little interference from the biomolecules in the EV´s lumen. We propose the use of XPS to analyze the membranes of small extracellular vesicles isolated from bodily fluids of cancer patients and compared them to those of healthy cells and donors. Notably, we have focused our analysis on the N chemical environment of EV membranes, as this can be related to previous complex chemical analyses of lipid and protein contents in EVs. We provide preliminary results on a fast and non-invasive novel strategy, as a method or tool for research-based EV investigations with potential application in cancer screening and monitoring. Small extracellular vesicle (EV) membranes display characteristic protein-lipidic composition features that are related to their cell of origin, providing valuable clues regarding their parental cell composition and real-time state. This could be especially interesting in the case of cancer cell-derived EVs, as their membranes could serve as valuable tools in liquid biopsy applications and to detect changes in the tumor malignancy. X-Ray Photoelectron Spectroscopy (XPS) is a powerful surface analysis technique able to detect every chemical element present, being also sensitive to their chemical environment. Here we explore the use of XPS as a fast technique to characterize EV membrane composition, with possible application in cancer research. Notably, we have focused on the nitrogen environment as an indicator of the relative abundance of pyridine-type bonding, primary, secondary and tertiary amines. Specifically, we have analyzed how tumoral and healthy cells have different nitrogen chemical environments that can indicate the presence or absence of malignancy. In addition, a collection of human serum samples from cancer patients and healthy donors was also analyzed. The differential XPS analysis of EVs collected from patients confirmed that the patterns of amine evolution could be related to markers of cancer disease, opening the possibility of their use as a non-invasive blood biomarker. Exosomes and extracellular vesicles (EVs) are membrane-based nanovesicles, that have been intensely investigated as excellent candidates for the diagnosis and the monitoring of several pathologies (such as cancer) due to their availability and established presence in most biological fluids . These eIt has been claimed that the membrane of cancer-derived EVs contains a fingerprint that on the one hand is responsible for their selectivity in targeting cells , and on In view of the above, a strong interest exists in the development of alternative methodologies analyzing EVs as biomarkers of pathological processes. X-Ray Photoelectron Spectroscopy (XPS) is a high sensitivity surface analysis technique able to detect the relative abundance of each single element and also to give information regarding their chemical environment. A highly advantageous feature is the fact that the effective penetration of XPS for analysis is determined by the element being detected, the matrix or the orbital, and is limited to around 10 nanometers , meaningWe have explored the use of XPS as a fast research tool to characterize the composition of EVs, with the objective of assessing its possible application in cancer screening. We propose the use of XPS to analyze the membranes of EVs isolated from bodily fluids of target patients. Notably we have focused our analysis on the N chemical environment of EV membranes, as this can be related to previous complex chemical analyses of lipid contents in exosomes . Specifi2-humidified atmosphere under hypoxic conditions (3% O2). For culturing B16-F1, B16-F10, and NIH-3T3 cells, DMEM with 10% of FBS (GIBCO), supplemented with 1% penicillin/streptomycin and 1% amphotericin  was used. Finally, monocytes were cultured in RMPI Medium 1640  supplemented with 10% FBS (GIBCO), 1% penicillin/streptomycin and 1% amphotericin . B16-F1, B16-F10 and NIH-3T3 cells were maintained under normoxic conditions. Human placental mesenchymal stem cells (hpMSCs) were obtained from Cellular Engineering Technologies (CET) , while B16-F1 (low metastatic variant) and B16-F10 (high metastatic variant) murine skin melanoma cells, were provided by cell services from Cancer Research-UK. NIH-3T3 murine healthy fibroblasts were obtained from Dr. Antonio de la Vieja’s group . hpMSCs were cultured in Dulbecco’s modified Eagle’s medium  supplemented with 5 μg mL−1 of FGF-2 growth factor , with 10% of fetal bovine serum , 1% penicillin/streptomycin and 1% amphotericin  and maintained at 37 °C in a 5% COg for 8 h at 4 °C. To obtain culture media free of EVs, they were depleted from serum by ultracentrifugation at 100,000× Comité de Ética de la Investigación de la Comunidad de Aragón, CEICA), and was conducted in compliance with the ethical standards formulated in the Helsinki Declaration of 1996 (revised in 2000), upon obtaining the informed consent from all patients. Human serum samples and data from patients included in this study were provided by the Biobank of the Aragon Health System, integrated in the Spanish National Biobanks Network (PT20/00112), and they were processed following standard operating procedures with the appropriate approval of the Ethics and Scientific Committees. The present study (PI18/198) was approved by the human research review committee of the Research Ethics Committee of the Community of Aragon (n = 10) and from stage III-IV pancreatic cancer patients (n = 10) using the first sample at diagnosis and two sub-sequential samples taken during disease progression. Ovarian cancer patients’ blood samples (n = 16) were obtained at diagnosis, including from patients in different stages . Ovarian EV samples were divided for analysis according to the different stages, considering the possible variations of EVs depending on the disease stage. Samples were collected in vacutainer sodium citrate tubes in Hospital Universitario Miguel Servet, Zaragoza (Spain). Samples were stored at −80 °C until use. Then, samples were defrosted and 500 µL of serum was employed for isolation of the EVs and further XPS characterization. Blood markers, CEA and CA 19:9, were quantified by the National Health System, following standard procedures. The pancreatic samples used here for analysis of atomic composition and nitrogen environment are samples extracted at diagnosis, meaning, samples extracted before starting any treatment. Additionally, we have studied the evolution of secondary amines  in relation to blood markers, CEA and CA 19:9. In this case, our purpose was to analyze if the variation of secondary amines occurs with the same trend as the evolution of blood markers, which are also modified depending on treatment. In this case, every patient has been studied independently, because we cannot compare secondary amine evolution during time among patients due to the differences in treatments and disease progression of each patient.Human serum samples were obtained from healthy donors (g and at 4 °C (to remove remaining debris). For the elimination of the microvesicles, another centrifugation step was carried out for 1 h at 10,000× g and at 4 °C. To obtain the exosome fraction, the samples were ultracentrifuged for 2 h at 100,000× g and at 4 °C. A further washing step of exosomes with PBS was carried out to discard the co-isolated and adsorbed proteins on exosomes . The exosomes pellet was finally resuspended in PBS for further analysis [EVs from cell culture samples were isolated following a protocol previously published and based on successive ultracentrifugation cycles from cell culture supernatants of the cells . Brieflyanalysis .n = 56) were divided into control samples (n = 10 samples/patients), ovarian tumor samples (n = 16 samples/patients) and pancreatic tumor samples . EVs from serum samples (n = 56) were extracted using Plasma/Serum Exosome Purification Mini Kit, 50 Preps . Following manufacturer instructions, the Plasma/Serum Exosome Purification Kit provides a reliable and convenient method to purify and enrich pure intact EVs factions from different plasma/serum samples, free from any cell-free circulating protein-bound RNA. Purification was performed according to the producer protocol. In brief, to 500 µL serum aliquots from each donor were added, in the following order, 3.5 mL nuclease-free water, 100 µL f ExoC buffer and 200 µL of Slurry E (resin). The mixture was mixed by vortexing and let stand at room temperature for 5 min, to allow the EVs to adhere to the resin. After incubation, the mixtures were centrifugated to recover the resin, the supernatant was discarded and the resin resuspended in 200 µL ExoR buffer; incubation was performed at room temperature for 5 min to release EVs from resin. After incubation, the mixture was centrifugated for 2 min at 500 rpm and the supernatant (containing free EVs) was recovered and filtered by centrifugation through a Mini Filter Spin column (1 min at 6000 rpm) to discard possible remaining resin (retained on the filter). After centrifugation, the EVs were ready for downstream applications. As negative control for following analysis, we used the same protocol starting with 4 mL nuclease-free water and the supernatant from the last step was analysed in the same way as the EVs from serum.Ultracentrifugation is currently the gold standard for exosome isolation; thus far, some in vitro separation methods, such as ultracentrifugation, polymer-based exosome separation kits and immune affinity-based isolation using antibodies against exosome surface proteins, have been used for tumor exosome isolation . We have®” objective lens allowing a point-to-point resolution of 2.4 A [The EVs’ morphology and the thickness of the EV membrane were characterized by transmission electron microscopy (T20-FEI Tecnai thermoionic transmission electron microscope) operated at 200 kV with a LaB6 electron source fitted with a “SuperTwinof 2.4 A . EVs werEV pellets isolated from cell cultures were finally resuspended in PBS and deposited drop by drop on a circular cover glass slip on a hotplate set at 30 °C. EVs from serum human samples and healthy controls were recovered in 200 µL ExoR buffer final volume. The resuspension was dried drop by drop (5 µL) on a circular cover glass slip on a hotplate set at 30 °C. The XPS analysis of all samples was performed 24 h after sample desiccation with an Axis Supra  apparatus to determine the atomic percentage and the relative abundance of the different nitrogen species analyzed . The spectra were excited by the monochromatized Al Kα source (1486.6 eV) run at 10 kV and 8 mA. For the individual peak regions, a pass energy of 20 eV was employed. Survey spectra were measured at 120 eV pass energy. The CasaXPS software was used to analyze the peaks. After the subtraction of Shirley background, a weighted sum of Lorentzian and Gaussian components curve was employed.t-test for binary comparisons. Analysis was performed using GraphPad Software. Statistically significant differences were indicated as follows: * p < 0.05; ** p < 0.01; *** p < 0.0001 and **** p < 0.00001. The results are expressed as mean ± SD (standard deviation). Statistical analysis of the data and the significant differences among the means were evaluated by two-way analysis of variance (ANOVA) and unpaired Student’s 