Ozonated glycerol is glycerol containing ozone, has no unpleasant odor, and has a long half-life. To apply ozonated glycerol for clinical use, ozonated macrogol ointment has been developed by adding macrogol ointment to ozonated glycerol to increase the retention in the affected area. However, the effects of ozone on this macrogol ointment were unclear. The viscosity of the ozonated macrogol ointment was approximately two times higher than that of ozonated glycerol. The effect of the ozonated macrogol ointment on the human osteosarcoma cell line Saos-2 (Saos-2 cells) proliferation, type 1 collagen production, and alkaline phosphatase (ALP) activity were studied. The proliferation of Saos-2 cells was assessed using MTT and DNA synthesis assays. Type 1 collagen production and ALP activity were studied using ELISA and ALP assays. Cells were treated for 24 h with or without 0.05, 0.5, or 5 ppm ozonated macrogol ointment. The 0.5 ppm ozonated macrogol ointment significantly elevated Saos-2 cell proliferation, type 1 collagen production, and ALP activity. These results also showed almost the same trend as for ozonated glycerol. Ozone is widely used in society for disinfection, deodorization, disinfecting , and cliTo apply ozonated glycerol for clinical use, ozonated macrogol ointment has been developed by adding macrogol ointment to ozonated glycerol to increase retention in the affected area. We have shown the anti-inflammatory effect of ozonated macrogol ointment on human gingival fibroblasts and its osteogenic potential on osteoblasts . HoweverThe ozonated macrogol ointment was provided by Mediplus Pharma Inc., Tokyo, Japan. The preparation procedure of the ozonated macrogol ointment is below. Ozone gas was administered to concentrated glycerol  to produce 2000 ppm of ozonated glycerol using an Ozonizer . In addition, a macrogol ointment (polyethylene glycol (PEG) 400 and PEG 4000: Yoshida Pharmaceutical Co., Ltd., Tokyo, Japan) and a water-soluble ointment (polyethylene glycol) were added to increase the viscosity of ozonated glycerol for the purpose of retention. The ratio of macrogol ointment to ozonated glycerol was 1:2 . The visThe shear rate in the B-type viscometer BM II  was calculated by measuring in advance a Newtonian fluid of known viscosity (viscosity standard fluid) at different rotor speeds .4) were cultured in DMEM containing 0.5% FBS (0.5% DMEM) for 24 h. Cells were cultured with 0.05, 0.5, or 5 ppm ozonated macrogol ointment for 2 min. The ozone concentration is the final concentration for each well. The culture medium was then removed. The cells were washed with 0.5% DMEM and cultured with 0.5% DMEM containing bromodeoxyuridine (BrdU) for 24 h. The level of DNA synthesis was determined by measuring BrdU incorporation using a BrdU cell proliferation assay kit . For the MTT -2,5-diphenyl tetrazolium bromide; Sigma-Aldrich, St. Louis, MO, USA) assay, cells were cultured with ozonated macrogol ointment at 0.05, 0.5, or 5 ppm in DMEM containing 10% FBS (10% DMEM) for 2 min, and the culture medium was then removed. After washing with 10% DMEM, cells were cultured with 10% DMEM for 24 h. The subsequent procedures were performed as previously described [For DNA synthesis, Saos-2 cells (1 × 10escribed . The mic4) were cultured with 0.5 ppm ozonated macrogol ointment in DMEM containing 1% FBS (1% DMEM) for 2 min. As a control, Saos-2 cells were cultured without ozonated macrogol ointment. Cells were washed and cultured with 1% DMEM. Levels of type 1 collagen in the media were measured by indirect ELISA using the biotinylated anti-type 1 collagen antibody  and samples (360 µg/mL protein). ELISA was performed as described in the user manual of a CytoSet kit  [For collagen production, Saos-2 cells  . Cells f4) were cultured with a 0.5 ppm ozone macrogol ointment for 2 min. The culture medium was then removed. Cells were washed with 0.5% DMEM and cultured with 10% DMEM for 24 h. Cells were lysed with 0.05% Triton X-100, and the ALP activity of the lysates was measured using a LabAssay ALP kit . Protein concentrations of cell lysates were also measured using a BCA protein assay kit. ALP activity was normalized to the protein content of the cell lysates. The microplate was used on a 96-well plate. Three wells were used for one sample to be analyzed. Samples were measured by the Microplate Reader model 550 using a wavelength of 405 nm. The analysis was performed three times for the same experiment.Saos-2 cells (1 × 10t-test (measurement of ALP activity and ELISA for collagen production) using the StatMate software . Differences were considered significant at p < 0.05.Quantitative data were statistically analyzed using either a one-way analysis of variance (ANOVA) followed by Tukey’s test (DNA synthesis and MTT assays) or the Student’s The viscosity of the ozonated glycerol and the ozonated macrogol ointment were 1499 and 3000 mPa·s, respectively. The viscosity of the ozonated macrogol ointment was about