Posidonia oceanica (L.) Delile, as model to evaluate the effect of ALAN on seagrass populations established in shallow waters, by taking advantage of a decreasing gradient of dim nocturnal light intensity (from < 0.01 to 4 lx) along the NW Mediterranean coastline. We first monitored the fluctuations of putative circadian-clock genes over a period of 24 h along the ALAN gradient. We then investigated whether key physiological processes, known to be synchronized with day length by the circadian rhythm, were also affected by ALAN. ALAN influenced the light signalling at dusk/night in P. oceanica, including that of shorter blue wavelengths, through the ELF3–LUX1–ZTL regulatory network, and suggested that the daily perturbation of internal clock orthologs in seagrass might have caused the recruitment of PoSEND33 and PoPSBS genes to mitigate the repercussions of a nocturnal stress on photosynthesis during the day. A long-lasting impairment of gene fluctuations in sites characterised by ALAN could explain the reduced growth of the seagrass leaves when these were transferred into controlled conditions and without lighting during the night. Our results highlight the potential contribution of ALAN to the global loss of seagrass meadows, posing questions about key interactions with a variety of other human-related stressors in urban areas, in order to develop more efficient strategies to globally preserve these coastal foundation species.Artificial light at night (ALAN) is a globally spreading anthropogenic stressor, affecting more than 20% of coastal habitats. The alteration of the natural light/darkness cycle is expected to impact the physiology of organisms by acting on the complex circuits termed as circadian rhythms. Our understanding of the impact of ALAN on marine organisms is lagging behind that of terrestrial ones, and effects on marine primary producers are almost unexplored. Here, we investigated the molecular and physiological response of the Mediterranean seagrass,  With a rate of 6% increase of sky brightness per year, ALAN is spreading globally, affecting over 23% of the world’s land surfaces between 75° N and 60° S4. In particular, European countries and United States are those most exposed to light-polluted nights, with 88% and almost 50% of the total surface, respectively4.Artificial light at night (ALAN) has come to the fore as one of the most impactful anthropogenic factors that affect wildlife1. Most life forms on Earth have evolved complex biochemical and molecular circuits that are entrained by light/dark cycles and allow memory and anticipative adaptation to environmental fluctuations5. Such regulatory processes display an oscillatory pattern of about 24 h, that is termed as circadian rhythms or the internal clock5. In addition to the joint role that light plays for all the organisms to entrain circadian rhythms and as a source of information from the surrounding environment, light radiation in plants is also the main driving force to fix carbon into organic compounds through photosynthesis6. While the threat of light pollution and its effect on terrestrial ecosystems are well documented, the implication that ALAN might have in marine habitats has become a relevant issue only over the last decade7. Indeed, several of the world’s largest metropolises are located along the coasts, where they contribute to illuminate both the shoreline and the adjacent shallow sea7. In this scenario, Europe holds the record of the continent with the highest percentage (54%) of light polluted coasts8.An alteration of the night skyglow by artificial light is expected to impact on the physiology of a wide range of organisms, including animals and plants, as well as the structure of ecological communities9. As for the light spectrum, recent advances in lighting technologies have boosted the use of broader-spectrum artificial lights  in urban areas, due to their improved energy efficiency and enhanced visual performances10. Despite their benefits, broader-spectrum lights have created more complex patterns of lighting as compared to natural moonlight, especially along the water column. In fact, the scattered blue component of the spectrum penetrates deeper than the red one, mostly affecting those species that are sensitive to short wavelengths8. As an additional source of spatial variability, the artificial skyglow extends the ‘sphere of potential influence’ of ALAN far beyond the localization of the point sources1, by creating gradients of light intensity tens of kilometres long, while moving away from urban centres along the coastline.Along the coastline, ALAN is mostly identified as a localized disturbance related to urban areas and originating from fixed light sources, either municipal or private. The spatial arrangement and type of lamps, however, create a quite heterogeneous light environment at the scale of meters, with higher and lower light intensity areas of variable extent and spectrum14, corals; e.g.16). To the best of our knowledge, effects on primary producers have been investigated only on microbial species19. This is at odds with the key role played by macroalgae and plants in marine habitats, both as oxygen producers and habitat-forming species .Recent advances in marine ALAN research focused mostly on the effect of direct sources of LED lights on the physiology and behaviour of animals, either in intertidal or shallow subtidal systems , which revealed species-specific sensitivity to artificial night lighting22. For instance, the proximity to streetlamps can greatly impact plant fitness and phenology in case of Platanus spp., leading to the retention of leaves into winter and the anticipation of budburst in spring23. Studies on herbaceous plants reported temporally variable, species-specific and light source dependent outcomes, both on growth-related variables and phenological aspects26. Interestingly, experimental exposure of bean plants to a gradient of artificial night light intensity showed a positive correlation of ALAN with plant biomass, suggesting potential effects of intensities as low as those experienced far from direct light sources27.The need for studies on ALAN effects on marine plants is further stimulated by research on trees commonly planted along roads (Posidonia oceanica (L.) Delile is the most important endemic seagrass along Mediterranean coastlines, covering about 2% of the seafloor28. It is a marine monocot species, whose meadows are adapted to grow across a broad depth range, from shallow waters down to 45-m depths, which expose them to different light and temperature regimes29. P. oceanica supports marine biodiversity through the provision of habitat and food and provides a wealth of services, ranging from the exportation of organic matter, carbon sequestration, sediment trapping and reduction of coastal erosion20. Like most seagrasses worldwide, occurrence along coastal areas poses P. oceanica meadows under threats related to urban/port development and activities, resulting, in the worst-case scenario, in their regression31. For those reasons, we chose P. oceanica  as model species to study the effects of ALAN on shallow-water seagrass populations next to urban areas, taking advantage of a decreasing gradient of nocturnal light intensity along the coastline south of the city of Leghorn .The seagrass Posidonia. Among several physiological processes, circadian rhythms regulate photoprotection and photosynthesis32 and induction of flowering33. We therefore investigated whether ALAN modified the oscillatory expression of putative circadian genes in Posidonia. We further extended our analysis to photosynthetic and photoprotection genes that could participate in the output of the circadian clock, and to the regulators of photoperiodic flowering in the land angiosperm Arabidopsis thaliana. To determine if ALAN effect on gene expression could affect performances of Posidonia plants, we monitored in situ the maximum photosystem II (PSII) efficiency on dark-adapted leaves along the gradient of nocturnal light intensity . Finally, to assess whether these effects were long-lasting, we monitored plant fitness in terms of growth under controlled conditions and without lighting during the night.We hypothesized that continuous exposure to artificial night lighting could impact the entrainment of gene expression rhythms in Posidonia; due to the shallow water depth (about 0.5–1 m), seagrass leaves could reach the surface under low tide conditions. Despite their geographical proximity, the three locations differ in (1) night sky brightness , (2) intensity of artificial light at night  and (3) distance from the industrial and merchant port  showed no statistically significant differences between the two sites (mean ± 1 SE (standard error); Red/Clear: Scalinata 1.7824 (1.0060), Rotonda 2.5103 (0.0036). Blue/Clear: Scalinata 0.3810 (0.2152), Rotonda 1.2067 (0.0071) (tested through one-way ANOVA).This study was carried out at three locations located south of the city of Leghorn : Rotonda (43°30′44.2″N 10°19′01.6″E), Scalinata (43°30′28.1″N 10°19′08.1″E) and Maroccone (43°29′03.9″N 10°19′51.9″E). All locations were characterized by a gently sloping rocky seabed, with occasional presence of pebbles and small boulders. Within a distance of 10–20 m from the shoreline, the biological assemblage was dominated by turf-forming and encrusting algae, with patches of Posidonia samples were collected at 4-h intervals over a period of 24 h at 19:00, 23:00, 3:00, 7:00, 11:00, 15:00 and 19:00 (GTM + 1), at 1-m depth, in mid-June 2017 (number of replicates at each time point: n = 10), characterised by an average of 15 h of light and 9 h of darkness (dawn and dusk approximately at 5:00 and 21:00 GTM + 1 according to https://www.timeanddate.com/). Each replicate consisted of one tip of 2 cm-length selected from the youngest, fully mature leaves collected from the same seagrass meadow, avoiding those covered by epiphytes, and subsequently stored in RNAlater tissue collection (Sigma-Aldrich). The study on plant complies with relevant institutional, national, and international guidelines and legislation. The collection of P. oceanica material was performed in line with the guidelines stated in the permit granted by the city of Leghorn to the University of Pisa in order to collect plant material for research purpose (Determinazione N.3030 del 29/05/2020).A total of 70 Cymodocea nodosa and Zostera marina38, but never in P. oceanica. Thus, we conducted a detailed phylogenetic analysis to retrieve the closest homologs of Arabidopsis thaliana circadian genes in Posidonia and compare their relatedness among various angiosperms, both monocotyledons and eudicotyledons, which thrive in terrestrial or aquatic environments. We focused on genes whose oscillatory expression leads to the establishment of the main endogenous circadian rhythm in A. thaliana: CIRCADIAN CLOCK ASSOCIATED 1 (CCA1), REVEILLE (RVE8), PSEUDO-RESPONSE REGULATOR 5 (the two orthologues PRR5-1 and PRR5-2) and GIGANTEA (GI), which constitute the morning loop, and LUX ARRHYTHMO (LUX1), EARLY FLOWERING 3 (ELF3) and ZEITLUPE (ZTL), which, instead, participate to the evening loop39. We did not find evidence of a TOC1 gene in Posidonia as the closest homologous of the Arabidopsis TOC1 was more similar to the PRR5 gene family. We also performed a similar phylogenetic analysis on the flowering regulator CONSTANS (CO), whose expression is tightly controlled by the endogenous clock in terrestrial angiosperms33.To the best of our knowledge, putative circadian clock genes and the daily fluctuation of the respective mRNAs have been only recently studied in two seagrass species, Posidonia and Zostera marina as examples of aquatic monocots, Nelumbo nucifera, the so-called ‘sacred lotus’, for the aquatic eudicots, Arabidopsis thaliana as terrestrial eudicot and Oryza sativa for the group of terrestrial monocots. Because of their key phylogenetic position in the evolution of plants on land, we also included the lycophyte Selaginella moellendorffii and the liverwort Marchantia polymorpha, whose homologs