3PW12O40 particles are adsorbed in EVs surfaces  .The above results seem to indicate that there are no intrinsic differences among EVs from healthy and cancer cell lines. In view of this, further analyses were focused on the N environment  since ouEXOS and NIH-3T3EXOS) and from cancer cell lines (B16-F1EXOS and B16-F10EXOS). Our results demonstrate that EVs from tumoral and healthy cells have different nitrogen chemical environments and that the N fingerprint can also be different among EVs from the same tumoral cell line depending on their malignancy degree. The results in EVs showed a nitrogen chemical environmental similar to stem cell derived EVs (hpMSCsEVs). This would be consistent with the known fact that aggressive cancer cells exhibit a phenotype similar to stem cells in terms of migration, proliferation and differentiation mechanisms. In this way, stem cells would be set apart from other healthy cells and this is also reflected in their nitrogen environment. Finally, pyridine-type nitrogen does not follow a clear trend and presents low but similar values for all cell types.XPS analysis allows investigation of atomic environments in a fast and accurate way, since different N-containing functional groups present themselves with different energies in the spectrum. It is obvious that EVs coming from tumoral cells only represent a small fraction of the EVs found in the blood of cancer cell patients. Since the methods available will isolate EVs released by all kind of cells from the body, a mixture of EVs from both cancer and healthy cells can be expected, with only a small fraction of EVs originating from tumoral cells. The dilution of cancer cell EVs by EVs from other origins represents the main difficulty when trying to detect their differential characteristics.However, it seems possible that in cancer patients the production of tumoral EVs may increase depending on the stage of the disease. For this reason, we studied not only the percentage of nitrogen in different chemical environments but also the percentage of oxygen, nitrogen, carbon, phosphorus and the phosphorus/nitrogen ratio in EVs isolated from the serum of healthy donors and patients suffering from different grades of ovarian or pancreatic cancer. n = 6), excluding male results, due to the possible influence of hormones in serum EV composition.The histopathological grade of ovarian epithelial carcinoma has generally been found to be of prognostic significance. The grading systems for epithelial ovarian carcinoma used most commonly have been those of the International Federation of Gynecology and Obstetrics (FIGO) and of the World Health Organization (WHO). The FIGO system is based on architectural features, and the grade depends on the ratio of glandular or papillary structures to solid tumor growth within an individual tumor; it has been related to molecular characteristics and clinical outcomes . The patAs expected from our previous results , carbon Encouraged by the in vitro results showing interesting differences in the nitrogen environment for tumoral and healthy cells , we carrPancreatic tumors were classified according the American Joint Committee on Cancer (AJCC) classification , which iTo compare the content of different nitrogen compounds in EV membranes, we selected the first isolated sample of every patient, to avoid the influence due to treatments. Regarding elemental analysis, again carbon and oxygen were the most abundant elements followed by nitrogen and then phosphorus present in all the samples at around a 1% level. There were no significant differences in the relative abundance of these four elements between EVs isolated from healthy donors and pancreatic cancer patients . 2-NH/N-C=O) where a significant increase was observed compared to healthy donor samples (3-N) amines was reduced on average by 40% in EVs isolated from pancreatic cancer patients compared to healthy donors  with time during the patient treatment time, and compared them to the evolution of clinical blood markers, CEA and CA 19:9 . AlthougComparative analysis of the secondary amines and both markers shows the same tendency for one or both markers and secondary amines in most patients. In summary, CEA was quantified in 9 out of 10 patients . In 6 ouCA 19:9 values were quantified in 10 patients, and 4 out of 10 patients (40%) showed the same relative trends for secondary amines and CA 19:9 during the disease progression. Although CEA and CA 19:9 are commonly used for the clinical diagnostic and prognostic analysis of pancreatic cancer, only 5 out of 9 (55%) cases showed the same tendency between the two of them. We can conclude that the correlation between CEA and secondary amines is higher than the correlation between the established clinical markers CEA and CA 19:9 (67% vs. 55%), at least for the cases studied here.The correlation between secondary amines and CEA has been shown to be more robust than between secondary amines and CA 19:9 (67% vs. 40%). Therefore, it seems reasonable to conclude that secondary amines evolution during pancreatic cancer disease correlates well with one of the biomarkers (CEA) and could be further investigated as a new blood biomarker with a higher number of samples.The cell membrane is composed of lipids and proteins. The lipid fraction of the cell membrane consists mainly of phospholipids, glycolipids, sphingolipids and cholesterol . The mosEVs are limited by a lipidic membrane, which encapsulates the cargo molecules in an inner aqueous core . Lipid, We have recently proposed a robust analytical procedure to isolate EVs and obtain a wide-ranging and reliable analysis of the phospholipid (PL) content of their membranes . Using tThese studies rely on the use of mass spectrometry protocols to analyze the lipidomic profile of EVs. While a powerful tool, these methods are rightly considered to constitute a complex technique, which requires the analysis of a complete lipidomic profile, distinguishing among the different lipids present in the EV membranes. The same could be said of our previous work involving EV isolation and analysis of the lipid EV extract by High-Performance Thin-Layer Chromatography-densitometry directly coupled to mass spectrometry, again a relatively complex procedure .Instead, here we propose the characterization of the nitrogen environment using a single technique (XPS) and a relatively standard measurement. We have focused our analysis on nitrogen as this single element can be present in a variety of environments that can be easily analyzed by XPS, and their relative abundances can be used to characterize the evolution of the EV membrane composition. As already mentioned, XPS is ideal not only as a fast and reliable technique to identify different chemical environments of nitrogen atoms based on their binding energies, but also because being a surface technique that only responds to signals from the uppermost atomic layers, it has obvious advantages when one wants to analyze the membrane composition with minimal interference from the inner content of the EVs. The atomic composition of EV membranes isolated from cell cultures by ultracentrifugation  did not To further study this trend, EVs were isolated from human serum from healthy donors and from ovarian and pancreatic cancer patients . Again, Different studies have shown significant differences in the lipid profile of EVs from healthy and cancer-suffering persons ,10,11,122-NH/N-C=01) and also a trend towards the decrease of pyridine-type bonding, although without statistical significance  and normal cell lines  were dysregulated between cancer-derived sEVs and normal sEVs, especially lipid species containing palmitic acid 16:0) and sphingomyelin . In a ge:0 and spificance . As withThe fact that these general alterations of the nitrogen environment can be ascertained from EVs sampled from the blood of healthy and cancer patients is in itself remarkable, because cancer cell-originated EVs are necessarily diluted with EVs from every other kind of cell shedding EVs to the bloodstream and also because the EV isolation method based on columns is far from perfect and the EVs collected are likely to be contaminated with foreign nitrogen-containing species, such as plasma proteins. Finally, the analysis of the nitrogen environment was also useful to analyze pancreatic cancer evolution. This is an area of great interest and in fact there is not a universally accepted marker to characterize prognosis and response to treatment. We have compared the XPS results regarding the content of secondary amines with two blood markers, CA19:9 and CEA. Our results show that 67% of analyzed patients showed the same evolution of values for secondary amines and CEA and 40% showed the same evolution of values for secondary amines and CA 19:9 during the disease progression. The agreement found with CEA is better than the agreement between those established markers, since only 55% of patients showed a tendency for accordance between CEA and CA 19:9. We can therefore conclude from these preliminary results that the analysis of secondary amines’ evolution during pancreatic cancer disease has potential as a new PDAC biomarker and should be further investigated with a larger number of individuals.In summary, analyzing by XPS the nitrogen environment of EVs isolated from the blood of two types of cancer patients reveals variations of pyridine-type bonding and primary amines in ovarian cancer, and secondary amines in pancreatic cancer, which can be related with significant changes in the lipidomic profile of their membranes, and provides analytical parameters that allow discrimination between cancer patients and healthy individuals. Furthermore, the analysis of secondary amines has shown potential as a marker of cancer evolution in pancreatic cancer. This type of measurement is attractive because it is relatively simple and could be used as a new, fast and non-invasive diagnostic tool. However, the differences found, while statistically significant in at least one nitrogen environmental parameter, do vary among the different types of cancer. It is known that exosome composition is dependent of cell origin ,37; for Another important point to discuss is the possibility of contaminants coming from other cell types, including EVs from other strains. The tumor EVs analyzed here are collected from the blood together with EVs from immune and other cells. Indeed, we isolate the total pool of EVs from the sample, including those from cancer cells and they will be mixed with the rest. This is a comparative study where all the samples were processed equally and the presence of EVs from non-cancer cells will happen in all the samples, adding to the “background noise” and reducing the statistical significance of the results. Fortunately, significant differences between healthy and cancer patient samples were shown, meaning that in tumoral samples the pool would have been highly enriched with tumoral EVs, leading to sufficient differences to achieve statistical significance between samples. Further work is needed to validate the results with a wider set of clinical samples and also to identify, for each type of cancer, the relevant analytical parameters that could be used in an EV-based liquid biopsy scenario.