two times greater than that of the ozonated glycerol.After 24 h of incubation, the ozonated macrogol ointment treatment increased Saos-2 proliferation in a dose-dependent manner . OzonateGiven the results of the proliferation assays, we further evaluated the effect of 0.5 ppm ozonated macrogol ointment on type 1 collagen production and ALP activity by Saos-2 cells. The medium containing ozonated macrogol ointment effectively increased type 1 collagen production  and ALP Ozonated glycerol has low viscosity and moderate fluidity because its main ingredient is glycerol. On the other hand, ozonated macrogol ointment is an ointment made by mixing ozonated glycerol with PEG 400 and PEG 4000, and has high viscosity and retention at the affected site. The viscosity of the ozonated macrogol ointment was about two times higher than that of the ozonated glycerol. The present study showed that although high concentrations of ozonated glycerol ointment, such as ozonated glycerol , also deType 1 collagen and ALP are widely known as markers secreted in the early phases of bone formation . In thisIn summary, we compared the ozonated glycerol and macrogol ointment. The effect of ozonated macrogol ointment on type 1 collagen production and ALP activity in Saos-2 cells was investigated. The results indicated that ozonated macrogol ointment controls the cellular metabolism of osteoblasts, resulting in the secretion of early bone-related biomarkers as well as ozonated glycerol. Currently, we shed light on the functional studies of ozonated macrogol ointment on long-term effects in terms of cellular damage toward clinical application.


Radiation-induced damage (RID) after radiotherapy (RT) of primary brain tumors and metastases can be challenging to clinico-radiographically distinguish from tumor progression. RID includes pseudoprogression and radiation necrosis; the latter being irreversible and often associated with severe symptoms. While histopathology constitutes the diagnostic gold standard, biopsy-controlled clinical studies investigating RID remain limited. Whether certain brain areas are potentially more vulnerable to RID remains an area of active investigation. Here, we analyze histopathologically confirmed cases of RID in relation to the temporal and spatial dose distribution.2) and the biologically effective dose (BED2) for normal brain tissue (α/β ratio of 2 Gy) and analyzed the spatial and temporal distribution using frequency maps.Histopathologically confirmed cases of RID after photon-based RT for primary or secondary central nervous system malignancies were included. Demographic, clinical, and dosimetric data were collected from patient records and treatment planning systems. We calculated the equivalent dose in 2 Gy fractions (EQD22 60 Gy) to a large planning target volume (PTV) (median 203.9 ccm) before diagnosis of RID. Despite the low EQD22 and BED2, three patients with an accelerated hyperfractionated RT developed RID. In contrast, brain metastases patients  were often treated with two or more RT courses and with radiosurgery or fractionated stereotactic RT, resulting in a higher cumulative EQD22 (median 162.4 Gy), to a small PTV (median 6.7 ccm). All (n = 34) RID lesions occurred within the PTV of at least one of the preceding RT courses. RID in the high-grade glioma group showed a frontotemporal distribution pattern, whereas, in metastatic patients, RID was observed throughout the brain with highest density in the parietal lobe. The cumulative EQD22 was significantly lower in RID lesions that involved the subventricular zone (SVZ) than in lesions without SVZ involvement .Thirty-three patients were identified. High-grade glioma patients (n = 18) mostly received one normofractionated RT series (median cumulative EQD22 and BED2 in high-grade glioma patients. The anatomical location of RID corresponded to the general tumor distribution of gliomas and metastases. The SVZ might be a particularly vulnerable area.Accelerated hyperfractionated RT can lead to RID despite computationally low EQD2The online version contains supplementary material available at 10.1186/s13014-023-02385-3. RID 2) > 100 Gy [Known risk factors for radiation necrosis include higher total radiation dose, larger fraction size and the use of concurrent and/or adjuvant systemic antineoplastic therapy –8. A bra> 100 Gy .The frequent need for invasive tissue biopsy to secure the diagnosis of RID has prompted substantial research efforts directed at the identification of imaging biomarkers capable of reliably distinguishing between RID and tumor progression. As such, a variety of diagnostic procedures have been explored, including standard magnetic resonance imaging (MRI) based methods, diffusion weighted imaging (DWI), perfusion-weighted imaging (PWI), magnetic resonance spectroscopy (MRS), positron emission tomography (PET), single photon emission computed tomography (SPECT), radiomics, and combinations thereof, with varying success –18. Histradiation-induced cellular atypia can result in further diagnostic challenges [Reported histopathological characteristics of radiation necrosis include coagulative and fibrinoid necrosis, gliosis, wall thickening and hyalinization of vessels, telangiectasia and calcium deposition, located predominantly in the white matter –22. For allenges , 26. As allenges .Treatment options for radiation necrosis remain limited and include corticosteroids, bevacizumab, surgical resection, laser interstitial thermal therapy (LITT) and hyperbaric oxygen therapy (HBOT) , 29.Several reports in the literature have suggested that certain areas of the brain, such as the subventricular zone (SVZ), a neural stem cell niche located along the lateral wall of the lateral ventricles , 31, migHerein, we provide an in-depth analysis of 34 histopathologically confirmed RID lesions after brain-directed RT with a focus on radiation parameters and spatio-temporal distribution with specific consideration of the role of the SVZ. Furthermore, we explore putative differences between RID in patients with primary and secondary brain tumors, i.e., high-grade gliomas (HGGs) and brain metastases (BMs). The aim of this study was to correlate the radiation dose with the spatial and temporal occurrence of RID.Patients with histopathologically confirmed RID who had undergone photon RT between January 2006 and January 2020 for malignant primary and secondary brain tumors were included in this retrospective monocentric study. Patients who had received proton or heavy ion RT and those treated for benign lesions were excluded. Diagnostic tissue samples had to contain RID . All samples of “mixed lesions” containing areas of solid tumor were excluded.The study was approved by the local institutional review board (EA2/056/20).Demographical, clinical, and dosimetric data, as well as imaging data were collected from the patient records and the radiation treatment planning system (TPS). The time-to-RID, defined as the interval between the day of the last fraction of RT and the first occurrence of RID on MRI imaging , was noted.2, BED2).The EQD2 and biologically effective dose (BED) were calculated for RT courses in the area of RID using previously described formulas –39. An αPreoperative  gadolinium-enhanced T1-weighted 3D Magnetization Prepared Rapid Acquisition with Gradient Echoes (MPRAGE) images of RID were imported into the TPS Varian Eclipse . The extent of RID (defined as the contrast-enhancing lesion) was manually delineated for each patient. All contours were reviewed by a board-certified radiation oncologist, specialized in neuro-oncology (DK). The radiation plans of prior RT courses were co-registered to the MRI using rigid image registration. The planning target volumes (PTVs) were then transferred to the MRI of the RID. RT plans that clearly had no relevant dose deposition in the area of RID  were excluded. Radiation plans created in Brainlab iPlan RT  and Accuray Precision TPS  were exported into Varian Eclipse and then processed as described. The MRIs with the corresponding structure sets were exported in DICOM/DICOM RT format.The exported MRIs and structure sets were imported into 3D Slicer, version 5.0.3 , 42 for The transform created during the ANTs registration was then used to register the structures (RID lesions and PTVs) to the MNI152 space. The structures were converted into binary label maps, exported and then summed using the ImCalc module of Statistical Parametric Mapping 12 (SPM12), revision 7771 , 54 in MIn order to assign the RID lesions to the different lobes, the lobe atlas provided with the ICBM 2009a nonlinear symmetric template , 50 was The coverage of the RID by the PTVs of the single RT courses was calculated within 3D Slicer using the intersect function for each PTV. Furthermore, in order to assess the cases that had more than one preceding RT in the area of the RID, a combined PTV was computed within 3D Slicer and coverage of the RID was calculated accordingly.2 values between lesions with and without SVZ involvement, the Mann-Whitney U test was used. For correlation analysis, the Spearman correlation coefficient r was calculated. P values ≤ 0.05 were considered statistically significant.All statistical analyses were performed using IBM SPSS Statistics, version 28.0 . Patient characteristics for the two main subgroups, HGG and BM, were summarized using descriptive statistics. For the assessment of differences in the cumulative EQD2A total of 33 patients with a diagnosis of RID met the inclusion criteria of our study. The diagnosis that led to brain-directed photon RT was HGG for 18 (55.5%) patients. This included glioblastoma WHO grade 4 (11 patients), oligodendroglioma WHO grade 3 (4 patients) and astrocytoma WHO grade 3 (3 patients). BMs were present in 15 (45.5%) patients. One patient in the BM group developed two independent RID lesions in different lobes of the brain. This resulted in 34 RID lesions in total. The