of the core clock genes have been recently identified40. Protein sequences were retrieved applying the BLAST algorithm41 to search the Phytozome (http://www.phytozome.net), DDBJ/ENA/GenBank  and NCBI (https://www.ncbi.nlm.nih.gov) databases  returned the percentage of trees in which the associated taxa clustered together.We selected es Table  and Arab40 Table . The phyPosidonia, we then selected five representative genes , from the original group already analysed in45, and which are known to be involved in different aspects of the photosynthetic process. To test the hypothesis that excessive night lighting could cause photo-oxidative damage and, consequently, activate photoprotective mechanisms in Posidonia tip leaves, we analysed the expression of two photo-protection marker genes, Zeaxanthin epoxidase (PoZEP) and Photosystem II 22 kDa protein (PoPSBS), and of one photosynthesis-related gene, Ferredoxin-1 (PoSEND33), during the usual time course. Finally, we analysed the expression of Ribulose-bisphosphate carboxylase small chain 5B, PoSSU5B  and the expression of Alcohol dehydrogenase (PoADH), considered as a marker for various stresses in plants.Given the capacity of a continuous source of light at night to modify the oscillatory expression of circadian genes in Posidonia leaf tips, 2 cm long, according to the procedure described in46. The addition of fifty milligrams of polyvinylpyrrolidone (PVP) to each sample during the grinding step was the only modification introduced to the original protocol. Half microgram of RNA was subjected to DNase treatment along with cDNA synthesis, both performed with Maxima First Strand cDNA Synthesis Kit (Thermo Scientific) for subsequent quantitative real-time PCR amplification (RT-qPCR).Total RNA was extracted from 47 were used for PoPSBS, PoZEP, PoSSU5B, PoLHCA4, PoPSAG genes; primers from48 were used for PoCAB-6A, PoCAB-151, PoLHCB4.2 and PoSEND33. RT-qPCR primers for PoPRR5-1, PoPRR5-2, PoELF3, PoLUX, PoREV1, PoCCA1, PoZTL, PoGI, PoCO and PoADH were designed on Posidonia transcriptome available at DDBJ/ENA/GenBank  and their sequences are listed in Table Ubiquitin (GeneBank: GO347619) and 60S ribosomal protein L23 (GeneBank: GO347779), Posidonia housekeeping genes described in49, were chosen as reference genes in our experimental set up as their raw Ct values remained constant during the time course sampling  method50. Data were reported as log2 x−FC, where x is a value based on primers efficiency E (x = 1 + E/100) and FC stands for expression fold change of the tested gene, calculated as difference in ΔCt from the reference (average ΔCt for samples collected in Maroccone). Primers percent efficiencies (E) are listed in Table x−FC values are listed in Table RT-qPCR primers froming Fig. . The avea fluorescence with a submersible fluorimeter , on fully mature leaves, avoiding those covered by epiphytes, at 12:00, in mid-June 2019. Maximum quantum efficiency of PSII was determined as Fv/Fm, where Fv indicates the variable fluorescence, calculated as the difference between the maximum fluorescence after the saturation of the light pulse (Fm) and the minimum fluorescence (F0) after 5 min of dark adaptation (Table Maximum efficiency of photosystem II (PSII) of plants from Maroccone, Scalinata and Rotonda was evaluated in the field by measuring chlorophyll on Table . MeasurePosidonia plants were collected from the three sites of Rotonda, Scalinata and Maroccone, in mid-June 2019, transferred in glass tanks (aquaria) where they were acclimated for 2 weeks and sequentially grown under the same and controlled environmental conditions . The aquaria were filled with water collected in Maroccone at the same time when plants were also harvested. Growth rate was measured on youngest fully mature leaves, newly emerged in the aquaria system, using a modified Zieman's method51. Each plant was marked by punching all leaves together with a hypodermic needle, just above the ligula of the most external leaf. Growth was monitored over 14 days, by tracking the distance of the puncture mark from the ligula. Five biological replicates (shoots) were considered for Maroccone and six for Scalinata and Rotonda plants.52. For each site, gene expression was modelled as a cubic regression spline in the fixed part of the model. To account for the relationship between gene-expression and time among locations (light intensity), Site and Time (hours) were also included in the random part as random intercept and slope, respectively. GAMMs were fitted using the gamm function of the package gmcv, in R 3.5.1. Model assumptions were assessed visually using plots of residuals vs. fitted values, box-plots of residuals vs. experimental conditions (Site and Time) and QQ-plots of standardized residuals vs. normal quantiles53. Because study sites may be characterized by additional confounding environmental factors aside from ALAN, we performed GAMM on residuals of gene expression. For this purpose, long-term means (2000–2014) of daily maxima of Surface Sea Temperature (SST), Chlorophyll a concentration, Photosynthetic Active Radiation (PAR), diffusion attenuation coefficient, nitrate concentrations, phosphorous concentrations, dissolved O2 and iron concentration were retrieved for each study site from Bio-ORACLE database54. We finally regressed the gene expression against the linear combination of all environmental variables and extracted the residuals that were used as response variables in the analysis with GAMM. A linear model was used to estimate differences in maximum photosynthetic efficiency and growth rate between sites characterized by different night light intensities. This analysis was performed using the lm function of the package stats, in R 3.5.1.Generalized additive mixed effect models (GAMMs) were used to evaluate the effect of light intensity on time series of residuals of gene expression derived from the regression-adjusted analysis. GAMMs allow flexibly in inferring the relationship between a response variable and a number of predictors and to deal with auto-correlated observations and complex variance–covariance structuresArabidopsis homolog showed a separation of Posidonia and Z. marina PRR5s from those of rice, lotus and Arabidopsis : Maroccone, Fig. PoLUX1 showed a rhythmic expression in its transcriptional level only under dim and intense night light conditions  appeared comparable in comparison to dark conditions , while the rhythmic expression completely disappeared with intense ALAN . A loss of rhythmic expression in presence of dim night light in Scalinata was observed in case of PoZTL   significantly fitted into a mathematical model describing an oscillatory curve only for SAG Fig. . Rhythmi6A Table . Unexpecse) Fig. .Figure 4Posidonia. Data collected in situ on dark-adapted leaves showed a significant decrease in Fv/Fm values measured on plants at Scalinata  in comparison to the control dark conditions in Maroccone   maximum efficiency of on) Fig. , Table 2Posidonia physiology when growth conditions are restored to dark nights. Therefore, we measured growth rates of Posidonia plants collected from the same three sites where we collected samples for gene expression analyses and measured chlorophyll fluorescence. To this end, the plants were collected from the three sites making sure that they retained a sufficient portion of their rhizome to enable survival, and they were subsequently installed and acclimatised at the bottom of tanks (aquaria) containing sea water from the Maroccone site. We aimed to collect plants when temperature, photoperiod and moon phase were similar to those at the time of our first sampling, for gene expression analyses. This was only possible 2 years later, in summer 2019. We confirmed that values of ALAN and sky brightness during new moon nights were comparable between 2017 and 2019. Indeed, the source of nocturnal urban lighting remained constant during the 2 years, both in terms of spectrum and intensity. The plants were grown for 2 weeks to measure the elongation rate of leaves produced after the transplant. In a controlled environment, with absence of any source of ALAN, plants harvested from Rotonda grew significantly less (with about 35% reduction) than those from Maroccone    in the absence of any source of nocturnal lighting, in our laboratory , we attested the acquisition of an oscillatory pattern by ion Fig. B. Since ory Fig. B. We con26 or negative25, reflecting the complexity of species-specific processes and mechanisms involved in plant growth. In the model plant Arabidopsis, sugar and starch consumption, as well as their allocation throughout the plant, are under the control of the circadian clock; in particular, sugar consumption is regulated antagonistically by the morning protein CCA1 and the evening complex ELF3–ELF4–LUX74. Due to the observed effect of ALAN on PoELF3–PoLUX pattern of expression, we hypothesise that, similarly to Arabidopsis, clock-controlled metabolic pathways might be responsible for the observed growth reduction in the seagrass Posidonia oceanica. PoADH could have a possible role in this, as we observed that intense nocturnal lighting negatively affected its expression , could indicate a metabolic impairment and a more susceptibility to other stressors , in response to ALAN.The few studies that have investigated the influence of ALAN on terrestrial plants reported temporally variable, species-specific and light source dependent outcomes. In particular, observed effects on growth-related variables were either positive33. Although the molecular regulation of flowering in Posidonia has not been characterised yet, and its occurrence in the Northern Mediterranean is far more variable and rarer than in southern populations, so far only heating has been correlated as an environmental stressor able to alter flowering time80. In our dataset, we observed that the best Posidonia homologous of the Arabidopsis CONSTANS was unlikely to be affected by ALAN at any of the study sites  under an irradiance of about 6.6 lx at night (80 nmol m2 s1)83. Our results showed a relation between an increase in intensity of dim artificial light and the pattern of expression of genes putatively related to the circadian clock in a marine angiosperm colonizing shallow coastal waters. In particular, they suggest an impairment in the mechanisms related to the control of gene expression during the day/night cycle , but not as dim as around 1–5 lxPosidonia oceanica, which provides habitat and food for invertebrates, fish, mammals, birds and sea turtles, nutrient recycling and sediment stabilization20. Seagrasses are key ecosystem engineers in both subtidal and intertidal coastal areas worldwide. A variety of human-related stressors have been recognized as causes of the loss of coastal seagrass meadows and the services they provide, with a rate of disappearance comparable to those of mangrove forests and coral reefs86. As ALAN frequently co-occurs with a number of human-related disturbances in urban areas, our results pose key questions about the potential for light pollution to cumulate additively or to interact synergistically or antagonistically with these stressors88, in order to develop more efficient strategies to preserve seagrass ecosystems in coastal areas.Artificial light pollution is currently impinging on more than 20% of world’s coasts, and this percentage is set to increase with the growth rate of coastal human population. The emergent interest on potential impacts of ALAN on marine environments is showing the wide variety of effects related to the disruption of circadian rhythms on different organisms and habitats, from the molecular to the community level. Here we present evidence on the molecular and physiological effects of light pollution on a model seagrass species, Supplementary Information 1.Supplementary Information 2.Supplementary Information 3.Supplementary Information 4.Supplementary Information 5.Supplementary Information 6.Supplementary Information 7.Supplementary Information 8.Supplementary Information 9.Supplementary Table S1.Supplementary Table S3.