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. In the vertebrate retina, photoreceptor cells perform the first step of vision by capturing light and generating a neuronal signal. This process, called phototransduction, occurs within a stack of disc-shaped membranes, or ‘discs’, confined within the ciliary outer segment  tomography, which allows for the imaging of thicker sections than other electron tomography approaches.z-section from this tomogram is shown in Using STEM tomography, we imaged ~750 nm plastic sections of mouse retinas cut tangentially at the region containing the interface between photoreceptor inner and outer segments. A representative example of a 3D reconstruction of such a section imaged at low magnification is shown in First and consistent with TEM data, no incisures were observed in the newly forming discs. Second, the newly forming discs are surprisingly lamellipodia-like in shape. While traditionally illustrated as round evaginations of the ciliary plasma membrane  form the disc rim. Together, rhodopsin and peripherin-2/ROM1 comprise ~99% of the total transmembrane protein material in normal discs . Therefords mice rds (rds/+) mice despite a reduction in the level of peripherin-2  mice, in which the molar fraction of peripherin-2 in discs is increased due to a reduction in rhodopsin expression. +/-Rho outer segments were previously shown to lack major morphological abnormalities apart from a reduced disc diameter ( +/-), increased incisure length -/-Rho) mice  mice  thought /-) mice . They ar/-) mice . TherefoTo further explore the idea that the sizes of discs and incisures are defined by the molar ratio between rhodopsin and peripherin-2, we measured this ratio in outer segments obtained from mice of all three genotypes. We also analyzed the levels of ROM1 because it is another tetraspanin contributing to disc rim formation by oligomerizing with peripherin-2. We used a quantitative mass spectrometry approach that we have recently applied to determine the molar ratio amongst multiple outer segment proteins, including these three proteins . Our meards/+ outer segments, the rhodopsin to peripherin-2 ratio was increased by ~1.7 fold (~31:1), whereas in +/-Rho outer segments it was decreased by +/-2 fold (~9:1). Both changes are consistent with these mouse lines expressing single copies of the corresponding gene , suggesting that more ROM1 is incorporated into disc rims when there is a deficiency in peripherin-2. Accordingly, the molar ratio between peripherin-2 and ROM1 shifted from ~2.3:1 in WT rods to ~1:1 in rds/+ rods. In contrast, the molar ratio between peripherin-2 and ROM1 was essentially unaffected in +/-Rho +/- (~2.6:1 vs. ~2.3:1 in WT rods), indicating that rhodopsin deficiency does not disrupt the balance between these two tetraspanins.In WT outer segments, the molar ratios of rhodopsin to peripherin-2 and ROM1 were ~18:1 and ~42:1, respectively, as in see also . Considerds/+ rods and~6:1 in +/-Rho +/-. These measurements are consistent with our qualitative conclusions from the ultrastructural analysis of these mice and suggest that these conclusions apply to both ratios of rhodopsin to peripherin-2 and the total tetraspanin content.Overall, the molar ratio between rhodopsin and total tetraspanin protein (peripherin-2 and ROM1 combined) changed from ~13:1 in WT rods to ~15:1 in +/-Rho +/- whose entire outer segment population is devoid of major morphological defects, we sought to determine whether the measured increase in relative tetraspanin abundance could explain the observed increase in the size and complexity of incisures. We calculated the theoretical incisure length in +/-Rho +/- based on the assumptions that the disc surface area is proportional to the number of rhodopsin molecules, whereas the entire length of the disc rim (including both the circumference and incisure) is proportional to the number of tetraspanin molecules. Unfortunately, rds/+ mice could not be analyzed this way because the majority of their outer segments did not produce proper discs  Xenopus tropicalis frog using CRISPR-Cas9 to create mutations within exon 1 of prph2 . Ultrastructural analysis of their retinas revealed a relatively normal outer segment morphology, except for occasional overgrowth of disc membranes sometimes shaped as whorls  is the frog peripherin-2 homolog (XenBase: XB-GENEPAGE-985593) while xrds36 and xrds35 are isoforms of ROM1 (XenBase: XB-GENEPAGE-962405). In addition, these genomes contain another peripherin-2-like gene, termed prph2l (XenBase: XB-GENEPAGE-5759091). Therefore, it is plausible that peripherin-2-like protein and/or ROM1 isoforms are sufficient or even play a primary role in supporting outer segment morphogenesis in frog photoreceptors.The formation of outer segments in these frogs was surprising considering the complete lack of outer segments in peripherin-2 knockout mice. This cross-species difference could be explained by d xrds35 . The cur-/-prph2 frogs displayed a nearly complete lack of incisures  due to an increase in ROM1 content. It is intriguing to speculate that the gross morphological defects of rds/+ discs may not be caused by this modest reduction in total tetraspanin content, but rather by the nearly 2-fold reduction in the relative peripherin-2 content. This would argue that ROM1 is unable to efficiently compensate for a deficiency in peripherin-2. Consistent with this line of thought, -/-Rom1 outer segments have relatively minor morphological defects  were C57BL/6 J (Jackson Labs stock #000664). rds mice are described in -/-Rho mice are described in rd8  are described below. Mice were used at 1 month of age. Frogs were used at either 7- or 14 days post fertilization. All experiments were performed with animals of randomized sex and, for each experiment, at least three biological replicates were analyzed.Animal maintenance and experiments were approved by the Institutional Animal Care and Use Committees at Duke  and the Marine Biological Laboratory . WT mice (d in rd8  or rd1 (rd8 (rd1  mutationprph2 knockout line (RRID:NXR_3003) was generated using CRISPR-Cas9. CRISPRScan prph2  with 5’ dinucleotides converted to GG for increased mutagenic activity . Each embryo was injected with 500 pg each of sgRNA1/sgRNA3 and 1000 pg Cas9. Founders were raised to sexual maturity and screened for germline transmission of mutations. A –20 bp mutation, which results in a frameshift mutation at amino acid 183 and a stop codon 7 amino acids downstream, was selected for generating the prph2 line. Intercrosses were done on –20 bp heterozygotes to generate prph2 mutants.The activity . sgRNAs For mice, tissue fixation was performed as described previously . In the For processing of longitudinal sections of mouse retinas, eyecups were dissected from fixed eyes, embedded in 2.5% low-melt agarose  and cut into 200-µm-thick slices on a Vibratome . Agarose sections were treated with 1% tannic acid  and 1% uranyl acetate (Electron Microscopy Sciences), gradually dehydrated with ethanol and infiltrated and embedded in Spurr’s resin (Electron Microscopy Sciences). For processing of tangential sections of mouse retinas, dissected retinas were treated with 1% tannic acid (Electron Microscopy Sciences) and 1% uranyl acetate (Electron Microscopy Sciences), gradually dehydrated with ethanol and infiltrated and embedded in Spurr’s resin (Electron Microscopy Sciences). For processing of frog samples, tadpoles were treated with 1% tannic acid (Electron Microscopy Sciences) and 1% uranyl acetate (Electron Microscopy Sciences), gradually dehydrated with ethanol and infiltrated and embedded in Spurr’s resin (Electron Microscopy Sciences).A total of 70 nm sections were cut from resin-embedded samples, placed on copper grids and counterstained with 2% uranyl acetate and 3.5% lead citrate . Samples were imaged on a JEM-1400 electron microscope  at 60 kV with a digital camera . Image analysis and processing was performed with ImageJ.Of the central retina, 750 nm sections were cut from resin-embedded samples and placed on 50 nm Luxel film slot grids. The grids were glow-discharged on both sides, and a mixture of 10 nm, 20 nm, and 60 nm gold particles were deposited on the sample surfaces to serve as fiducial markers. Electron tomography was conducted on a Titan Halo  operating at 300 kV in STEM mode. A 4-tilt series data acquisition scheme previously described  was foll2, 1.2 mM CaCl2, and 10 mM HEPES, pH 7.4) that was adjusted to 314 mOsm. The preparation was briefly left on ice to allow the remaining retinal tissue to settle. The supernatant was removed and centrifuged at 20,000 x g. Pelleted outer segments were gently washed with mouse Ringer’s solution before lysis with 2% SDS in PBS. Protein concentration was measured with the Bio-Rad Protein Assay kit . Samples containing 20 µg of protein were mixed with 0.5 µg BSA  and cleaved with 1 µg trypsin/LysC mix  using the SP3 beads protocol described in A crude preparation of rod outer segments was obtained from dissected mouse retinas that had been vortexed in 8% OptiPrep in mouse Ringer’s solution  is available at the National Xenopus Resource.The  This study describes the importance of the stoichiometric relationship between rhodopsin and peripherin-2 in mouse rod outer segments. The authors use genetic manipulations to vary the ratio of these two proteins to demonstrate that excess peripherin leads to excess perimeter, which then leads to the infolded structures known as incisures. These data illustrate a fundamental principle that relates factors that control the area of a structure to factors that control the perimeter of a structure. In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Review Commons.][Editors' note: this paper was reviewed by  General Statements [optional]We are very pleased that our manuscript was well-received by each of the three reviewers. Reviewer #1 found our study to be “interesting”, “clear” and “convincing”. Reviewer #2 found our study to be “unprecedented” and of “considerable interest to cell biologists in the vision community and likely to the broader cell biology community”. Reviewer #3 believed that we reported “novel and significant findings into an important cell biological problem” and that our study “should be of broad interest to cell biologists and vision scientists”. This reviewer also stated that “it is strongly recommended for publication”.Point-by-point description of the revisionsReviewer #1:The paper of Lewis et al. presents an interesting study describing new information about the morphology of nascent discs and the role of peripherin in determining disc and incisure structure. I have only a few comments mostly about presentation.We are happy that the reviewer liked our study.1. Because this study employs both frog and mouse, the authors should be careful to give the species when describing their results. The naming of the species would be particularly important in the first paragraph of the Results section and the legend to the first data figure, Figure 2.We have clarified the text and figure legends to allow the reader to better follow which species each result came from.2. It is unclear what Movie 1 adds to Figure 3. This movie could perhaps be omitted.We prefer to include the source data that the image in Figure 3 is derived from, particularly to stress that the pattern shown in a single z-section in this figure can be seen throughout the entire tomogram. We hope that the reviewer would agree.3. Movies 3 and 5 either don't work or consist of single frames, which would be better illustrated as figures in the text rather than as supplementary movies.We appreciate the reviewer catching that there were some technical issues with video playback. We have recompressed these videos and ensured that they will now play appropriately across a wide variety of computer specifications and video player applications.4. The incisures in Figure 4 will be difficult for many readers to visualize. My experience was that once I saw one of them, I began to see the others. The incisures in Figure 5 are, on the other hand, very easy to see. If Figure 5 had come before Figure 4, I would have had no problem. The authors may wish to exchange these two figures or to supply a cartoon for one of the rods in Figure 4, so that the reader can more easily understand what he or she should be trying to see.We thank the reviewer for pointing out that some readers may have difficulties in fully appreciating the structure of incisures in this figure. We made two changes to improve the presentation of these images. First, we pseudo-colored several examples of enclosed discs in Figure 3, which highlights the structure of incisures. We also indicated one example of an incisure in these images with an arrowhead. Second, we pseudo-colored the example shown in Figure 4A to illustrate the same point, while still allowing the reader to view the three remaining examples in Figure 4 without any overlaid modifications.5. It is