median age at resection of the RID was 56 years (interquartile range (IQR) 49–64.5 years). Details on patient and lesion characteristics are summarized in Table Pathological diagnosis was made based on surgical resection (88.2%), biopsy (8.8%) or autopsy (2.9%). In 70.6% of the lesions only RID was observed, whereas in 29.4% single tumor cells were present in addition to RID. The proportion of lesions with tumor cells present was higher in the HGG subgroup (44.4% vs. 12.5%).18F-FET PET/CT), but the lesion was resected because of progressive symptoms. One RID lesion was diagnosed by autopsy after death of the patient from systemic tumor progression.The main reasons for performing resection or biopsy were suspected tumor recurrence or progression or ambiguous results of the obtained imaging, either in combination with symptoms associated with the RID lesions (63.6% of the lesions) or without any symptoms caused by RID  and the BM subgroup (median 46.4%). The median coverage of the lesion by combined PTV, i.e., the percentage of the RID that was covered by any of the PTVs of the preceding RTs, was 97.8%, with slightly higher values in the HGG subgroup than in the BM subgroup  and among the HGG subgroup (72.2%). In the BM subgroup frontal lobe involvement was less frequent (37.5%), while still being the second most common location after the parietal lobe (43.8%). Overall, lesions in the BM group were more evenly distributed throughout the whole brain, whereas in the HGG subgroup no involvement of the occipital lobe and cerebellum was found. Only one lesion was found in the cerebellum in the BM group and none of the lesions were located in the brainstem.52.9% of the lesions involved the SVZ; the proportion was higher for the HGG subgroup (77.8%) than for the BM subgroup (25.0%). All RID lesions involved the white matter, and the vast majority (94.1%) involved the cortex as well. Only two RID lesions were exclusively located in subcortical compartments, one each in the HGG and BM subgroup, respectively. Basal ganglia were involved in 29.4% of all lesions, more frequently in the HGG than in the BM subgroup (50.0% vs. 6.3%).2 administered was 103.2 Gy for the whole cohort, with higher values in the BM subgroup (median 162.4 Gy) than in the HGG subgroup (median 60 Gy). Furthermore, higher fraction size (median 8 Gy vs. 2 Gy) and single course EQD22 (median 71.6 Gy vs. 60 Gy) were found in the BM subgroup. On the other hand, PTV volumes, excluding whole brain radiotherapy (WBRT), were smaller in the BM subgroup (median 6.7 ccm vs. 203.9 ccm). The relationship between single RT course EQD22 and PTV volume is illustrated in Fig. Table 2 > 100 Gy, except of one patient with a cumulative EQD22 of 98.1 Gy. The majority of RT courses (90.6%) were administered on five days per week, whereas one RT course was performed with only three fractions per week and four courses were performed with two fractions per day (b.i.d.), i.e., using an accelerated hyperfractionated RT (AHFRT) regimen, three of these in the HGG group and one in the BM group. The three AHFRT courses in the HGG group were used in patients that received only one RT course in the area of the RID and the EQD22 for these lesions was among the lowest in the whole study population (53.3 Gy for each lesion).The patients that received two or more RT courses all had a cumulative EQD22 values were considerably lower in RID lesions that involved the SVZ  than in lesions without SVZ involvement ; the differences in distributions were statistically significant (p = 0.01).As illustrated in Fig. 2 and the time-to-RID  = -0.095, p = 0.594).The median time-to-RID  was 5.8 months (range 0.7–29.0 months). There was no statistically significant correlation found between the cumulative EQD2In 73.5% of the lesions systemic therapies were administered during or close to (± 1 months) the RT for at least one of the RT courses in the area of the RID. The proportion of cases receiving systemic therapy was higher in the HGG subgroup (83.5%) than in the BM subgroup (56.3%). An overview of the different drugs used is provided in Additional file 2: Table The diagnosis of RID, including pseudoprogression and radiation necrosis, remains a common neuro-oncological challenge, with several important aspects to discuss. Despite its status as the diagnostic gold standard, histopathological evaluation may yield ambiguous results, especially in “mixed lesions” which include both RID and vital tumor cells and/or foci of solid tumor , 25, 57.For instance, Patrizz et al.  classifiradiation-induced cellular atypia (reminiscent of viable tumor cells) in RID lesions constitutes another confounding factor [In our study, we chose to include only cases with “pure” RID and cases with single tumor cells present, if RID accounted for most of the sample. All cases with solid tumor present in addition to RID were excluded. We deemed that this was the best compromise, as excluding all cases with tumor