Cytotoxicity test results depend on the composition of the scaffolds and showed satisfactory cell growth with visible cell accumulation on the hybrid surfaces. The constructed hybrid scaffolds have suitable high-performance properties, suggesting potential for further in vivo and clinical studies.The idea of this study was to create a new scaffolding system based on 2-hydroxyethyl methacrylate, gelatin, and alginate that contains titanium(IV) oxide nanoparticles as a platform for the controlled release of the bioactive agent curcumin. The innovative strategy to develop hybrid scaffolds was the modified porogenation method. The effect of the scaffold composition on the chemical, morphology, porosity, mechanical, hydrophilicity, swelling, degradation, biocompatibility, loading, and release features of hybrid scaffolds was evaluated. A porous structure with interconnected pores in the range of 52.33–65.76%, favorable swelling capacity, fully hydrophilic surfaces, degradability to 45% for 6 months, curcumin loading efficiency above 96%, and favorable controlled release profiles were obtained. By applying four kinetic models of release, valuable parameters were obtained for the curcumin/PHEMA/gelatin/alginate/TiO The topic of developing advanced biomaterials that play an irreplaceable and highly effective role in improving the healing process and tissue regeneration has been a very intense scientific field for decades. In this field of research, hydrogels are considered leading materials that can provide many benefits and excellent outcomes for healing ,2,3,4,5.The creation of efficient polymeric systems for the controlled release of bioactive agents is inextricably linked with the design of potent scaffolds embedded with biomolecules. Therefore, such systems should exhibit advanced biological and therapeutic activities. For these purposes, both natural and synthetic origin polymers are the most used due to their superior properties for the ability to form a 3D hydrophilic structure with a favorable water/biological fluid content and to enable the population cells into their porous structures, as well as load and controlled release various bioactive agents . Polymer2) has become the focus of scaffold design, with improved biological activity due to non-toxicity, high biocompatibility, bioactivity, ability to support adhesion and proliferation of cells, excellent mechanical stability, and availability in large quantities [Inorganic compounds have been used to obtain special classes of biomaterials for hydrogel scaffolds capable of loading and releasing bioactive agents. The incorporation of a rigid inorganic compound may enhance the mechanical strength and, in some cases, the biodegradability of biomaterials. In recent years, as an important inorganic compound, titanium(IV) oxide  and active agent-curcumin (from Curcuma longa (Tumeric), powder, quality level 200, assay > 65%, Sigma–Aldrich, St. Louis, MO, USA). N-Ethyl-N′-(3-dimethyl aminopropyl)carbodiimide hydrochloride  as the crosslinking agent of gelatin was purchased from Sigma–Aldrich, St. Louis, MO, USA. RPMI-1640 medium and supplements for cell proliferation as well as 3--2,5-diphenyltetrazolium bromide (MTT) reduction assay components were purchased from Sigma–Aldrich, St. Louis, MO, USA. Potassium hydrogen phosphates  were used for buffer preparations. All experiments were performed using lab-produced, ultra-distilled water.Synthetic monomer 2-hydroxyethyl methacrylate  and natural origin polymers-alginate  and gelatin  were purchased from Sigma–Aldrich, St. Louis, MO, USA. N,N′-methylene bisacrylamide  was used as a crosslinking agent of HEMA. Pluronic F-172 (reagent grade 98%), ammonium persulfate  and N,N,N′,N′-tetramethylene diamine  were obtained from Sigma–Aldrich, St. Louis, MO, USA and used in all polymerizations as a foaming stabilizer, initiator, and activator, respectively. Porogen agent-sodium bicarbonate, inorganic compound titanium(IV) oxide nanoparticles   and washed with deionized water for 7 days to remove unreacted components. Samples were placed in a deep freezer (−80 °C) and lyophilized (−55 °C) for further characterization.Hybrid scaffolds based on synthetic monomer HEMA, polymers-gelatin (G), and alginate (A) and TiOte ratio . HEMA, 2−1 on an FTIR Nicolet 6700  diamond crystal spectrometer with attenuated total reflectance (ATR) sampling technique.The chemical structure of hybrid scaffolds was determined by FTIR spectra recorded over the wavelength range of 700–4000 cmThe morphology of hybrid scaffolds was observed by a scanning electron microscope EOL JSM-7600F. The lyophilized samples were previously sputtered with gold by BAL-TEC SCD 005 and dried in a VC 50 SalvisLab Vacucenter vacuum chamber.0 = 1.2038 g/cm3) was used as a wetting medium [The porosity of hybrid scaffolds was calculated by the solvent replacement method, using the true and bulk density of the hybrid scaffolds as well as the density obtained by the Archimedes method, where glycerol  on cylindrically shaped specimens that were accurately measured for their dimensions (diameter and height) before testing. The specimens were compressed uniaxially with a 100-N load cell at a crosshead speed of 0.5 mm/min at room temperature. The elastic moduli were determined from the slope of the linear elastic region of the stress-strain curves.The swelling capacity of hybrid scaffolds was monitored in a simulated physiological conditions-phosphate buffer of pH 7.40 at 37 °C, calculating the degree of swelling by gravimetric method ,32,33. AThe static water contact angle measurement was analyzed on a Theta Lite-Biolin Scientific Contact angle meter in a measuring range from 0 to 180 deg. and accuracy ±0.1 deg., ±0.01 mN/m, applying the sessile drop method by putting approximately 1 μL drop of MilliQ water on the scaffold’s surface. All experiments were performed in triplicate.In vitro degradation studies were conducted by the gravimetric method. Samples were placed into a 10 mL buffer pH of 7.40 at 37 °C for 3 and 6 months, removed from the buffer and dried at room temperature to constant weight. Degradation was expressed as a percentage of sample weight loss . All expv/v) scaffold extracts and 200 µg/mL of the ground and filtered scaffold and incubated for an additional 48 h. Control cultures were treated with a growth medium. Cell proliferation was calculated by measuring absorbance at 540 nm using the Tecan Infinite 200 Pro Multiple Reader  [Cytotoxic/antiproliferative activity of the hybrid scaffolds was determined using human lung fibroblasts (MRC5 cells) and the standard MMT assay . MRC5 cezerland) ,36. ImagCurcumin was selected as a bioactive agent and loaded into hybrid scaffolds using the swelling-diffusion method. The hybrid scaffolds were immersed in 5 mL of curcumin solution to reach an equilibrium swelling state . The potmax value of 430 nm in determined time intervals.Hybrid scaffolds loaded with curcumin were placed in a basket stirrer containing 15 mL of release medium (phosphate buffer pH of 8.00) at 37 ± 0.5 °C. UV/VIS spectrophotometer (Shimadzu UV/Vis Spectrophotometer UV-1800) was used to measure the concentration of curcumin released from the hybrid scaffolds by taking the absorbance of the released media at λTo reveal the transport mechanisms and curcumin release kinetics from hybrid scaffolds, the first 60% of the in vitro obtained curcumin release data were fitted using different release kinetic models Higuchi, Ritger–Peppas, Peppas–Sahlin and Peppas–Sahlin model (where m = 0.5) ,38,39,40The Origin Pro 9.0 software was used to analyze data. The data are expressed as mean ± SD.New hybrid scaffolds loaded with bioactive agent curcumin were prepared by modified porogenation method ). Gelatin was crosslinked by EDC and intertwined with linear alginate chains  to creat2 was detected by Fourier transform infrared spectroscopy (FTIR). 2. The FTIR spectra of all samples confirmed the presence of PHEMA component by typical peaks for O–H stretching vibration at around 3432 cm−1, C–H stretching vibrations at 2930 and 2886 cm−1, and strong C=O vibration at 1717 cm−1. The FTIR spectrum of PHEMA/A/TiO2 showed peaks for PHEMA, peaks of alginate C–O stretching at 1278 cm−1, C–C stretching at 1160 cm−1 and C–O–C stretching at 1017 cm−1 [2 at 3354 cm−1, 3003 cm−1, and 1630 cm−1 related to N–H stretching vibration, C–H stretching vibration, and N–H stretching vibration for the amide II, indicating the presence of gelatin in the scaffold [2 and PHEMA/G/TiO2 around 3500 cm−1 corresponded to the stretching vibration of the hydroxyl group O–H of the TiO2 and the appearance of the peaks at 1630 cm−1 and 1300 cm−1 corresponded to bending modes of water Ti–OH and Ti–O modes, confirming an embedding of TiO2 in the samples [The chemical structure of hybrid scaffolds based on PHEMA, gelatin, alginate, and TiO017 cm−1 ,42. TypiThe porous morphology is a leading property for the scaffold design and capability for loading bioactive agent and releasing it. This porous morphology is imperative for scaffolding biomaterial to provide host-cell population, neovascularization, cell migration, and flow of nutrients and oxygen for cell proliferation and tissue regrowth ,45,46,472, indicating that the morphology of the hybrids can be modulated by varying their content. Thus, the morphology of the hybrid scaffolds can be easily adapted to the specific requirements of their final biomedical applications.Since the goal of this study is to design new scaffolding biomaterials with great potential to load and release bioactive agents, the morphology of the hybrid scaffolds was investigated by SEM. The obtained SEM micrographs of the cross-section for the hybrid scaffolds  show wel2 hybrid scaffolds are in the range of 52.33% to 65.76% (2 and PHEMA/A/TiO2 samples) into the hybrids leads to an increase in porosity because segments of the linear chains affect the structures of the polymeric networks of PHEMA/gelatin in terms of increasing the free space within the scaffold. Hybrid scaffolds containing alginate have a porosity above 60%, which is very suitable for their biomedical applications.The porosity offered by these types of hybrid scaffolds results in improved tissue binding ability due to the high surface-to-volume ratio inducing bioactivity. Interconnected pores provide a scaffold for tissue growth in the scaffold matrix and anchor them to the surrounding tissue, preventing micro-movements which in turn increase further tissue growth. Interconnected porosity is also a source of oxygen and nutrient supply and helps in vascularization and waste removal. Porosity is an essential property for assessing the internal structure and free spaces of scaffolds and is expressed as the percentage of free space within the total volume of biomaterial construct. It was noticed that inadequate porosity of the scaffold leads to inappropriate cell function inside the scaffold . The appropriate porosity of scaffolds for successful cell populations is above 50% and depends on the type of cells ,47,48,49o 65.76% . The infThe swelling capacity of hydrogels is an essential characteristic for evaluating the potential of hydrogel scaffolds for the design of a platform for the efficient release of bioactive agents for biomedical and tissue engineering applications and considering its influence on the transport of nutrients and oxygen within the scaffold . Swellin2 hybrid scaffolds are presented in 2 hybrid scaffolds and the water drop immediately disappeared after placing it on the hybrid scaffold surface. This behavior classifies the PHEMA/G/A/TiO2 hybrid scaffolds as highly convenient scaffolding biomaterials for efficient adhesion, proliferation, and differentiation of various types of cells as well as for tissue engineering applications.The hydrophilic/hydrophobic nature of the surface of biomaterial is a significant property that affects the interaction and response of cells and can be analyzed by the water surface contact angle. In the case where obtained water surface contact angle is above 90°, the surface of the scaffold is considered hydrophobic while when the obtained angle is less than 90° degrees, the surface is considered hydrophilic. Versatile scientific studies confirmed that cells thrive greater on scaffolding biomaterials with hydrophilic surfaces