unclear to me why the authors are so fond of their untested theory that incisures "likely serve to protect the flat lamellar disc membranes from undesirable deformations" but seem skeptical of the notion that incisures are present and especially numerous in rods of large diameter to aid longitudinal diffusion. The later notion is supported not only by theoretical calculations but also by common sense.We appreciate this comment and, in fact, feel agnostic about both of these not mutually exclusive ideas. We removed the statement that the deposition of peripherin-2 in incisures likely serves to protect the flat lamellar disc membranes from undesirable deformations from the Introduction and rephrased the text in Discussion to stress that both functions are plausible and not mutually exclusive.This manuscript presents a clear and convincing description of disc formation and the role of the protein peripherin in the formation of disc incisures.Thank you for your kind comment.Reviewer #2:Summary: The manuscript by Lewis et al. focuses on the potential mechanisms underlying formation of incisures in rod photoreceptors. Incisures refer to the indentations that occur on the rim of the photoreceptor disc membranes. The presence of incisures has been noted for decades and have been identified across a number of species. The role of incisures is not entirely clear and the mechanisms governing their formation have largely been inferred from early transmission electron microscopy studies 40-60 years ago. More recent ultrastructural studies of rod outer segment discs from mice carrying mutant alleles of rhodopsin or periperhin-2 described changes in the length or presence of incisures, suggesting that these proteins likely play a fundamental role in incisure formation in mouse. The authors take advantage of advances in electron tomography to provide unprecedented analyses of incisure formation, size, and structural complexity in stacked discs within mouse photoreceptors. They also use genetic models to explore how rhodopsin and peripherin-2 contribute to incisure formation and length. The authors find that new discs are highly irregular in shape and do not contain incisures during disc formation. Incisures are only formed are discs are enclosed. They find that the incisures in adjacent discs always align adjacent to the ciliary axoneme. Intriguingly, they find evidence of physical connections on opposing sides of the incisure. Critically, they find that elevated levels of peripherin-2 increase incisure size and complexity while low levels of peripherin-2 prevent incisure formation. In contrast, reduced molar ratios of rhodopsin lead to smaller disc surface area but increased incisure complexity. These results lead the authors to conclude that incisure formation is mechanistically linked to the relative molar ratio of peripherin-2 to rhodopsin and that rods make a slight excess of peripherin-2 in order to drive proper disc closure. The excess peripherin-2 within the disc rim forces formation of an incisure.We are happy that the reviewer liked our study.Major comments1. Line 145-146: the location of the incisure adjacent to the ciliary axoneme is an interesting observation indeed. As frogs have a number of incisures, is this a similar observation in species with multiple incisures or more exclusive to those species with a single incisure?Indeed, we did observe that one of the many incisures in a frog disc is aligned with the ciliary axoneme. We have now included Supplementary Figure S2 to highlight this observation using an example of two adjacent cells.2. While the presence of non-axonemal microtubules aligned with incisures in frog rods may provide an explanation for the number of incisures, the correlation with peripherin and rhodopsin content was lacking. In other words, do frog rods have considerably more peripherin-2 per disc than mouse rods?This is a great question and one that we are interested in pursuing in the future. However, adapting the mass spectrometry-based protein quantification approach that we used to determine the absolute numbers of peripherin-2, ROM1 and rhodopsin molecules per disc was a significant undertaking that took several years . This approach is currently applicable to only a particular set of outer segment proteins in the mouse and cannot be automatically re-purposed to quantification of proteins in other species that have different amino acid sequences. Thus, designing, validating and employing this quantification protocol to all peripherin-2 and ROM1 isoforms along with rhodopsin in the frog would be a major undertaking that cannot be completed in the context of this revision. Nonetheless, we are very appreciative for the reviewer’s enthusiasm for this topic and plan to address this question in the future.Minor comments1. The location of the incisures are difficult to see in Figure 4. The arrowhead is pointing to a very low contrast area of the disc and the thin incisure can be seen, but it's difficult. If it is possible to pseudocolor the image in some way to highlight the disc vs the extramembrane space, it would be helpful.We thank the reviewer for noticing this issue, which was also commented by another reviewer. As described above, we pseudo-colored several examples in Figures 3 and 4.2. Line 138-142: As with any descriptive narrative of cell structures, it is important to ensure the reader can fully understand and appreciate the interpretation of the authors. The shape of the newly forming discs can be difficult to appreciate in Figure 4. The authors are strongly encouraged to perhaps take 1-2 examples and provide a drawing or schematic of the image that can be more clearly annotated to assist readers in finding the outline of the discs and incisures.We appreciate this point and pseudo-colored the surfaces of new forming discs in the examples shown in Figure 4A. We feel that pseudo-coloring helps the reader better visualize not only the structure of incisures, but also the irregular shape of the newly forming discs as in this specific example.Overall, the paper is well-written and organized logically. The figures are generally easy to interpret although some additional annotations would help readers identify incisures in some low-contrast images (see comments). The authors utilized state-of-the-art electron tomographic data and mouse genetics to address a fundamental question. This will be of considerable interest to cell biologists in the vision community and likely to the broader cell biology community on how peripheral/rim proteins can shape membrane.It is needless to say that we are very pleased by these comments and that the reviewer found our study to be of considerable interest to the broader cell biology community.Xenopus were limited and only found that a CRISPR knockout of one peripherin-2 ortholog prevented incisure formation. While this result agreed with the general model and how molar ratios of peripherin-2 contribute, the knockout phenotypes are different than that of mouse. Some hypotheses are mentioned to explain this, but none were tested. The authors provide a model that agrees with mouse data, but is this generalizable? The model should permit several predictions for incisure formation beyond that of mouse rods. It would be most helpful to look in a species with multiple incisures and calculate the molar ratios of rhodopsin and peripherin-2. Do Xenopus require significantly more peripherin-2 to form multiple incisures? Alternatively, is it possible for the authors to mine publically available proteomics studies to assess rhodopsin and peripherin-2 content from other species  and correlate to incisure number and/or length? The study overall is interesting and thought-provoking, but the overall impact would be greatly enhanced if additional evidence was provided that their model is broadly generalizable given the variety in incisure number and photoreceptor disc morphology  across species.The authors provide a well-reasoned model for how incisures form in mouse rod photoreceptors: a relative excess of peripherin-2 drives incisure formation. This agrees with their mass-spectrometry data and molar ratios of peripherin-2 and rhodopsin. The main concern and outstanding question is whether these results are specific to mouse photoreceptors? The experiments in Related to a comment above, we are interested in pursuing each of these directions in frogs and other species in future studies, although it is not feasible to accomplish the required body of work in the context of manuscript revision. There are three other photoreceptor tetraspanins homologous to peripherin-2 that remain to be quantified and knocked out in frogs, alone and in combinations (the xrds36 and xrds35 isoforms and the peripherin-2-like protein). Testing the function of each of these in incisure formation would be an endeavor spanning several years of work. Additionally, there are no publicly available proteomic datasets that would contain information allowing an accurate quantification of rhodopsin and these homologous tetraspanins in other species. This question would require us to adapt our protein quantification approach, which would indeed be valuable, but would take significant time to complete.Reviewer #3:Summary. This work explores the formation of incisures in rod outer-segment (OS) disks. The visual pigment rhodopsin is the major lamellar protein in rod OS disks, while peripherin is the major structural protein of the disk rim. The authors used wild-type, Rho+/- and Rds+/- mice to vary the ratio of rhodopsin to peripherin in vivo, and compared these ratios to incisure length and complexity in rod OS.Xenopus tropicalis, an animal containing large OS disks with multiple incisures and three homologs of peripherin. They used gene editing to generate Xenopus tropicalis with a null mutation in the xrds35 gene, which is most like mammalian peripherin. OS disks from xrds35-/- frogs contained no incisures by EM tomography, further supporting their hypothesis.Comments. This study presents several new findings. The authors convincingly show by EM tomography that incisures only form after each OS disk has reached maturity (fully separated from the plasma membrane). This new finding corrects an earlier published observation. Next, they examined disk morphology in Rds+/- heterozygous null-mutant mice and showed that an ~50% reduction in peripherin levels resulted in rod OS disks with no incisures. They performed a similar study on Rho+/- heterozygotes mutants. This time they observed that an ~50% reduction in rhodopsin levels resulted in OS disks with excessively long incisures. MS analysis of rod OS proteins and quantitative analysis of the EM images showed that incisure length varies with the ratio of peripherin to rhodopsin. They further showed that wild-type rods contain a small excess of peripherin over the amount required to form mature disks with normal incisures. Finally, the authors examined the effects of peripherin levels in rods from Thank you for this nice summary of our study.Another protein in the rims of rod OS disks is ABCA4, an ATP-driven flippase that translocates PE conjugated to retinaldehyde from the lumenal to cytoplasmic leaflets of the disk membrane. Retinaldehyde is a toxic photoproduct of rhodopsin bleaching. It has been suggested that the large number of incisures in frog disks is due to the larger diameter of frog versus mouse rod OS, and hence the greater number of rhodopsins per disk. This relationship is thought to ensure sufficient ABCA4 flippase activity to process the larger flux of retinaldehyde released by rhodopsin in these wide disks during light exposure, and possibly to minimize the diffusion distance of retinaldehyde from the disk lamella to the rim. The authors' findings seem in conflict with this explanation. They may wish to comment on this facet of their results.We agree that it is possible for incisures to promote the encounter rate between retinaldehyde and ABCA4 in the disc. We do not find this idea to conflict with any of our interpretations; rather, this may be a complementary function of incisures. However, we failed to find any place in the literature where this hypothesis has been explicitly proposed and feel uneasy to present it as a new idea of our own in discussion. Fortunately, these reviews are public and truly interested readers could appreciate this idea. It is also worth noting that the correlation between incisure number and disc diameter is not perfect. For example, owl monkey discs appear to have a large number of incisures despite having a similar diameter to the mouse .Significance. The manuscript presents novel and significant findings into an important cell biological problem, development of the rod OS. It is clearly written and the data are of high quality. The manuscript should be of broad interest to cell biologists and vision scientists. It is strongly recommended for publication.We are glad that the reviewer found our study to be of broad interest.