cells present would have led to the exclusion of almost all glioblastoma patients, but including cases with solid tumor present would have had negatively affected the diagnostic accuracy, as tumor necrosis is often present in HGG. Moreover, the potential presence of g factor , 26, cirThe main reasons for performing resection or biopsy in our study were suspected tumor recurrence or progression or ambiguous results of the obtained imaging, either in combination with or without symptoms associated with the RID lesions. Two thirds of the patients developed symptoms, similar to those commonly seen in brain tumors and in some cases severe such as hemiparesis or epileptic seizures. This highlights the current diagnostic and therapeutic challenges clinicians are confronted with in the management of RID.2 for healthy brain tissue instead, as it is better suited to reflect the differences in the fractionation schemes. Using this approach, we could not find a statistically significant correlation between the cumulative EQD22 in Gy and the time-to-RID.Regarding the temporal occurrence pattern of RID, Campos et al.  reportedAll RID lesions in our study occurred at least in part within the area of the PTV of one or more of the preceding RTs and in most cases coverage of RID by the PTVs was high. This is in line with the findings of Winter et al.  who descThe location pattern of RID in our study differed between the two subgroups. In the HGG subgroup RID lesions occurred preferentially in the frontotemporal areas of the brain, while posterior parts and the cerebellum were spared. If we compare our data to studies that examined the location frequency of glioma or glioblastoma, very similar location patterns are found –63. ThusIn the BM subgroup, RID lesions were more heterogeneously distributed throughout the whole brain in our cohort. Studies that investigated the location of BM , 65 also2 values were considerably lower in RID lesions that involved the SVZ, possibly suggesting a higher vulnerability of this region.SVZ involvement of RID lesions was highly frequently observed within the HGG subgroup of our study (77.8%), substantially more often than in the BM subgroup (25.0%). However, SVZ involvement and radiation of it is more common for gliomas, too, given the necessary safety margins and respective contouring guidelines. For example, a study from our institution comprising 200 glioblastoma patients found that 66.0% of the tumors had contact to the SVZ . Similar2 (median 60 Gy) in comparison to the BM patients. The three patients in this subgroup that received AHFRT had relatively low EQD22 (53.3 Gy) and BED2 (106.6 Gy) values. This was also noted in the Quantitative Analyses of Normal Tissue Effects in the Clinic (QUANTEC) publication by Lawrence et al. [Patients with HGG mostly received one course of normofractionated RT or AHFRT, partially with a SIB, to a large PTV (median 203.9 ccm) with a more moderate cumulative EQD22 values compared to the HGG subgroup. The median PTV size was substantially smaller (6.7 ccm).BM patients, on the other hand, received two or more RT courses in more than 80% of the cases. All patients in this subgroup received hypofractionated RT (single dose ≥2.5 Gy) in at least one of their RT courses, resulting in higher cumulative (median 162.4 Gy) and single course (median 71.6 Gy) EQD22 higher than 100 Gy, except for one patient with a cumulative EQD22 of 98.11 Gy. This finding is supported by another recently published study from our institution on the reirradiation of IDH wild type glioblastoma using moderate cumulative EQD2 values that did not find any cases of confirmed radiation necrosis [Mayer et al.  reportednecrosis .2 values compared to the BM cases, might be the more frequent involvement of the SVZ in this subgroup. In the guideline for radiation necrosis published by the DEGRO in 2022 [2 values were significantly lower in RID lesions that involved the SVZ than in lesions without SVZ involvement. There are several possible confounders, such as the much larger PTV volumes and the frequent concurrent administration of temozolomide in HGG patients that constitute inherent risk factors for RID [One possible explanation for the occurrence of RID in the HGG subgroup in spite of the lower EQD2 in 2022  it is st in 2022 –35. Van  in 2022  reported in 2022  pointed  for RID –8, but fOur study has several limitations. Data collection was carried out retrospectively and at a single institution. With 33 patients and 34 lesions included, the sample size might be regarded as relatively small. However, to our knowledge, this is still one of the largest samples of RID lesions diagnosed by histopathologic confirmation. The lack of a control group without RID in this study causes some difficulties in interpreting the data, especially regarding the location analysis. Limiting the study to patients diagnosed by histopathologic confirmation bears the risk to only include the more severe and symptomatic