which promote better cell adhesion and proliferation ,51,52. I2 hybrid scaffolds was monitored in simulated physiological conditions (37 °C and pH of 7.40) for 3 and 6 months. The obtained results of degradation (2 samples) leads to increased degradation compared to PHEMA/G/TiO2 scaffold. This phenomenon could be explained as an effect of linear alginate chains (which are not crosslinked) leaching out of the polymeric network. Also, the influence of hydrophilic groups from the components that build the scaffolds and the interaction with the water medium contributes to the degradation process. Therefore, the degradation behavior of the hybrids can be tuned by varying scaffold composition. Scaffold degradation data are correlated with porosity values. These results are in agreement with the finding that adjusting the gelatin crosslinking can influence the degradation of biomaterials [The degradability of scaffolding biomaterials is also a significant property for biomedical applications to enable cell and/or tissue regeneration in vitro/in vivo conditions ,54. Duriradation  are in taterials .2 hybrid scaffolds were tested and obtained results of Young’s modulus are presented in 2 hybrid scaffolds due to the higher crosslinked gelatin content and lower porosity, which contributes to the mechanical strength. The addition of linear polymer alginate to the composition of the hybrid scaffolds results in lower values of Young’s modulus, which may be suitable for soft tissue regeneration. The obtained data for the mechanical properties follow the porosity results  but limited clinical uses due to poor water solubility and low bioavailability. Because the formulation of active agents poorly soluble in water represents a current challenge in modern pharmacy and biomedicine, this study tends to create an efficient platform for the successful formulation of a bioactive agent without the use of toxic organic solvents. Obtained data for curcumin loading (DL) and entrapment efficiency (EE) of the PHEMA/G/A/TiO2 hybrid scaffolds are presented in 2 can load the lowest amount of curcumin 52.01 mg curcumin/g scaffold because of its lower swelling capacity compared to the scaffolds based on PHEMA/A/TiO2 better loading capacity (a higher amount of curcumin 53.54 mg curcumin/g scaffold) due to favorable swelling performances. By introducing gelatin into the hybrid scaffolds (gelatin as component crosslinked PHEMA/G network), the swelling capacity becomes lower resulting in a reduced capacity of the polymeric network to absorb curcumin. The better swelling capacity of scaffolds facilitates the diffusion of the bioactive agent through the polymeric network and improves embedding into pores which results in higher values of DL and EE. Obtained values of EE for the hybrid scaffolds are in the range of 96.02% to 98.84%, suggesting the efficacy of the hybrids to load curcumin where desired formulation can be achieved by varying the composition of scaffolds.A new generation of scaffolding biomaterials for biomedical engineering applications tends toward sophisticated scientific advances in the tissue regeneration process, not only by supporting cell proliferation but also by targeted/local delivery/release of bioactive agents essential as a natural additional support for tissue formation of targeted tissues. In that regard, the ability of PHEMA/G/A/TiO2 hybrid scaffolds as release platform wasevaluated by in vitro curcumin release study in a simulated physiological microenvironment. Obtained release profile data are presented in 2 hybrid scaffolds. As shown in 2 and PHEMA/A/TiO2 samples due to significant differences in their swelling capacity. The curcumin release rate increases with increasing the alginate content which is consistent with obtained swelling data. Curcumin releases up to 24 h with a short initial “burst” period (around 2 h) for all hybrid scaffolds. It is also observed that the hybrid scaffolds loaded with a higher amount of curcumin showed faster release due to the presence of different curcumin loading levels inside scaffold pores, which facilitate the diffusivity of the bioactive agent [The efficiency of PHEMA/G/A/TiOve agent .To determine the mechanism of curcumin release from the hybrid scaffolds, four kinetics models were applied on obtained release data . Calcula2 hybrid scaffolds, the sum of the squared residuals (SSRs) and Akaike Information Criterion (AIC) was calculated and analyzed using four release kinetic models  point out that the curcumin transport mechanism is Fickian diffusion for prepared PHEMA/G/A/TiO2 hybrid scaffolds. Additionally, to determine the contribution and synergistic effect of two different mechanisms (Fickian diffusion and polymeric chain relaxation) of curcumin release from hybrid scaffolds Peppas–Sahlin model was used. The calculated values of the kinetic constants K1 (define Fickian transport contribution) and K2 (define polymeric chains relaxation contribution) indicate that the Fickian transport is the dominant mechanism for the curcumin release from all obtained PHEMA/G/A/TiO2 hybrid scaffolds (K1 > K2).To determine the mechanism and kinetics of curcumin release, various release models were applied ,66,67,68c models . The mod2 were synthesized by a modified porogenation method. The physicochemical testings of the PHEMA/G/A/TiO2 hybrid scaffolds revealed that the composition variation permits tailoring of all properties of scaffolds for different areas of biomedical applications. The porous structure with interconnected pores and tunable porosity values up to 66%, and in vitro degradation grows up to 45.89% after 6 months. Fully hydrophilic surfaces and in vitro cytotoxicity assay on MRC5 cells with visible cell accumulation on the surface of the scaffolds were obtained. Excellent curcumin loading and release performances with entrapment efficiency above 96% are powerful properties for their potential biomedical application as scaffolds with improved bioactivity. The efficient curcumin/PHEMA/G/A/TiO2 release platform was designed. Valuable release parameters were obtained for the curcumin/PHEMA/G/A/TiO2 release platform using four release kinetic models. Favorable properties of new PHEMA/G/A/TiO2 hybrid scaffolds for biomedical applications recommend further detailed testings with a variation of TiO2 fraction, as well as in vivo and clinical studies in the future research.New hybrid scaffolds based on PHEMA/G/A/TiO


N = 2229; 50.7% girls) from the longitudinal TRacking Adolescents’ Individual Lives Survey (TRAILS) in the Netherlands were used . At T1, adolescents reported on their mothers’ and fathers’ overprotection. From T1 to T3 adolescents and teachers reported about internalizing problems, academic achievement, prosocial, and antisocial behavior. The results showed concurrent associations between higher levels of perceived overprotection and higher levels of internalizing problems, antisocial behaviors, and  lower levels of academic achievement. Perceived overprotection was positively associated with decreased internalizing problems over time. This longitudinal association disappeared after controlling for baseline levels of internalizing problems, suggesting that this result was less robust than expected. Mothers and fathers did not differ in their associations between perceived overprotection and adaptive functioning. The findings showed that perceived overprotection is mainly concurrently associated with adaptive adolescent functioning. Future research recommendations are discussed in terms of stability and bidirectional relations.Although parental overprotection is theorized to have lasting negative effects throughout a child’s life, there is limited empirical evidence available on its long-term significance on adolescent well-being. This preregistered, three-wave longitudinal study investigated the association of maternal and paternal perceived overprotection in early adolescence with the development of adaptive psychological, academic, and social functioning throughout adolescence. Data ( This preregistered longitudinal study investigates associations between maternal and paternal perceived overprotection in early adolescence (mean age 11) and the development of adaptive psychological, academic, and social functioning throughout adolescence (11 to 16 years).It is well-established that parental involvement, support, and protection are related to better wellbeing in adolescents , adolescence is characterized by a growing desire for autonomy and independence. Over time, adolescents become more independent of their parents during adolescence as they spend an increasing amount of time outside the family context .Through a multidimensional approach with a focus on both maladaptive and adaptive adolescent functioning, addressing overprotection of both fathers and mothers, this study aims to better understand some of the heterogenous findings on how overprotection relates to adolescent functioning. This preregistered three-wave study examined the longitudinal relation between maternal and paternal overprotection (as perceived by adolescents) and the development of psychological functioning , academic functioning (represented by academic achievement), and social functioning  in adolescents. The first research question entailed how levels of mothers’ and fathers’ perceived overprotection at age 11 are related to (a) the initial level of and (b) the change in adolescent outcomes from age 11 to 16. Perceived overprotection is expected to be related to higher initial levels of internalizing problems (hypothesis 1a), lower initial levels of prosocial behavior (hypothesis 2a), and higher initial antisocial behaviors (hypothesis 3a) as well as stronger increases in internalizing problems (hypothesis 1b), stronger decreases in prosocial behavior (hypothesis 2b), and stronger increases in antisocial behavior (hypothesis 3b) between age 11 to 16. No a priori hypotheses were formulated regarding academic achievement. The second research question pertained to the exploration of parental gender differences in the associations between perceived overprotection and (a) the initial level of and (b) the change in adolescent outcomes. This was answered without a priori hypotheses. This study was preregistered at the Open Science Framework  , Time 2 , and Time 3 . The total sample in this study consisted of 2229 participants (50.7% girls), with different sample sizes based on different time points and measures , 66.4% of the mothers and 59.1% of the fathers had a medium educational level (finished lower or higher tracks of secondary education), and 26.8% of the mothers and 35.2% of the fathers were highly educated . Of the total sample, 74.2% of the parents were married and lived together, 13.1% were divorced, 8.3% were never married, 1.4% were widowed, and 1.0% was married but did not live with their spouse. In total 89.4% of the sample consisted of adolescents from which both parents were born in the Netherlands, and for 10.6% at least one parent was born in another country than the Netherlands .No, never to 4 = Yes, always), for mothers and fathers separately. Cronbach’s alphas in this sample were 0.70 for mothers and 0.71 for fathers. Sufficient support for the factorial and construct validity of the EMBU-C has been established  through the overprotection subscale of the short version of the Egna Minnen Beträffande Uppfostran (My Memories of Upbringing) for Children (EMBU-C) Markus, . The 12  Markus, , also us Markus, . The one Markus,  was repl(not true), 1 (somewhat/sometimes true), or 2 (very/often true). Cronbach’s alphas in this sample were 0.87 (T1), 0.88 (T2), and 0.89 (T3). The YSR was found to be adequately valid and reliable , work pace , and effort , by means of five items in T1 and nine items in T2 and T3. This teacher questionnaire was developed for TRAILS. The different number of included items in the different waves accommodate for the different number of subjects in the Dutch school system in T1 compared to T2 and T3. The questionnaire was filled out by the primary school teacher at T1, and the secondary school teacher (“mentor”) at T2 and T3. Generally, a mentor also teaches a specific subject, but is assigned to a specific class to keep track of the student’s progress and to provide guidance through additional class