To report our experience on the use of antibiotic coated nails (ACN) and cement beads for the management of bone infections.Infected nonunion (INU) cases were classified as: Type I (mild infection with no gap), Type II , Type III . Treatment involved either the insertion of ACN and cast (Type I), insertion of ACN, beads and external fixator (Type II), or Ilizarov methodology (Type III). A subset of 28 open fractures were admitted with severe contamination or delayed presentation with established infection and treated with debridement, ACN insertion, and antibiotic beads placed in soft tissue dead space areas.Results of 133 cases were classified excellent, good, and poor. Type I INU reported 40 excellent and 22 good results. Type II INU reported 28 (39%) excellent, 30 (43%) good, and 13 (18%) poor results. Poor results were due to uncontrolled infection and knee stiffness. Three patients required knee fusion and 1 required amputation. Fracture union was reported in 68 cases. Four of the 28 Gustilo grade III open fractures treated with ACN developed infected nonunion and had poor function caused by stiff knees.An antibiotic impregnated cement nail (ACN) fills the dead space and elutes high concentrations of antibiotics providing some mechanical stability. We recommend the adjunct use of an ACN for the management of INU cases and for use in select cases of Gustilo grade III open fractures. Once biofilm is formed at the infection site, it is extremely difficult to eradicate the infection as the biofilm protects the bacteria (antibiotics do not penetrate the biofilm) thus contributing to the chronicity of infection.Dead space management remains an essential step in the eradication of infection. The dead space created after debridement is usually filled with body fluid which could be a culture media for pathogens. BuchhlozPMMA cement can be customized to different shapes and forms. Currently, PMMA cement spacers loaded with antibiotics are used as follows: in the treatment of joint infection after arthroplasty and in spine surgery as a space filler; in the 2-stage Masquelet technique for the control of infection and induction of the induced membrane; in open fractures as bead pouches (antibiotic beads placed in dead space areas in soft tissue); in the treatment of intramedullary sepsis (antibiotic impregnated cement nails [ACN]); and in the treatment of chronic osteomyelitis cases.The main purpose of this study is to discuss the techniques and major indications for the use of cement spacers in our practice, and to present the clinical results of patients treated with ACN.2The indication for ACN is intramedullary (IM) infection, a well-known complication of treating a fracture with IM nailing, plating, or external fixation. IM infection might also occur after conversion of an external fixator to IM nailing, limb lengthening over a nail, delayed presentation of open fracture with established infection, and hematogenous spread to the canal causing osteomyelitis. Part of the treatment process includes removal of the infected metal implant, reaming and irrigation of the medullary cavity, and the insertion of the ACN rod. The ACN is an ideal space filler offering some mechanical stability with good bony contact and high local release of antibiotics.3Pus from the infected area is sent for culture and sensitivity of organisms. Culture-specific antibiotics are used. A combination of vancomycin and gentamycin or tobramycin is used as a broad-spectrum antibiotic combination to treat both gram-negative and gram-positive organisms. We commonly use 2 to 3 g of vancomycin and 2 to 3 g of gentamycin or tobramycin. The antibiotic rod is prepared using PMMA cement  mixed with 4 to 6 g of antibiotic powder. When a soft dough is formed, it is wrapped around a 6 to 9 millimeter (mm) K-nail and is smoothened by rolling over a smooth surface such as a stainless-steel screw box. Herzog's bend is created for the tibial nail. While making the rod for the femur, a slight anterior curvature is made. Bumps on the nail, if any, are shaved off with a knife blade after the cement has hardened. Similarly, the diameter of the nail can also be reduced if necessary. The nail is kept in the air to help with polymer evaporation and to harden the cement. Leftover cement is then used for preparing beads.Beads are threaded over 16-guage wire to prepare the chains and ends of the wire are bent to prevent beads from coming out of the chain. Beads should be round in shape and approximately 7 mm in size. The local concentration of antibiotics after the insertion of ACN and beads is substantially higher than that achieved with serum concentration.4We conducted a retrospective review of patients treated in our institution from January 2008 to December 2019. The original technique described by Paley et alCases were classified as Type I (62 patients), Type II (71 patients treated by ACN), or Type III (45 patients treated by Ilizarov technique). A subset of 28 open fracture cases admitted with severe contamination or delayed presentation with established infection were also treated with debridement, ACN insertion, and beads in pouch technique. The patient subset included 6 Gustilo type I fractures, 12 Gustilo type II fractures, 6 Gustilo type III fractures, and 4 patients with infected nailing after primary surgery, which was performed elsewhere.5The authors have developed a classification based on the severity of infection, apposition of fragments, and presence or absence of gap, shortening and deformity. Severity was judged by the quantity of pus drained, scarring of soft tissue, gap between fragments, and presence of callus. The classification indicates guidelines for treating INU. The INUs are classified into 3 types.Type I (mild): Fragments are in opposition (no gap). There may be an implant in situ. On pressing around the wound, discharge of pus may be observed at the sinus or wound. Type I also includes a nondraining, dry wound for at least 3 months. Treatment is usually by a 1-stage surgical procedure with debridement, insertion of ACN, and plaster cast for tibial or hinged knee brace for femoral INU. Three-stage surgical procedures may be required to treat recurrent infection.Type II (moderate): Fragments are in alignment with severe infection and no gap ≤3 centimeters (cm), shortening, or deformity. A small or large wound or sinus and a large amount of pus drainage will be present. An abscess deep down at the nonunion site may exist, which should be drained and debrided. An external fixator, preferably an Ilizarov ring fixator, gives additional stability to the nonunion after nailing, allowing compression or distraction and also deformity correction. If the wound is large, a plastic surgical reconstructive procedure may be needed to cover the wound. An active, nondraining wound with abscess and presence of fever is included in this type. Treatment is by a 3-stage procedure described below, and bone grafting may be necessary.Type III (severe): Indicates severe infection with a gap >3 cm and deformity, shortening or combination of both. The Ilizarov method of treatment is required to correct the deformity and to treat the gap by bone transport. Forty-five patients classified as Type III were treated locally with antibiotic beads instead of ACN. Therefore, Type III is not discussed in this paper.66.11.Thorough debridement: The first and single most important step in all 3 types of INU is thorough radical debridement. Debridement consists of excising the sinus tract plus all infected and necrotic or avascular tissues. Dead sclerotic bony ends are also removed. A 3.2 mm drill bit is passed at every centimeter from the end of each fragment, until blood oozes out from drill holes indicating the line of vascularity. The long axis of the bone is cut at a right angle by an oscillating saw. Punctate bleeding determined by the Paprica sign, should be seen at bony ends.2.Reaming: The next important step of debridement is to ream the medullary canal to remove infected granulation tissue and small sequestrii. Frequent cooling by saline irrigation is important. A drill hole is created in the distal fragment to flush out the infected material which is collected for culture and sensitivity. Copious irrigation of the nonunion site and the medullary canal of both fragments by pulsed lavage with normal saline is performed.3.Insertion of antibiotic intramedullary nail and beads, and wound care: An ACN with a 6 to 9 mm K-nail is inserted proximally into the distal fragment. The technique for inserting the ACN is the same as for antegrade interlocking nails. Dead space around the nonunion site is filled with antibiotic beads. If possible, the wound should be closed primarily. Open wounds with infection are treated with a vacuum-assisted closure (VAC) system after initial debridement. Healthy granulation tissue is evident in most cases after the first 3 or 4 VAC dressings. Large wounds require soft tissue reconstruction by muscle flap and/or skin grafting.4.Stability: If stability is satisfactory after antibiotic rod insertion, Type I INU tibial fractures are treated with a full leg, knee in extension, plaster cast, which is inherently stable. ACN alone is not stable enough in Type II, especially to protect rotational stability, so additional external fixation is needed. A 2-ring Ilizarov construct or monolateral external fixator is used to further stabilize the ACN.5.Insertion of pins and wires for Ilizarov frame: Metaphyseal areas of the femur and tibia are large, making it possible to insert Schantz pins and wires for llizarov frames 1 to 2 cm away from the path of the nail to prevent pin tract infection from spreading into the intramedullary canal. The distal tip of the nail stops 3 cm short of the ankle joint and the wires of the Ilizarov fixator are 1 cm away from the ankle joint. There is a clear space of 2 cm from the tip of the nail to the wires. Instead of an Ilizarov frame, monolateral external fixation may be used to stabilize the construct.The first stage consists of radical debridement, insertion of ACN, and stabilization by plaster cast or external fixator . Steps include:6.2After the first surgery, patients are reassessed at 4 to 6 weeks. The status of infection is evaluated