cases of RID that had to be resected or biopsied. Furthermore, it is possible that in the location analysis RID lesions occurring in certain areas are underrepresented, because performing resection or biopsy in these areas was not clinically feasible and/or contraindicated. However, as to date there is still no other method that can provide the same diagnostic accuracy for RID as pathologic confirmation and it is still regarded the gold standard, studies based on pathology-proven samples remain an indispensable and highly valuable tool for investigating the mechanisms leading to RID after brain-directed RT.2 and BED22. The actual risk in comparison to other fractionation schemes has yet to be evaluated. Our data supports the notion that the SVZ is potentially more vulnerable to the development of RID, given that lesions there developed after a lower cumulative EQD22. The larger irradiated volumes as well as the more frequently performed concurrent administration of temozolomide in these lesions might have to be considered as independent risk factors for RID; thus, further research on the topic should ideally address these potential confounders.In this analysis of histopathologically confirmed RID, the location of RID mostly followed the general distribution pattern of the tumors irradiated. A previously reported correlation between the radiation dose and the period between RT and the occurrence of the RID could not be confirmed. High-grade glioma patients that receive twice-daily AHFRT schemes might develop RID despite a presumably lower EQD2Below is the link to the electronic supplementary material.Supplementary Material 1Supplementary Material 2


We examined internet searches on psoriasis in Germany and found that in weeks with high search volume, mean temperature and humidity were lower and sunshine level was higher compared to weeks with low search volume. A connection between psoriasis severity and weather has long been suspected. Cold, dry weather has been noted anecdotally to exacerbate symptoms, whereas sunshine improves the condition . PsoriasIn this study, we examined internet searches related to psoriasis in Germany, looking at whether patterns in searching were weather related. The internet search volume for the search term “psoriasis” (disease) for Germany was obtained from Google Trends . This weWeather data from the German Wetterdienst  was used. Daily data for mean temperature, percentage relative humidity, and total hours of sunshine was obtained for 6 weather stations: Berlin, Cologne, Frankfurt, Hamburg, Munich, and Stuttgart. Weekly mean values for temperature and humidity as well as total weekly hours of sunshine were calculated. A value for all of Germany was calculated using data from the 6 stations.The RSV ranged from 53 to 100. Although there was only a slight correlation between searching and specific weather features , 3D scatter plots suggested differences between weeks of high and low searching. Low search weeks blue in A occurret test: P<.001).Notable differences were apparent when comparing mean values between the lowest  and highest  search weeks. The mean temperature was higher during low rather than high search weeks , as was humidity , but sunshine hours was lower . The difference for humidity was significant (2-tailed P<.001), they were logged. Separate regressions were performed with logged values for temperature, humidity, and sunshine as dependent variables. The resulting coefficients indicated that higher temperature and humidity led to lower search volumes but more sunshine was associated with increased searching . Regression fitting suggests an RSV of 78 at 10 °C, declining to an RSV of 40 at 15 °C. At 65% humidity, an RSV of 93 is expected; when humidity rises to 80%, the RSV should drop to 47. With 40 hours of sunshine per week, an RSV of 68 is expected.These trends were further explored using linear regression. Since the search data were not normally distributed (Shapiro-Wilk W=0.96; This study revealed that weeks of high internet searching related to psoriasis were associated with cold, dry weather conditions. Differences were most apparent when comparing the highest and lowest search weeks. The possible association between high searching and more sunshine is of interest; this is contrary to anecdotal evidence but supports recent survey work , which uGoogle Trends data allowed for the examination of search volume trends over long time periods and on a weekly basis. However, only national data were available, meaning regional assessment was not possible. Local differences may exist. We assumed that internet searching is constant throughout the year, but increases in winter months may occur when people spend more time on computers due to longer, darker evenings. Future studies could control for this problem by calibrating search volume with results from nonseasonal conditions. Another assumption is that internet searching reflects the severity of psoriasis. One would expect this to be the case because people are more likely to search when their symptoms worsen and discomfort is felt. It is well known that internet searching reflects the incidence of a condition . The eff