hours. Exploratory factor analyses in earlier work on the same dataset . Mean scores on the items were calculated. Cronbach’s alphas in this sample were 0.93 (T1), 0.92 (T2), and 0.93 (T3), comparable to previous TRAILS studies  rated adolescents’ prosocial behavior with the Prosocial Behavior Questionnaire (PSBQ) which consisted of eleven items from an earlier prosocial behavior questionnaire . Mean scores on the items were calculated. Cronbach’s alphas in this sample were 0.86 (T1), 0.85 (T2), and 0.86 (T3), comparable with previous TRAILS studies  were included in the analyses . Background information about the family and the adolescent, were added as covariates in the main and sensitivity analyses. Background family information  was measured through the parent interviews at T1. Family socio-economic status (SES) was measured as the z-standardized mean score of mothers’ and fathers’ occupational position  and rejection  at T1 using the subscales Warmth (18 items) and Rejection (17 items) of the EMBU-C Markus, . Cronbacχ2 = 2136.60, χ2/df = 1.38). As the chi-square test is quite sensitive to sample size, the normed (or relative) approach was applied by dividing the chi-square by the df  of the variables of interest was used. To assess the missing data patterns . To address multicollinearity issues, the analyses for research question 1 were performed with a composite (combined) variable for perceived overprotection of father and mothers.To answer the research questions as preregistered, we performed Latent Growth Curve Modeling  was also tested separately to examine parent gender differences more closely. Child and family background characteristics were included as covariates in the models by estimating regression paths with the intercept and slope factors of the outcomes. Sensitivity analyses were performed for research question 1 and research question 2 (as preregistered) by adding the subscales Warmth and Rejection from the EMBU-C.2), the Comparative Fit Index (CFI), the Tucker-Lewis Index (TLI), and the Root Mean Square Error of Approximation (RMSEA). A good model fit is indicated by CFI and/or TLI values close to 0.950 and not below 0.900, and/or RMSEA values below 0.080  was used. Overall fit of each model was determined by the chi-square  was used as an indicator of effect size Cohen’s d.Hypotheses were confirmed if the regression paths from perceived overprotection to the intercept and/or slope factors of each outcome were significant on a wilowsky  . In additional analyses adolescent sex differences in initial levels and development of internalizing problemsTo answer research question 1, parental perceived overprotection was added as predictor of the estimated intercept (T1 level) and slope (rate of change) of the outcomesPerceived overprotection was not related to initial levels or the rate of change in adolescents’ academic achievement (no a priori hypotheses), or prosocial behaviors (hypothesis 2a and 2b). Finally, perceived overprotection was positively related with initial levels , and not related to the change (hypothesis 3b) of antisocial behavior.Preregistered sensitivity analyses in which parental warmth and rejection were controlled for , demonstrated similar results for the association between perceived overprotection and  adolescent internalizing problems (hypothesis 1a), but a somewhat different pattern for the other outcomes . No significant group differences were retrieved when comparing the overall model fit of the models Table . When exSensitivity analyses  showed that mothers and fathers did not differ in their pathways from perceived overprotection to adolescent functioning, when controlled for maternal and paternal warmth and rejection. Comparing overall model fit (paths freely estimated versus constrained) yielded no significant group differences Table . MothersNormative developmental changes during adolescence require that parents relinquish guidance, protection, and control over their child to promote their child’s healthy functioning later in life. However, certain levels of protection may not be developmentally appropriate, and may frustrate adolescents’ psychological needs and may thus be detrimental for adolescent functioning on various developmental domains. Empirical research demonstrated that overprotection was concurrently linked to maladaptive functioning in adolescents, but longitudinal research is still scarce  at age 11. After controlling for parental warmth and rejection, perceived overprotection was also associated with lower levels of adaptive functioning (academic achievement) at age 11, but no longer associated with antisocial behavior. Except for internalizing problems, parental perceived overprotection was not related to longitudinal changes in adolescent functioning . This longitudinal association disappeared after controlling for baseline levels of internalizing problems, suggesting that this result was less robust than expected. Moreover, mothers and fathers showed no differences in their associations between perceived overprotection and adolescent functioning. These key findings, the potential implications, and directions for future research will be discussed below.To yield a more complete picture of adolescent functioning, both maladaptive and adaptive outcomes of adolescent functioning were examined. Altogether, it was found that at age 11 perceived overprotection was positively related to maladaptive and, when controlling for related positive and negative parenting behaviors, negatively related to adaptive functioning. As such, this study confirmed many of the findings of earlier work on overprotection and maladaptive functioning  are fulfilled through positive parenting practices, such as warmth, but not met through negative parenting practices, such as rejection. Sensitivity analyses showed that parental warmth and rejection were related to initial levels and development of adolescents’ psychological, academic, and social functioning. Moreover, the results showed that the associations between perceived overprotection and maladaptive functioning were attenuated, and its associations with adaptive functioning were strengthened when accounting for other positive and negative parenting behaviors (warmth and rejection). These findings highlight that accounting for other parenting behaviors is important, as it may provide additional knowledge on the extent to which overprotection is related to adolescent functioning.Between ages 11 and 16, adolescents, on average, showed lower levels of internalizing problems, academic achievement, prosocial behavior, and antisocial behavior. Yet, the effect sizes of the decreases were rather small. Although the decrease in internalizing problems was unexpected, additional analyses showed that this decrease was mainly observed in  internalizing problems in boys. Girls showed an increase in anxious and affective problems over time. These findings are in line with research on sex differences in internalizing problems throughout adolescence, namely, that internalizing problems from childhood on continue to increase in girls, but in boys they decline from adolescence on adaptive functioning in adolescents, also when accounting for parental warmth and rejection. These findings are in contrast to previous studies in which associations between overprotection and maladaptive functioning differed in mothers and fathers  for the T3 measures were included, the use of solely questionnaires could have been strengthened by employing a multi-method study design. Overprotection was measured through one source (self-reports) only. Yet, adolescent self-reports may serve of great significance, as associations between overprotection and adolescent well-being may highly depend on the extent to which adolescents actually feel that their parents are overprotective.Some methodological and theoretical notions should be taken into account when interpreting the results of teacher-reported prosocial behavior and academic achievement. Between T1 and T2, adolescents transitioned from elementary school to secondary school. Hence, different teachers reported on adolescents’ prosocial behavior and academic achievement throughout the study. Primary school teachers have more opportunities to get to know and to observe their students, as they are the students’ parttime or fulltime teacher. The “mentor” in secondary school (which reported at T2 and T3) meets their students a few hours per week. Thus, the results may be confounded by the fact that teachers at T1 differed in their opportunities to observe adolescents’ social interactions and academic behaviors compared to the teachers at the later assessments.A last limitation concerns the generalizability of the results. A large proportion (approximately 90%) of the current study sample had a native Dutch background. In some cultures overprotection may be perceived as less autonomy-limiting, and more as legitimate involvement . Some of the associations disappeared or emerged when adolescents’ perceived warmth and rejection from their parents were taken into account, underscoring the multidimensionality of overprotective parenting. Future research using a more extensive longitudinal design, is needed to explore the development of parental overprotection and to unravel bidirectional relations between overprotection and adolescent functioning.Longitudinal research on the role of parental overprotection in relation to adolescent functioning is limited. By examining both fathers’ and mothers’ perceived overprotection in relation to maladaptive Supplementary InformationSupplementary Information


The aim of this study was to evaluate clinical results and prognostic factors in a cohort of patient with oligometastatic esophagogastric adenocarcinoma treated with stereotactic radiation therapy (SRT).This retrospective study included patients affected by 1–3 metastases treated with SRT from 2013 to 2021. Local control (LC), overall survival (OS), progression-free survival (PFS), time to polymetastatic dissemination (TTPD) and time to systemic therapy change/initiation (TTS) were evaluated.Between 2013 and 2021, 55 patients were treated with SRT on 80 oligometastatic sites. Median follow-up was 20 months. Nine patients had local progression. 1 and 3 years LC was respectively 92 and 78%. 41 patients experienced further distant disease progression, median PFS was 9.6 months, 1 and 3 years PFS was respectively 40 and 15%. 34 patients died, median OS was 26.6 months, 1 and 3 years OS was respectively 78 and 40%. During follow-up, 24 patients changed or initiated a new systemic therapy; median TTS time was 9 months. 