clinically, radiologically, and with laboratory tests . If exchange nailing with an interlocking IM nail is decided on, the external fixator is removed. A cast or brace is applied for 2 to 3 weeks for healing of the pin site wounds. In Type II INU cases, 15 patients had a persistent small gap and shingling, and bone grafting was carried out. In 5 patients, infection persisted with a draining sinus. These patients were treated with repeat debridement and a fresh ACN of a different antibiotic combination, such as tobramycin and ciprofloxacin.6.3If the infection is controlled or minimal, the wound is explored at 6 weeks. ACN beads are removed, the wound is debrided, and the canal is reamed. An IM interlocking nail is inserted and static locking is done. Cancellous bone grafting is performed if needed. If infection still persists, the wound is debrided and an ACN loaded with a different antibiotic is inserted.6.4Patients are allowed to weight bear as tolerated as early as possible. Immediate joint mobilization is encouraged to avoid stiffness and contractures of soft tissue. Stage I lasts for a period of 4 to 6 weeks. Radiographs are taken every 4 weeks. If the fracture shows signs of healing, the same treatment of ACN with cast is continued. External fixation should be removed as early as possible to avoid restriction of joint movements, to prevent pin tract infection, and for the comfort of the patient.7Case follow-up ranged from 1 to 24 years, with an average follow-up of 5 years and 7 months. Eight patients who could not travel to our institution participated in a telephone-based follow-up session with the help of a local orthopaedic surgeon. Results were classified into 3 groups: excellent, good, and poor. Excellent results indicate the fracture united, infection was controlled, and full function was regained. Good results indicate the nonunion was healed and infection was controlled, although patients had some restricted mobility of the knee and mild limp. Poor results indicate a stiff knee, persistent infection, nonunion, or amputation if required. Out of 133 cases reviewed, 72 cases involved the tibia and 61 the femur.Sixty-two cases were classified as Type I. Forty cases united without reoperation and were rated as excellent. Twenty-two cases needed a second-stage procedure of exchange nailing with an IM interlocking nail and were rated as good. In 4 of the 22 cases, bone grafting from the iliac crest was required. Eight cases had restricted mobility of knee, but could flex the knee >90 degrees. Sixteen cases had a mild limp.Of the 71 cases classified as Type II, 38 cases united and 15 cases required shingling and bone grafting. Eight cases had limb shortening of 3 to 4 cm. The patients were advised of limb lengthening over a slotted plate. Three patients agreed to limb lengthening and the procedure was successfully carried out. Five patients had a shoe raise. Twelve cases had a persistent or recurrent sinus for a duration of >1 year. Six patients healed, 6 had repeat debridement surgery; in 5 of those cases the sinus healed, and in 1 case, the sinus persisted. In 10 patients there was mild flexion deformity with flexion being 0 to 90° degrees or more. In 6 cases knee fusion was done because of a stiff painful knee (3 cases had septic arthritis of the knee). In 3 cases, infection could not be controlled and nonunion persisted. These patients were advised repeat debridement but refused further surgeries. One case developed severe infection and septicemia, and was treated with amputation. Thirty patients had some restriction of movements of the knee ranging from 5° to 90°. They were grouped as good results. Of the 71 Type II cases, 28 cases were rated as excellent, 30 cases were rated as good, and 13 cases were rated as poor. Cases and complications are summarized in Tables 8Indications for ACN and beads in open fractures of the tibia and femur were: wound was severely contaminated with mud and sand deeply embedded in the muscles and wound required multiple debridements and plastic surgery; late arrival of patient ; late arrival of patient (1 or more months after infection after nailing or plating); farmyard injuries with high risk of infection.Radical debridement is the most important step to improve outcomes. The ACN is inserted with the addition of an Ilizarov external fixator . Once the infection is under control, ACN and external fixator is removed and replaced with an interlocking IM nail or plate with an interval of 2 to 3 weeks allowing time for the pin tract infection to settle. Culture-specific antibiotics are added in the cement used to prepare ACN and beads. Antibiotics are given systemically for 3 to 4 weeks . All 28 cases had additional external fixation .Six Gustilo grade I open fracture cases were reported with a puncture wound and delayed presentation of >4 days. Two united without second surgery, 4 had exchange interlocking IM nailing. All 6 united within 6 to 10 months.Twelve Gustilo type II open fracture cases were reported. All had ACN and external fixation. External fixation was removed at 4 to 6 weeks and exchange interlocking IM nailing was done. Three cases required a third surgery, consisting of shingling and cancellous bone grafting from the iliac crest at 4 months after interlocking IM nailing.Six cases of Gustilo open type III injuries presented 1 to 3 weeks after injury. All were treated elsewhere with nailing or plating and all had infection. Three cases required plastic surgery in the form of a local or distant flap and were treated with ACN and unilateral external fixation. The other 3 cases were treated with ACN and Ilizarov external fixation. After 4 to 6 weeks exchange interlocking IM nailing was performed. Two of these cases required bone grafting.Four cases were closed fractures treated elsewhere with nailing and were admitted to our institute 1 to 3 months after the first surgery with frank infection. These patients were treated with debridement, ACN insertion, and external fixation. After 4 to 6 weeks, ACN was exchanged to an interlocking IM nail. Two needed bone grafting. Three cases united within 6 to 8 months, and 1 had nonunion which was treated with exchange nailing with an additional 8-hole 4.5 mm pate and bone grafting.99 were the first to use a cement stick that did not provide any stability. Paley and Herzenberg1012131415121314In this study we found that results of using ACN for treating INU and a subset of open fractures were very good. Klemm and Seligson17Intramedullary infection is a well-recognized complication of internal fixation by nailing or plating, with the infection spreading throughout the medullary canal. Multiple points or the entire canal may be involved in pin tract infections after external fixation.We have classified INU into 3 types based on the severity and gap. We found the classification helpful in our treatment strategy. Shyam et al7 which is adequate to kill even resistant organisms. ACN along with cast, brace, or external fixation provides adequate stability and also fills the dead space of the medullary canal. Noteworthy, the high concentration of antibiotics has minimal influence on new bone formation.In our opinion there are distinct advantages of using ACN and beads. A high local concentration of antibiotic is established, 200 times more than the serum concentration,We are aware that there is some controversy regarding the rate of antibiotic elution. It is biphasic with a high early elution rate followed by slow and sustained release as time progresses. Elution shows exponential decline after day 1 of implantation.After a period of 6 weeks when most of the elution has subsided, ACN acts as an inert implant. Therefore, ACN should be removed before 6 to 8 weeks and then an interlocking IM nail is inserted. After 8 weeks, if infection still persists, a fresh ACN with different culture-specific antibiotics is inserted. If the infection is mild or minimal, interlocking intramedullary nailing is used alone.After a prolonged period of implantation, bacterial colonies are detected on the antibiotic cement and even have the potential ability to develop resistance to gentamycin despite preoperative susceptibility to local antibiotic.KlemmAntibiotics commonly used for ACN are vancomycin, gentamycin, and tobramycin. Other antibiotics that have been used include cefazolin, ciprofloxacin, clindamycin, teicoplanin, erythromycin, colistin, and cefotaxime.29 In our cohort of patients, we did not observe a single case of cement debonding after use of a 6 to7 mm K-nail, which was routinely used. Cement gets locked into the canal and around the nail due to its clover leaf configuration. In 1 series of 21 cases treated with ACN, there was nail breakage in 2 cases.31 The worst scenario is a combination of active infection with instability, which perpetuates infection creating a vicious circle which ultimately leads to failure.Despite the advantages of ACN, one should also consider potential complications related to its usage. One of the common complications is cement debonding, as reported by Thonse.Overall, complications in this series were mostly related to the anatomical location around the knee . Persistence of infection requiring repeated debridement was the cause of treatment failure.This study had certain limitations. It is a retrospective study with possible bias for the selection of patients and the mode of treatment. Twenty-two patients were lost to follow-up. We have not compared our method of preparation of antibiotic rods with other methods. However, we think our method is simple and is not associated with any debonding of cement because of K-nail use. Strengths of this paper are the number of patients recruited and 90% good to excellent results. All cases were operated on under supervision of one surgeon (first author).In conclusion, the use of ACN and beads in INU achieved good infection control. Complications, however, are to be expected in these challenging cases. Our classification of INU into 3 types according to the severity of infection and gap provides guidelines for treatment. Finally, the use of ACN and beads in a subset of open fractures, where infection has settled due to late presentation or farmyard injuries, appears to be a good option of treatment.