Dirinara applanta and Parmotrema tinctorum, and compared their microbial communities to those found in the pine bark to which the lichens were attached. Our hypothesis was that the endolichenic communities would exhibit distinct diversity patterns, community structures, network structures, and specialist composition compared to the surrounding epiphytic community.Lichens represent not only the mutualism of fungal and photosynthetic partners but also are composed of microbial consortium harboring diverse fungi known as endolichenic fungi. While endolichenic fungi are known to exert a remarkable influence on lichen ecology through their crucial roles in nutrient cycling, bioprospecting and biodiversity, the enigmatic community structures of these fungal inhabitants remain shrouded in mystery, awaiting further exploration and discovery. To address knowledge gap, we conducted metabarcoding on two lichens using 18S gene amplification, Our investigation has shed light on the clear demarcation between the endolichenic and epiphytic fungal communities, as they exhibit markedly different characteristics that set them apart from each other. This research demonstrated that the endolichenic communities are less diverse as compared to the epiphytic communities. Through community similarity analysis, we observed that two endolichenic communities are more similar to each other in terms of community composition than with the adjacent epiphytic communities. Moreover, we unveiled a striking contrast in the network structures between the endolichenic and epiphytic communities, as the former displayed a more modular and less nested features that is evocative of a potent host-filtration mechanism.Through our investigation, we have discovered that lichens harbor less intricate and interconnected fungal communities compared to the neighboring epiphytic environment. These observations provide valuable insights into the metagenomic architecture of lichens and offer a tantalizing glimpse into the unique mycobiome.The online version contains supplementary material available at 10.1186/s40793-023-00531-8. Lichens are a symbiotic mutualism comprising lichen-forming fungi and their photosynthetic partners . MutualiPrevious research about the community structure of ELF found that these fungi are a diverse group with a majority belonging to the Ascomycota phylum , 16. TheDirinaria applanta and Parmotrema tinctorum, as well as the EPF community of Pinus thunbergii, using metabarcoding techniques. Precisely, our aim was to investigate fundamental inquiries regarding the distinctions between ELF and EPF communities, focusing on aspects such as diversity patterns and network architecture. To accomplish this, we compared the fungal communities across six different aspects: alpha diversity, taxonomic composition, community similarity, network structure, core microbiome, and host specialists.To gain a better understanding of the distinct structure of ELF communities, it would be valuable to conduct a comparative study between lichen communities and the surrounding environment using metabarcoding techniques. The goal of this study is to uncover the unique architecture of the ELF community in comparison to the neighboring epiphytic (EPF) community. We selected the EPF community as a suitable comparator to ELF because it is likely that the tree bark represents the original fungal pool from which the ELF community was established. This is supported by the fact that the tree bark is physically closest to the epiphytic lichen and existed prior to lichenization. To explore the differences between ELF and EPF communities, we analyzed the fungal communities within the epiphytic lichens Pinus thunderbergii bark, and the epiphytic lichen Dirinaria applanta and Parmotrema tinctorum from five sites (n = 75) on Jeju Island in the southern part of South Korea  , the five collection sites did not show significant climatic differences  as described by Yang et al. [We collected twenty five samples of g et al. .Fig. 1CoThe bark and lichen segments were homogenized, and DNA was extracted using a PowerSoil DNA isolation kit . We conducted a polymerase chain reaction (PCR) of the fungal ITS1 region  using thhttps://docs.qiime2.org).Amplicon library sequencing was performed using the Illumina MiSeq platform . Raw sequences were processed using QIIME2 v.2021.4  by demulhttps://forum.qiime2.org) and rarefied to 3,000 sequences per sample using the “phyloseq” package [The following analyses were performed using R v.3.5.3 . For the package . The subp value of the permutation test [Cws, Nestedness metric based on overlap and decreasing fill (NODF), Betweenness, Closeness, InDegree, OutDegree, and Expected influences. Cws represents the level of