27 patients experienced poliprogression, 44% after 1 year and 52% after 3 years. Median TTPD was 8 months. The best local response (LR), tyming of metastases and PS were related with prolonged PFS on multivariate analysis. LR was correlated with OS at multivariate analysis.SRT represents a valid treatment for oligometastatic esophagogastric adenocarcinoma. CR correlated with PFS and OS, while metachronous metastasis and a good PS correlated with a better PFS.In selected gastroesopagheal oligometastatic patients, SRT can prolong OS Local response to SRT, metachronous timing of metastases and better PS improve PFS.Local response correlates with OS. Despite advancements both in local and systemic treatment, approximately half of patients still relapse after primary curative treatment, particularly in node positive disease, and one-third of patients has already metastatic disease at diagnosis. Prognosis of Stage IV disease is generally bad, ranging from approximately 4 months with best supportive care to 12 months with chemotherapy.Esophageal and gastric cancer represents a major epidemiological issue and a relevant cause of cancer death. by definition histology agnostic. Recent prospective trials validated the use of local ablative treatments (LATs) including stereotactic radiation therapy (SRT) for these patients, with most data focused on lung, prostate and colorectal cancer. For esophagogastric cancer, randomized control trials are ongoing and the available evidences are derived from few non-randomized studies and retrospective series, mainly of surgical metastasectomy. A recent systematic review and meta-analysis, done by the oligometastatic esophagogastric cancer (OMEC) consortium, identified 16 studies reporting survival outcomes in patients receiving also LAT as part of their therapy. The same group analyzed 97 studies to derive a shared definition of oligometastatic esophagogastric cancer. The authors’consensus defined as OMD disease one organ with ≤3 metastases or one extraregional lymph node station; on the contrary, no consensus was reached about treatment strategies for this disease in a parallel project of the same consortium.Oligometastatic disease (OMD) is a distinct clinical state between localized disease and widespread metastatic disease, as first theorized in 1995,Moving from this definition of OMD esophagogastric cancer, we conducted this analysis on our patients, focusing on adenocarcinoma patients, to evaluate the impact of SRT in this scenario. We evaluated survival outcomes  and possible patients and treatment factors related to prognosis. We also estimated time to polymetastatic dissemination (TTPD) in patients experiencing a further disease progression and time to systemic therapy change/initiation (TTS), as novel endpoints of interest in the OMD setting. ; treatments received for Stage IV disease before SRT; date, dose and number of fractions of SRT; number and site of irradiated metastases; concomitant systemic therapies.From an institutional database collecting data of oligometastatic patients treated with SRT from 2013, we extrapolated data on esophagogastric adenocarcinoma patients affected by less than three metastases in one distant organ or one organ and one nodal station. For each patient, the following information were collected: date of birth; sex; date of primary cancer diagnosis; primary cancer site ; adenocarcinoma subtype; treatment of primary cancer; clinical or pathological stage of primary cancer; date of Stage IV diagnosis; imaging performed for OMD diagnosis; comorbidities; performance status (PS) according to Eastern Cooperative Oncology Group (ECOG); smoking habits; timing of metastases occurrence (synchronous metastases were defined as occurring <6 months from primary diagnosis); type of oligometastases according to ESTRO-EORTC classificationThe study was conducted with the approval of institutional review boards, and each patient signed an informed consent at SRT time to use data for future research.Patients were all treated with SRT with volumetric modulated arc therapy in its rapid arc form (VMAT-RA). Different dose and fractionation schedules were employed according to site and size of the lesion(s) and to regional organs at risk (OARs) dose constraints. A balance to maximize efficacy while minimizing toxicity was pursued in all cases.Biologically equivalent dose (BED) was calculated assuming a α/β ratio of 10 Gy. In case different metastases were treated with different doses in the same patient, the lowest BED per patient was used for the analysis. and according to PET Response Criteria in Solid Tumors (PERCIST). Acute and late toxicities were recorded and scored according to Common Terminology Criteria for Adverse Events (CTCAE) v. 5.0.Patients were regularly followed-up, generally with CT scan and or MRI 3 months after SRT and a PET/CT after 6 months. Thereafter, follow-up was scheduled according to response, disease status and international guidelines. Response to radiotherapy was scored according to European Organization for Research and Treatment of Cancer Response Evaluation Criteria In Solid Tumours (EORTC-RECIST) criteria v. 1.1Last day of SRT was utilized for survival analysis. OS was calculated until last follow-up visit or death from any cause, whatever it occurs first. LC was calculated until last follow-up visit or local recurrence. PFS included as events both local and further distant recurrence whatever occurs first. Further distant progression was classified as oligo- or polyprogression according to number of new/progressing metastases and number of involved organs  and date of polyprogression was used to estimate the TTPD. Subsequent systemic therapies received by patients were recorded and the date of change/initiation of the new therapy was used to estimate TTS.p-value < 0.05 obtained from UVA were included in the multivariate analysis (MVA) to search for independent predictive factors. Statistical analyses were performed using MedCalc for Windows v. 20.110 .All event occurrences were analyzed with the Kaplan–Meier method and the long-rank test was used for univariate analysis (UVA) to compare subgroups. Further, factors with Between 2013 and 2021, 55 patients were treated with SRT on 80 oligometastatic sites. Median age was 69 years (range 43–91). Most frequently, patients were treated on nodal metastases , followed by liver  and lung lesions . Only three patients were treated on one organ and one nodal station, precisely adrenal gland in two cases and liver in the other one. Considering nodal metastases, in all but two cases these were outside the field of a previous RT, if received. In most cases, patients were irradiated for a single metastasis, 2 lesions were simultaneously treated in 13 patients (23.64%) and 3 lesions in 6 cases (10.91%). More than half patients  received SRT as upfront therapy, with no other previous systemic treatment for Stage IV disease. Primary tumor, patients and oligometastatic disease characteristics are shown in During follow-up, we observed as best response 21 complete response (CR); 24 partial response (RP); 8 stable disease (SD) and 2 progressive disease (PD). An example of CR of brain metastases from esophageal cancer is shown in Treatment was generally well tolerated. No G3 or higher toxicity was recordedOne patient developed G2 pneumonitis few weeks after lung SRT. As acute side-effects, four other patients reported G1 toxicity . During follow-up, three other patients complained late toxicity .Toxicities linked with irradiated site and RT dose and fractionation are shown in With a median follow-up of 20 months (range 3–96), 34 patients died . At last follow-up, seven patients had no evidence of disease, five patients were alive with persisting disease in the irradiated lesion(s) and nine patients were alive with further distant metastases. Median OS was 26.6 months, 1, 2 and 3 years OS was respectively 78%, 56% and 40% .vs PR vs SD + PD [p < 0.0001]) and BED >75 Gy (p 0.0353) correlated with OS. At multivariate analysis, best local response maintained a predictive value, both considering CR compared with PR+SD+ PD  and comparing SD + PD vs CR+PR     .During follow-up, nine patients experienced a local progression, after a median time of 13.9 months (range 2.6–36.1). Median LC was not reached. 1 and 3 years LC was respectively 92 and 78% . At univFurther distant progression was the main pattern of failure. Indeed, 41 patients had a further disease diffusion . Median time to distant progression was 8 months (range 2.6–36.1). More than half patients  progressed initially again in an “oligo” way. Thirteen patients received a second course of local therapy (SRT or surgery), while 15 of them were treated with systemic therapy. The remnant patients were candidate to best supportive care.Considering both local and distant progression, whatever occurs first, median PFS was 9.6 months (range 7.9–12.9), 1 and 3-years PFS was respectively 40 and 15% .p 0.02), timing of metastases (p 0.0092), DFI > 493 days (p 0.0260) and best local response (p 0.0003) (LR) correlated with PFS. At multivariate analysis, best LR, PS and timing of metastases maintained statistical significance with a p-value < 0.001 . Polymetastatic progression free survival was 56% after 1 year and 48% after 3 years .24 patients change or initiate a new systemic therapy after SRT due to disease progression. Median TTS time was 9 months (range 5–43). New systemic therapy free survival was 68% at 1 year and 45% at 3 years . with the only exception of three patients that were affected by metastases in a visceral organ and in a single nodal station. Our data confirmed the excellent toxicity profile of SRT, high LC rates (78% at 3 years) and a promising OS (median 26.6 months), longer than the historical prognosis of patients treated with systemic therapy only. As commonly described in oligometastatic series, the major pattern of failure was further distant dissemination, with a median PFS of 9.6 months, similar to those reported with other histology in prospective Phase II trials.We report a single institution experience on the use of SRT for OMD from esophagogastric cancer, as defined by the OMEC consortium,Few data are available regarding esophagogastric cancer, compared to other primary tumors. Most of them come from surgical series or miscellaneous LAT. A pattern of failure analysis presented in 2020 analyzed 1438 patients, finding that 197 (13.7%) of them were oligometastatic (with a broader definition of the one used in our study). Interestingly, they also found that, of 152 pts who progressed, 91 (60%) had isolated initial site failure, 17% had isolated new site failure, and 23% had both initial and new site failure. Only 11% of patients were treated with RT to metastatic site, i.e. this high percentage of original site failure (83%) represents a good rationale to support LAT in this selected population. Indeed, original site failure in our experience occurred only in 16% of cases , suggesting that the use of SRT at least changed the trajectory of the disease, apart from prolonging survival.It is quite difficult to estimate the proportion of OM patients in esophagogastric carcinoma compared with all Stage IV patients. In the Agamenon surgical registry, the proportion of patients who underwent surgical metastasectomy compared to all patients in the registry with advanced adenocarcinoma of the stomach, distal esophagus, or gastroesophageal junction was only 5%. 34 patients were treated on 40 OMD, defined as 3 or fewer metastases amenable to SRT with primary tumor controlled. All patients were affected by squamous cell carcinoma of the esophagus. Authors reported OS at 1 and 2 years comparable to our (76.2 and 58 vs  78.1% and 56%), with a similar absence of relevant toxicity. The median PFS was 13.3 months; the 1- and 2-year PFS rates were 55.9 and 33.8%, respectively. These results are slightly better than ours and probably could reflect the prospective selection of patients, the different histology and disease, and also the fact that half of patients received “adjuvant” chemotherapy in their experience (compared with 13% in our series). Moreover, half of our patients were not pre-treated with systemic therapy before SRT, generally due to comorbidities or advanced age. Anyway, in our opinion, a better integration of local and systemic approaches should be pursued and could be the key to reduce distant failure. Ongoing trials, like the Renaissance trial (NCT02578368) and the ECOG-ACRIN trial (NCT04248452), could better clarify the benefit of this combination, since both consider an “induction” chemotherapy followed by local approach (surgery or radiotherapy) in the experimental arm.In the Phase II trial by Liu et al, The similar OS in our series deserves to be highlighted, considering the unfavorable selection of our patients when compared to surgical candidates. General condition of patients is a well-known prognostic factor in oncology, indeed also in our study a better PS correlated with longer PFS. In any case, these survival data should be taken very carefully due to the significant risk of biases due to the retrospective nature of the studies.A median OS of 25 months and a median 1-year and 5-year OS rates of 75 and 44% were found by the systematic review of 16 retrospective studies for a total of 740 analyzed patients, including patients treated with all LAT. identified the duration of chemotherapy before surgery as correlated to mortality, suggesting that any delay before LAT should be kept at minimum. They also found a survival benefit in HER2+ patients, although this could simply reflect the availability of more effective systemic therapies in this category of patients. In our experience, systemic therapies before or during SRT were not related with survival outcomes.In our analysis, we also tried to identify prognostic factors predictive for outcome, since a key in the oligometastatic world is the selection of patients and the identification