Background and objectiveMany international students often face challenges regarding their mental health, finances, and academics. The coronavirus disease 2019 (COVID-19) outbreak may have presented unprecedented challenges to many foreign students in these aspects. Our study examined the academic, financial, and mental health challenges encountered by international students residing in the United States due to the COVID-19 pandemic. It also examined the association between the mental health of the respondents and the academic and financial challenges they encountered.MethodThe study involved international students enrolled at Texas A&M University, who identified themselves as non-US citizens or non-permanent residents. We conducted a cross-sectional study by using Qualtrics® to explore the three domains of the study. Questions included in previous studies were modified to assess the academic and financial status while the Patient Health Questionnaire-4 (PHQ-4) score was used to assess the mental health of the respondents. We presented descriptive statistics for all domains and used an ordered logistic regression to further analyze the effect of the other domains on the mental health of the respondents.ResultsOf the 281 respondents, the majority (79%) experienced challenges with online classes; 91% reported having negative emotions and some students (24%) lost funding due to the pandemic. The inability to pay bills resulted in a three-fold increase in the likelihood of reporting higher mental distress , and experiencing academic challenges led to a seven-fold increase in the likelihood of reporting higher mental distress .ConclusionThe COVID-19 pandemic posed a major challenge to international students and its impact on the mental health of the participants was aggravated by concurrent academic and financial hardships. The coronavirus disease 2019 (COVID-19) pandemic is a global public health emergency that has profoundly affected international students living in the United States. About 1.1 million international students studied in the United States of America during the 2018-2019 school year ,4. To beInternational students routinely experience stressors such as language barriers, and financial, cultural, and academic difficulties, as well as challenges in forming new relationships, which may adversely affect their academic performance ,6. They While financial security is a problem for all students, it is even more pronounced for international students . In the In the US, economic stimulus packages were distributed to alleviate some of the financial difficulties brought on by the pandemic; however, international students do not qualify for such financial assistance programs . ResearcWith most colleges transitioning to online instruction, students staying at home might have to adopt a different sleeping pattern leading to reduced sleep quality, thereby elevating stress levels and anxiety about their academic and employment prospects . A studyGiven these various challenges faced by international students, it is important to identify them and develop actionable insights to improve the well-being of international students during and after the coronavirus pandemic. In this study, we aim to examine the academic, financial, and mental health challenges experienced by international students at a large academic institution in the United States. Furthermore, we aimed to assess the relationship between the mental health challenges experienced by students and the academic and financial challenges during the pandemic.Study designA convenience sampling technique was used to administer an online cross-sectional survey by using Qualtrics® to assess the mental health, financial, and academic status of international students at a large US university. The survey was made available in the English language and was disseminated using the Texas A&M University bulk email system, which allows for the targeting of international students. In this study, we recruited individuals enrolled at the undergraduate or graduate levels who were non-US citizens or non-permanent residents. Recruitment emails contained links to the online survey and were sent out five times between November 16, 2020, and January 14, 2021, to maximize response rates. Prior to the surveys being administered, a pilot version was pretested among 20 international students from the same university to determine the degree to which the survey questions were comprehensible and to enhance the validity of the study. The pretested survey questionnaire was improved based on the feedback provided by the pretest subjects, but the pretest result was not included in the final analysis.Study participantsThe inclusion criteria were as follows: students currently enrolled at Texas A&M University at undergraduate, master's, or Ph.D. levels and registered as international students. US citizens or permanent residents in the US were excluded from the study. After the recruitment emails were sent, 281 participants who met the study criteria responded to the survey. Study participation was voluntary and before participants began the survey, they were provided an electronic informed consent form, information about the nature and purpose of the study, and information about the ethical approval obtained from the Texas A&M Institutional Review Board.Study variables and measuresThe study consisted of 42 questions and was required to be completed in approximately 10 minutes. The questions can be categorized into the following sections: (1) 10 questions about participants’ demographics, (2) Eight questions assessing the academic challenges associated with the COVID-19 pandemic, (3) 12 questions assessing the financial challenges posed by the COVID-19 pandemic, and (4) 12 questions assessing the mental health challenges linked to the COVID-19 pandemic. To assess the effect of the pandemic on respondents, questions assessing mental health were framed using the Patient Health Questionnaire-4 (PHQ-4), while questions on academic and financial challenges were adapted from previous studies -19.Specifically, the mental health challenges were explored by employing PHQ-4-types questions such as the following: "Since March, how often have you felt depressed due to the pandemic?" and non-PHQ-4-type questions such as the following: "I feel the pandemic has affected my mental health". These had to be answered with the categorical options of "none of the time", "some of the time", "most of the time", and "all of the time" and graded on the Likert scale from "strongly agree" to "extremely disagree", respectively. In addition, a composite PHQ-4 score was created by combining the responses to each PHQ-4 component question, which was subsequently used as a dependent variable in an ordered logistic regression. The PHQ-4 has been tested and validated in previous studies. All four questions in the PHQ-4 were used. The PHQ-4 has been previously tested and validated in similar populations and other populations where it has been shown to have a 0.8 association with mental health . PHQ-4 oData analysisDescriptive statistics (sample size=281) were used to report the distribution of the demographic factors and all other survey component responses. Furthermore, an ordered logistic regression analysis was conducted using the PHQ-4 scores as a categorical dependent variable in four levels. The total score ranged from 0 to 12, with the ordered categories identified as "none" (0-2), "mild mental distress" (3-5), "moderate mental distress" (6-8), and "severe mental distress" (9 -12) . The samFurthermore, since all the questions in a questionnaire/survey do not always directly translate into variables to be included in a model, the reasonableness of the questions to be included in the model was assessed besides including it as a question in the questionnaire. To develop a parsimonious model, we initially ran a univariate analysis for the 42 variables (39 after combining the PHQ-4 variables) and selected the variables that were either statistically significant with p ≤0.05 or were deemed necessary to answer our research question. The final 13 variables included to create a parsimonious model were as follows: PHQ-4 score; current marital status; gender; race; ethnicity; the degree the student is working toward; current employment status; the current status of funding provided by Texas AM; annual family income; the way in which COVID-19 has affected the student's finances; whether the student is unable to pay past/upcoming bills due to COVID-19; whether COVID-19 has negatively affected the student's academic performance; and whether the student has lost a specific funding source due to COVID-19.A total of 281 international students  were included in the analysis; 51% were female, with Asians representing the largest proportion of international students in the survey (56%)  have reported fewer mental health challenges as TAMU has one of the biggest international student communities in the US. Also, these two groups come from a more communal society where limited resources are shared, and the focus is on the collective rather than the individual, in contrast to White students, who may come from a relatively more individualistic society with an emphasis on the individual over the group and with less sharing of resources. Hence, their societal norms carried over to the US in a location with a sizable international student representation may have further enhanced the propensity for ethnic connectedness . This coCOVID-19 also had adverse impacts on the economy of the United States as well as on individuals. A previous study reported a similar economic impact due to national health crises during epidemics and reported that loss of income sources could lead to anxiety about paying rent and tuition fees . This fiOf note, only 9% of international students reported feeling no form of anxiety and 19% reported no uncontrollable worry in this study. However, another study conducted to assess the psychological impact of COVID-19 among university students  in the United States found that a much smaller proportion (1%) of students reported positive changes due to the COVID-19 pandemic . Some COOverall, it appears that academic and financial challenges affected students’ mental states. It is more likely for a student with academic and economic challenges to be anxious and depressed, emphasizing the association between mental health status and academic/financial status. We recommend that universities boost their outreach to vulnerable populations at risk of high levels of negative impact from COVID-19 . FurtherThere are several limitations to this study. Firstly, this study was based on a convenience sample and may not be representative of the entire international student population at Texas A&M University. Out of over 5,000 international students on campus, only 281 responded to the survey, as participation was voluntary. For the ordered logistic regression, 243 respondents completed the response with regard to the specified dependent variable - PHQ-4. Since certain sensitive survey questions may not always receive a full response, and as per the ethical considerations relating to survey administration, respondents were provided the option not to respond to questions they may not wish to answer. Hence, for the descriptive analysis table, we used a sample size of 281 to preserve vital and valid information provided by the respondents, while a complete cases analysis (243) was used for the ordered logistic regression. As this was our dependent variable, it was important that the respondents gave full responses to the four component variables that were later combined to form the PHQ-4 score variable used in the analysis. Furthermore, adhering to the algorithm by Detorri et al., a complete case analysis as conducted was deemed best for our analysis due to the proportion of missing responses and other limitations of different impartation methods in relation to our analysis .The low level of response rate to our study may be attributed to various reasons; previous studies have shown that students who are more vulnerable socioeconomically are less likely to participate in surveys . It is pOur analysis showed that the COVID-19 pandemic posed a significant challenge to international students at TAMU across all three domains examined. The pandemic posed significant challenges to the students’ mental health as well as financial and academic well-being. Furthermore, respondents’ mental health challenges were further aggravated by academic and financial difficulties, such as the perception that the pandemic had negatively impacted their academic performance and their inability to pay their bills. Even though the majority of study participants indicated that they were satisfied with the University’s response to the pandemic, the government and the University need to provide more efficient and robust social and financial support to international students during crises like public health emergencies.


Two limitations of the clinical use of 3-dimensional (3D) reconstruction and virtual reality systems are the relatively high cost and the amount of experience required to use hardware and software to effectively explore medical images. We have tried to simplify the process and validate a new tool developed for this purpose with a novel software package.Five patients with right partial anomalous pulmonary venous return with adequate preoperative images acquired with magnetic resonance imaging were enrolled. Five volunteers with no previous experience in the field of 3D reconstruction were instructed to use the software after viewing a short video tutorial. Users were then asked to create a 3D model of each patient's heart using DIVA software. Their results were compared quantitatively and qualitatively with a benchmark reconstruction performed by an experienced user.All our participants recreated 3D models in a relatively short time, maintaining a good overall quality . The overall trend of all the parameters analysed showed a statistical improvement between case 1 and case 5, as users became more and more experienced.DIVA is a simple software program that allows accurate 3D reconstruction in a relatively short time . In this study, we demonstrated the potential use of DIVA by inexperienced users, with a significant improvement in quality and time after a few cases were performed. Further studies are needed to confirm the potential application of this technology on a larger scale. Computer-generated 3-dimensional (3D) reconstruction is emerging as a growing technology in the world of congenital heart disease (CHD). Computer-generated 3-dimensional (3D) reconstruction is emerging as a growing technology in the world of congenital heart disease (CHD). The benefits of virtual reality (VR) or 3D printed models have been clearly demonstrated, especially when dealing with complex anatomies or in the case of planning minimally invasive procedures . Indeed,Nevertheless, one limitation of the clinical use of these systems is the relatively high cost and the degree of expertise required to use the currently available software. In addition, the spread of these technologies has been limited by the lack of standardized approaches, long processing times and a lack of dynamic representations of the cardiac cycle. As this field develops, new options are becoming available with a potential simplification of the complexity of the platforms required to obtain virtual models.The DIVA software  is a new VR technology allowing fast and user-friendly 3D reconstruction of CHD . We prevPatient data were completely anonymized so that neither informed consent nor institutional review board approval was required.A cohort of patients diagnosed with right partial anomalous pulmonary venous return (PAPVR) and associated superior sinus venous defect was selected from among all patients who underwent surgery at the University of Padua between January 2015 and December 2020. The exclusion criteria were the unavailability of medical imaging, imaging performed after surgical correction, any imaging techniques other than cardiac magnetic resonance imaging (MRI), and patients whose imaging was otherwise unsuitable for 3D reconstruction due to poor quality or lack of 3D acquisitions.Cardiac MRI data from 5 patients were selected and used to build the reconstructed 3D models. Cases 1, 2, 3 and 5 were 8-, 31-, 26- and 4-year-old females, respectively. Case 4 was a 10-year-old boy.We then recruited 5 participants with little or no expertise in 3D reconstruction, including 2 medical students and 3 cardiac surgery residents, all with little experience in the field of CHD. An expert user (FB) with years of experience in the field of 3D reconstruction of CHD and time spent with the DIVA software recorded a 40-min video tutorial for the participants. The participants were allowed to review the video as often as they wanted a few days before the actual reconstructions. For the tutorial, a sample case was selected for didactic purposes among the patients excluded from the study, to avoid any simple emulation among test users. This tutorial was designed to guide completely novice users through the complete pipeline of the reconstruction process. After this, we asked the participants to generate PAPVR VR models of the 5 patients using the DIVA software. The expert user also made 3D reconstructions of all the cases.The data were then evaluated, and the results of the expert user were compared with those of the 5 participants Figs. –3. The qCardiac MRI was performed using a Philips Achieva 1.5 T scanner . Images were acquired with a 5-channel phased-array heart coil. The 3D SSFP sequence, electrocardiogram and gated navigator in free breathing were performed for whole heart acquisition.3D reconstructed models of the patients' anatomies were generated using DIVA software. This software was developed using the popular Unity game engine and includes 2 native interfaces: a desktop mode for volumetric viewing on a standard computer monitor (Video 1) and a VR mode for the interaction with medical images in an artificial environment through the use of a VR headset. Patient imaging data were loaded directly from Digital Imaging and Communications in Medicine (DICOM) files, and MRI volumetric renderings were generated instantly by the software, without any human intervention.Three different reconstruction tools were available in the software, namely, Maximum Intensity Projection, AVATAR Filter (a threshold drawn on the intensity histogram using different curves) and 2F transfer function . Users were encouraged to experiment with them and find which one gave the best results. In particular, for the 2F transfer function, multiple combinations of X and Y axes were available to plot the pixel space in a 2-dimensional matrix of grey values, but very little explanation was provided on the exact meaning of each option. This was done on purpose, because a thorough understanding of those tools requires a mathematical and engineering background that is simply not available to most surgeons and doctors. Our goal was to demonstrate the feasibility of quick and easy training for naive medical staff; therefore no in-depth technical discussion was deemed necessary. Most users chose the 2F transfer feature as their preferred tool, but this was not subjected to judgment Figs. –3.The reconstruction pipeline starts with a simple folder containing the raw, unprocessed, DICOM images. After importation into the software, a Maximum Intensity Projection rendering is automatically generated, and the user has the option to crop the model in all 3 dimensions to isolate the region of interest as much as possible. Because a 3D rendering is already present, all following steps are optional. Finally, the user can pick among the tools available as well as customize the tools themselves to find the best combination of parameters to achieve the optimal reconstruction using a trial-and-error approach.P-value for statistical significance was set at 0.05. When a significant test result was obtained, a Bonferroni-adjusted post hoc analysis was used to assess pairwise differences between groups.All the scores were converted into a five-point Likert scale. Due to normal distribution, numerical variables were presented as mean ± standard deviation. A one-way repeated-measures analysis-of-variance (ANOVA) was used to compare the different cases. The Case 1: An 8-year-old female with PAPVR of the right superior pulmonary vein into the superior vena cava (SVC) well above the origin of the azygos vein. The right middle pulmonary vein drained into the junction between the SVC and the right atrium (RA). This defect was associated with a rather large sinus venosus defect , time (P < 0.001), time score (P < 0.001), adjusted score (P < 0.001).The variation of the scores over time was significant for all the parameters analysed: quality score . The models were then uploaded to a VR environment. The accuracy and precision of the anatomical details were excellent. These features allow wider use of 3D technology even in small hospitals or centres without 3D modelling experts, to implement and improve potential surgical planning. The scores collected by users varied. However, the overall trend of all the parameters analysed showed a statistical improvement between case 1 and case 5. This result suggests that the learning curve is linear and that, although the results have improved gradually, the former were already of sufficient quality to be useful. This is particularly important in setting up accelerated model generation. Indeed, long reconstruction protocols that guarantee accuracy only after considerable experience would defeat the main purpose of this software, which is to allow “lay” users to reconstruct 3D DICOM images themselves. The main advantages of this technology lie in speed and realism, the latter mainly attributed to the immersive 3D experience that can be achieved through a VR headset. Despite the cost barrier, the required hardware is widely available and mass-produced as gaming peripherals, with the base system not even requiring a VR headset, thus allowing for gradual upgrades if needed. The cross-platform compatibility of the software is guaranteed by the use of the Unity engine. Our surgeons have found the VR experience to be superior to the 2D images offered by most 3D segmentation software, primarily due to the internal navigation and manipulation that become possible using a VR headset. This feature adds information and details about the anatomical conformation of internal structures without adding too much additional complexity, if any, to the user's experience.Our selection of a simple extracardiac defect was intentional, and our goal was to create the optimal conditions for evaluation of software performance. However, we have also evaluated the effectiveness of this tool with more complex intracardiac defects, such as an atrioventricular septal defect or double outlet right ventricle, which presented a challenging set-up problem. The senior author  later reported that he found the tool useful, because it allowed for intracardiac navigation and familiarity with various proportions and structural orientations. However, quantifying such outcomes presents significant challenges, requiring a case series or, preferably, a clinical study capable of measuring improved clinical outcomes such as operating time and patient morbidity.Finally, we made a conscious decision to limit our investigation to cardiac MRI only, to achieve maximum homogeneity of the data set. Although it is conceivable to use other imaging modalities such as CT scans, we have refrained from such practices to rule out potential bias. Using a heterogeneous data set could give an unfair advantage to similar cases, as demonstrated by the sample case used in the training video tutorial, which would have inevitably been captured using CT or MRI imaging.3D models and VR are becoming important technologies for preoperative assessment and surgical planning in the treatment of CHD. DIVA software is a very simple software that allows accurate 3D reconstruction in a relatively short time (“fast-track VR”). In this study, we have demonstrated the potential use of DIVA software by inexperienced users, with a significant improvement in quality and time after performing a few cases. Further studies are needed to confirm the potential application of this technology on a larger scale.The whole project was supported by general funding, thus unrelated to the specific topic addressed in our paper.Conflict of interest: Mohamed El Beheiry and Jean-Baptiste Masson are cofounders, shareholders and, respectively, Chief Technology Officer and Chief Scientific Officer of AVATAR MEDICAL SAS, a start-up company that commercializes software for surgery planning in virtual reality. The DIVA software used in this study is not being commercialized by AVATAR MEDICAL SAS, although the company’s technology is based on the same technology. The DIVA software, which serves as base for this study, is freely available and has been reported previously in the literature [terature . All dev