of predictive biomarkers. This predictive ability could be even more relevant in an aggressive tumor like esophagogastric cancer with a limited proportion of “true” oligo patients. In surgical patients, Carmona-Bayonas et al identified as predictive factor for OS at univariate analysis the clinical stage of the primary tumor, type of recurrence (i.e. oligometastasis or not), and DFI. However, in the multivariate analysis, only oligometastasis was an independent predictor of OS. Also Ghaly et al found that time to recurrence was a predictive factor for survival of recurrent esophageal cancer patients after curative esophagectomy. In our experience, a longer DFI was correlated to a longer PFS after SRT, but only at UVA. At the same time, we found a worse PFS for patients affected by synchronous metastases when compared to patients with metachronous occurrence. The same authors also found a better prognosis for patients who underwent surgical resection for nodal metastases compared with visceral. In our study, location of the irradiated site did not influence any outcome.Still remaining in the surgical scenario, another study found on multivariate analysis a better PFS correlated with no smoking history and no fistula in a series of metastatic esophageal squamous cell cancer patients. We also analyzed smoking habits of our patients but did not found a statistical correlation, possibly due to the partially different carcinogenesis of esophagogastric adenocarcinoma which was the focus of our work.Li et al with a dose cut-off of 60 Gy. Also in the general analysis by Hong et al on OMD by various histologies, a BED higher than 75 Gy was included in the model as predictor for survival. Whenever feasible, an ablative dose is necessary to have a real impact on these patients prognosis. Indeed, we found that the LR of the irradiated lesion(s) strongly correlated with PFS and OS, with the best outcome in patients experiencing a CR. This could be regarded as an indirect proof that an effective local treatment can really have an impact on patients‘ outcome.We found that BED higher than 75 Gy correlated with OS at univariate analysis. A similar result was reported in the esophagogastric cancer scenario by Li et al survival outcomes are similar. Indeed, they report a median OS in patients local treatment alone of 24 months and in those receiving local plus systemic therapy of 35 months. On the contrary, PFS was significantly longer (16 and 28 months), representing an excellent results may be linked with a strict patients’ selection.Comparing our results with those reported by Kroese et al in a recent work,We must acknowledge the limitations of the present study. First of all, this is a retrospective analysis, so selection biases cannot be ruled out. It is likely that the patients represent a selected subpopulation of the whole Stage IV scenario; therefore the good survival rates could at least partially reflect this selection. However, we should highlight the good homogeneity of our series, since all patients were selected for histology, and also how our results are in line with available literature. Also number of patients in our study is quite low; however, this still represents one of the larger published experiences and the only one, at the best of our knowledge, focusing on adenocarcinoma. Lastly, heterogeneity of disease stage, treatments received and SRT dose and fractionations represents a further limitation particularly for the identification of prognostic factors.SRT is a valid, non-invasive and non-toxic treatment for OMD in esophagogastric adenocarcinoma. Survival outcomes were satisfactory, although further distant failure still represents a major challenge. Response of the irradiated lesion(s) correlated with better OS and PFS, highlighting the opportunity of an ablative local approach in these patients. Further research is necessary to better define the role of local approaches and to better integrate them with systemic therapies.


CNGA3; CNGB3; GNAT2; PDE6C and PDE6H; and novel variants were identified in CNGB3 and PDE6C. Progressive deterioration of BCVA only attributable to achromatopsia was found in three of 58 patients. Progressive phenotype was seen with variants in CNGB3 and PDE6C. The results indicate that myopia could be more frequently occurring with variants in GNAT2, PDE6C and PDE6H and support the evidence that achromatopsia is a predominantly stationary condition with respect to BCVA. Although a clear genotype-phenotype correlation can still not be concluded, there may be differences in phenotypical characteristics with variants in different genes.Achromatopsia is a rare congenital condition with cone photoreceptor dysfunction causing color blindness, reduced vision, nystagmus and photophobia. New treatments are being developed, but the current evidence is still conflicting regarding possible progression over time, and there is no clear genotype-phenotype correlation. This natural history study aimed to further explore the course of disease and potential clinical differences between various genotypes. The retrospective design allowed for the study of a large cohort with a long follow-up. Patients were identified from the Danish national registries. If not already available, genetic analysis was offered to the patient. Clinical data from 1945–2022 were retrieved from medical records and included best-corrected visual acuity (BCVA), color vision, refractive error, nystagmus, visual fields and fundoscopic findings. We identified variants believed to be disease causing in five of the known achromatopsia genes:  Achromatopsia (ACHM) is a rare congenital, autosomal recessively inherited retinal condition with an estimated prevalence of 1 in 30,000–50,000. Since it is caused by dysfunction of cone photoreceptors, it is also known as rod monochromacy. Gene therapy trials are ongoing and have shown promising results , but uncClinically, ACHM is characterized by an inability to see colors, low visual acuity of around 20/200, photo-aversion and nystagmus. Complete ACHM is the most common form, but incomplete ACHM with a milder phenotype has also been described . FundoscAchromatopsia has classically been described as a stationary condition, but some studies have found that the severity of certain phenotypic characteristics correspond with increasing age, indicating progression in some patients. These characteristics include the loss of cone inner and outer segments on OCT , macularCNGA3 (MIM#216900) [CNGB3 (MIM#262300) [GNAT2 (MIM#613856) [PDE6C (MIM#600827) [PDE6H (MIM#601190) [ATF6 (MIM#616517), which encodes an activating transcription factor essential for cone photoreceptor development [CNGA3 and CNGB3 have been completed [The molecular basis for ACHM is most often found in cone phototransduction since five of the six identified genes involved in ACHM encode proteins involved in this cascade: #216900) , CNGB3 (#262300) , GNAT2 (#613856) , PDE6C (#600827) , and PDE#601190) . The sixelopment ,21. GeneWe aimed to include all Danish patients with genetically verified ACHM in the study. Patients were identified from the Danish Family Archive for Genetic Eye Disease, the Danish Register for the Blind and Partially Sighted Children and electronic medical charts. Briefly, these registries hold information about Danish patients with hereditary eye diseases and visually impaired and blind children. The registries are located at the National Eye Clinic for the Visually Impaired at the Kennedy Center, which also has medical records dating back more than a hundred years.Patients with a clinical diagnosis of ACHM, cone dysfunction and/or variants in one of the known ACHM genes were identified. If genetic analysis was not already available, or previous results needed confirmation, and the patient was still alive, he/she was contacted and offered genetic testing to confirm the diagnosis. Only patients in whom the diagnosis could be genetically confirmed were included.https://clinicalgenome.org/working-groups/sequence-variant-interpretation/, accessed on 6 January 2023). Achromatopsia was considered genetically confirmed if two pathogenic or likely pathogenic variants in one of the six known genes  were identified in the patient or an affected sibling. In a few cases  one pathogenic or likely pathogenic variant was identified together with a variant classified as a variant of unknown significance (VUS). These cases were still included since the genetic findings overall were believed to be a likely cause fitting with the phenotype. It was not possible to confirm that the variants were in trans for all patients because parental samples were not always available.Blood samples from patients were collected from 1999 to 2021. Before 2017, DNA was mainly analyzed by Sanger sequencing of coding regions and adjacent intronic sequences of known ACHM genes. After 2017, genetic analysis of ACHM genes was performed using customized SureSelect libraries , followed by next-generation sequencing (NGS) using Illumina technology  and a MiSeq machine. Genes associated with ACHM were included in a larger panel of genes associated with inherited retinal disease. Alignment and variant calling were performed using SureCall (Agilent) and interpretation using VarSeq  and Alamut . For some patients the genetic diagnosis was already published in previous studies ,26,27,28Clinical data from 1945 to 2022 were retrieved and recorded. They included best-corrected visual acuity (BCVA), refractive error, color vision, visual fields, history of childhood nystagmus and fundoscopic findings. Results of electroretinography exams were not included in this study since different test modalities have been used over the years, and ISCEV standards were only introduced in recent years. Optical coherence tomography scans (OCT) were not consistently available for review. For a few patients some clinical findings have been published in previous studies, either along with genetic findings ,27,28,30Best-corrected visual acuity measurement was converted from Snellen to logMAR for analysis. For small children and patients with developmental delays, visual acuity was measured by symbol recognition (Østerberg or Kay acuity charts). Measurements based on preferential looking were not evaluated in the current study since they were not precise enough to compare with later measurements using symbol recognition. To evaluate changes in BCVA over time, the last available measurement was compared to the first measurement. Only data from patients older than 9 years old were analyzed since these patients were able to cooperate with Snellen visual acuity measurement with a reliable result. A change in BCVA of ≥0.2 logMAR was considered clinically significant .The latest refractive data were noted. Cycloplegic autorefraction was preferred for patients younger than 30 years of age and non-mydriatic autorefraction for patients older than 30 years old, but alternatively, the prescribed refractive correction was noted if autorefraction measurements were not available. Spherical equivalent refractive error  was calculated and analyzed. Myopia was defined as SER ≤ −0.5 diopters, hyperopia as SER ≥ +0.5 diopters and emmetropia as SER < + 0.50 and >−0.50 diopters. Statistical analysis included Fisher’s pairwise exact test comparing refractive data with variants in two different genes for three outcome calculations . Calculations were performed using R statistical software, version 4.2.2 .Color vision tests were reviewed. Throughout the years, different tests have been used and included Ishihara pseudoisochromatic plates, Farnsworth–Munsell (FM) 15 or 28 saturated and/or Nagel anomaloscopy. Complete colorblindness was concluded if the patient was only able to see the first Ishihara plate, had multiple diagonal errors >3) on FM and/or achromatic color matching on anomaloscopy. Some patients were tested using more than one modality. In these cases, the reliability of the test was considered. In accordance with previous definitions  on FM an, anomaloA mild phenotype consistent with incomplete achromatopsia was recorded if the patient had a combination of BCVA < 1.0 logMAR and residual color vision.Fundoscopic findings were reviewed, and it was recorded if there was central chorioretinal atrophy at the latest follow-up. There was some variation in the description of the fundoscopic findings from visit to visit and between different clinicians, especially if findings were subtle, but the presence/absence of central atrophy was a consistent description. When available, fundus photographs were reviewed to confirm the findings reported in the medical record. Goldman visual fields were reviewed when available. If these measurements were not available, descriptions of confrontation visual fields were used.We identified 89 patients carrying one apparent homozygous or two heterozygous variants believed to be disease causing in known ACHM genes. Eighteen patients had a diagnosis of ACHM or cone dysfunction but were unavailable for genetic testing and were not included. Both clinical and genetic data were available for 85 patients. The median age at the last follow-up was 31 years old . The median follow-up was 17 years . Six patients only had clinical data from one visit. In 21 families more than one sibling had ACHM, and eight of these families were consanguineous.CNGB3 were the most common cause of ACHM ; see n = 51) of patients. In seven patients, a clinically significant deterioration of more than 0.2 logMAR was observed in one eye (three patients) or both eyes (four patients). In three patients, the visual decline was unrelated to ACHM: cataract (n = 2) and corneal ulcer (n = 1). In one patient, BCVA deteriorated from 0.8 to 1.0 logMAR from age 11 to 17 years old in one eye, which also had exotropia. In three patients, the visual decline could not be explained by ocular comorbidities; one of these patients had deterioration of BCVA in one eye only.Fifty-eight patients had at least two BCVA measurements available after the age of 9 years old, enabling longitudinal analysis with a median follow-up of 22 years ; see Color vision testing was performed in 57 patients, 49 of these patients had complete color blindness, and eight had residual color vision see also . Five ofn = 72) had a history of childhood nystagmus, but eight had no history of nystagmus; two of these patients also had the other characteristics of incomplete ACHM.Most patients  had visual field constriction to <30 degrees. Central scotoma was not recorded in any of the patients.CNGA3 and CNGB3 with and without central chorioretinal atrophy are shown in Fundoscopic findings varied from no pathologies to central chorioretinal atrophy. In 14 patients (19%), chorioretinal atrophy was noted at the latest follow-up, the median age for these patients was 56 years old , and eight of these patients had BCVA of 1.00 logMAR or better. Representative fundus pictures of patients with variants in Refractive data were available for 83 patients; see Generally, the clinical characteristics were similar in patients with causative variants in the five identified genes, but some differences were observed  and c.1208G > A;p.(R403Q), and the genotype of the third patient was c.1148delC;p.(T383fs) and c.886–896del11insT;p.(T296fs).Median visual acuity was slightly better for patients with variants in CNGA3 n = 2, CNGB3 n = 1, GNAT2 n = 1, PDE6H n = 1), but not in patients with causative variants in PDE6C. Two patients with incomplete ACHM and variants in CNGA3  and c.1694C > T;p.(T565M)) were twins. Incomplete and complete ACHM was observed in a sibling pair with variants in CNGB3  and c.886–896del11insT;p.(T3296fs)). One patient with residual color vision had BCVA < 1.0 logMAR in the left eye only and deterioration of BCVA in the right (exotropic) eye; the genotype for this patient was CNGB3 c.1148delC;p.(T383fs) and c.1208G > A;p.(R403Q). One other patient shared this genotype and had residual color vision but demonstrated a clinically significant worsening of BCVA in both eyes during follow-up. Two other patients with causative variants in CNGB3 and residual color vision and demonstrated clinically significant worsening of BCVA throughout follow-up, but for these patients, the deterioration could also be explained by ocular comorbidities. Deterioration of BCVA was also seen in two patients with variants in PDE6C, but in one of them, the deterioration was unrelated to ACHM. Worsening of BCVA was not seen with variants in GNAT2 and PDE6H. Complete ACHM was seen in a single patient with variants in PDE6H.A combination of BCVA < 1.0 logMAR in both eyes and residual color vision at the latest follow up consistent with incomplete ACHM was found in five patients, with variants in four different genes  and only in patients with causative variants in CNGA3 and CNGB3. For the patient with visual field constriction and variants in CNGA3, pigmentary changes were noted in the retinal periphery; this patient also had worsening of BCVA, which could also be attributable to cataract. The patients with CNGB3 variants did not have peripheral retinal changes. One had a pale optic disc noted on fundoscopy; the other had normal fundoscopic findings.Visual field constriction <30 degrees was rarely seen . Refractive errors varied among the five gene groups . Differences in refractive error with variants in different genes using pairwise analyses was statistically significant when comparing patients with variants in CNGB3 and PDE6H . Severe myopia (SER ≤ −6.00 diopters) was observed with variants in all five genes but was more frequently occurring in patients with variants in PDE6H , PDE6C  and GNAT2  than in patients with variants in CNGB3  and CNGA3 .Data on refractive errors at latest follow up was available for 82 patients, with variants in the five different genes . This finding supports the notion that ACHM is a predominantly stationary condition, at least as far as it is experienced by patients. Other studies have found similar results ,43,44, a.4 years  and a siCNGB3 being the most frequent cause and c.1148delC being the most frequent disease-causing variant [PDE6C and PDE6H were more frequent in this study than the <2% previously documented [PDE6H came from two different consanguineous families originating from Syria and Turkey, so it is unlikely that there is a higher prevalence of PDE6H variants in the general Danish population. In contrast, patients with variants in PDE6C were unrelated, except for two patients, so it is possible that the prevalence of PDE6C variants in Denmark is higher than in other Northern European populations. We did not identify any patients with causative variants in ATF6.The genetic background of Danish ACHM patients found in this study is overall consistent with that from other studies in Northern Europe, with variants in  variant . Variantcumented ,17,18. PCNGA3 variants were predominantly missense variants, and the majority of variants in CNGB3 were small deletions. Variants in GNAT2 all resulted in a premature stop codon. We identified novel variants in CNGB3 and PDE6C. The single base pair substitution identified in PDE6H is the only variant associated with ACHM identified in this gene to date [In agreement with earlier studies ,36 we fo to date ,45.CNGA3, CNGB3, GNAT2, PDE6C and PDE6H [PDE6H is exclusively associated with incomplete ACHM [CNGA3, CNGB3, GNAT2 and PDE6H but not in PDE6C. Incomplete ACHM was most frequent with variants in CNGA3, but data on color vision were only available for the few patients with variants in PDE6H. One patient with variants in PDE6H had a phenotype consistent with complete ACHM, thereby broadening the phenotypic spectrum for this gene.Genotype-phenotype correlations are typically sparse in ACHM with regard to severity of the disease . A mildend PDE6H ,24,27,46ete ACHM . The DanCNGA3, CNGB3, GNAT2 and PDE6C [GNAT2 and not at all with variants in PDE6H [GNAT2 and PDE6H.Progressive disease has been described in patients with variants in nd PDE6C ,24,27,37in PDE6H . In concCNGA3 and worsening of BCVA, this outcome could also be explained by cataract, so in our study, a progressive phenotype only attributable to ACHM was only seen in patients with variants in CNGB3 and PDE6C. This finding is consistent with previous findings indicating that progressive cone [For patients with variants in ive cone ,37 and cive cone  are moreCNGB3, we identified three unrelated patients with the c.1208G > A;p.(R403Q) missense variant in conjunction with c.1148delC. One of these patients also had a variant in CNGA3. The c.1208G > A variant is classified as pathogenic but presumably mild, likely representing a hypomorphic allele since it has also been found homozygous in 24 controls out of 140,050 (gnomAD v.2.1.1). It is, however, also a frequently occurring variant in ACHM [CNGA3 variant with the two CNGB3 variants, one being c.1208G > A;p.(R403Q), was recently described as exacerbating CNG-channel retinopathy [Regarding  in ACHM ,48 and i in ACHM ,49. The GNAT2, PDE6C, and PDE6H. The small sample sizes makes it difficult to draw strong conclusions on possible genotype-phenotype correlations. Detecting significant differences in refractive outcomes across various groups based on causative genes may necessitate larger sample sizes. Additionally, the multiple statistical testing required for small sample sizes could also pose issues. Future studies systemically documenting refractive error with variants in different genes in different populations could be useful, not only to further explore a possible genotype-phenotype association, but if such an association does exist, it could also provide insights into the complex and multifactorial process of emmetropization.With respect to refractive error, patients with ACHM have classically been described as being predominately hyperopic , but we With the development of new treatments for ACHM, natural history studies such as the present one are relevant to establish which age groups are the best candidates for treatment. In case of progressive disease, younger patients may benefit more than older patients. In this current study, progressive disease was concluded from a worsening of BCVA, both because it is a clinically meaningful parameter for patients and because OCT data were not consistently available due to the retrospective nature of the study. Other studies have found that structural measures, such as OCT staging, do not necessarily correlate well with more functional measures, such as BCVA, in ACHM ,10. DiffThe retrospective nature of the current study allows for a large cohort of patients to be studied with a longer follow-up period compared to other studies. However, it also has limitations, namely the variable follow-up time, the lack of OCT to investigate morphological changes and the inconsistency in the methods used to evaluate color vision and fundoscopic changes. Full-field ERG was used to establish the diagnosis, but since it was performed years ago, in some cases before the development of ISCEV standards, it could not be evaluated systematically. CNGB3 and PDE6C. A mild phenotype was more commonly seen with variants in GNAT2 and PDE6H. We do, however, also describe a patient with complete ACHM and variants in PDE6H, thereby broadening the phenotypic spectrum for this gene. We also found indications of differences in refractive error with variants in the different genes associated with ACHM, which could also provide potential insights into the process of emmetropization. To further explore this possibility, it would be beneficial for future studies on the phenotypic characteristics of ACHM to also report refractive error systematically.In conclusion, the current study describes the genetic background and phenotypical characteristics of Danish patients with ACHM. We present data from a large cohort, and the retrospective nature allows for a long follow up. In concordance with other studies, we found genotype-phenotype correlations in the severity of the condition to be sparse, but evidence presented here supports that progressive disease is more frequently occurring with variants in With the development of new gene therapeutic treatments for ACHM, natural history studies with long follow-ups are important. The results presented here support the evidence of ACHM being a predominantly stable condition with respect to BCVA, which is highly relevant to patients. However, future large, prospective studies enabling the study of other factors, such as work using high-resolution imaging, are also important in establishing possible treatment response and timing of treatment.