modularity in the network, while NODF quantifies the extent of nestedness. Betweenness and Closeness indicate vectors that contain the betweenness centrality and closeness centrality of each node, respectively. InDegree and OutDegree indicate vectors that encompass the inward and outward degrees of each node. ExpectedInfluences indicates the sum of incoming or outgoing edge weights connected to a node. Identifying the core mycobiome [p < 0.05. Chi-squared tests [2 fold change > 2 and p < 0.05.The alpha diversity indices, Chao1 richness , Fisher’ion test   and Parmotrema . For a comparative analysis of the fungal communities, a rarefaction process [Dirinaria and ten Parmotrema communities were excluded from the further analyses because they were below this cutoff values.After denoising and filtering out the host lichen fungal sequences, the ITS amplicon data yielded 571,273 total fungal sequences in the  process  was implDirinaria and Parmotrema, but not significantly different between the two ELF communities. Shannon’s diversity and evenness did not vary significantly between host types. Thus, the data suggested that fungal diversity was lower in lichens than in pine bark.The alpha diversity of the ELF and EPF communities was assessed using four diversity indices Fig. b. ResultDirinaria and Parmotrema. In contrast, Basidiomycota accounted for 27.9% of the EPF communities and were less abundant in the ELF communities. At the class level, the Ascomycota-dominant pattern diverged in the two ELF communities, with Dothideomycetes being the dominant fungal class in both communities. Sordariomycetes were more abundant in Parmotrema than in Dirinaria. The most abundant order in all fungal communities was Capnodiales.Taxonomic compositions of the ELF and EPF communities were distinguishable, with differences in Basidiomycota frequency being particularly pronounced  than to that of Parmotrema (Pi. vs. Pa.: 0.983 ± 0.030)  and diversity (Shannon)  Fig. c. The neon) Fig. d. Notables Figs. b and d. ity Fig. e. Taken Dirinaria and Parmotrema were smaller, with 8 and 17 ASVs, respectively.  to identify host specialists. We observed host-specific ELF and EPF at the genus level (shown in Fig. alTraits , showed Dirinaria communities, there were 52 EPF and 20 ELF specialists identified (Additional file Parmotrema, 64 EPF and 42 ELF specialists were observed. These findings were consistent with the LDA analysis and showed that the abundance of plant-pathogen fungi was higher in the ELF communities than in the EPF communities.To further analyze the differences between the fungal communities, pairwise comparisons were conducted using a chi-squared test Fig. b. The reWhile the diversity and structure of fungal communities within lichen thalli play a critical role in their ecological functioning , 17, thePinus bark [D. applanta [P. tinctorum [The fungal communities of EPF were found to exhibit higher richness and diversity compared to ELF communities, suggesting that epiphytic fungi might serve as a potential source of fungal diversity in lichen thalli attached to the bark. Moreover, the considerable overlap of ASVs between the EPF and ELF communities further strengthens this proposition. The structure of ELF communities appeared to be influenced by the filtering effect of lichen hosts, as their taxonomic compositions were more strongly associated with host identity than with geographical location. The occurrence of Basidiomycetes in the EPF communities was a frequent finding, corroborating previous reports on the occurrence of wood-decay fungi on nus bark . The lowapplanta  and chloinctorum , which iinctorum . CapnodiDirinaria were found to be more similar to the EPF communities than those of Parmotrema, possibly due to the physical distance between the lichens and the bark. This is consistent with a previous study by Noh et al. [P. tinctorum is relatively loosely attached to Pinus bark with its rhizine. Furthermore, this might be attributed to the similarity in physical properties of the hosts. The resemblance between Dirinaria ELF and EPF could potentially be elucidated through subsequent experimental approaches.The hypothesis of host-filtration was supported by community similarity analyses, which showed that the ELF communities of two different host lichens were more similar to each other than to the EPF communities. Notably, the ELF of h et al.  which shThe network analysis revealed that the ELF communities exhibited a more modular and less nested architecture compared to the EPF communities. A higher modularity suggests that the ELF communities are more robustly sorted out by the host lichen compared to the pine bark, as discussed by Chagnon et al. . This spDirinaria were found to have a higher level of core mycobiome overlap with the EPF communities compared to the ELF communities of Parmotrema. The core member of ELF, Pestalotiopsis, a fungus known for its role as both a plant endophyte and a pathogen was observed in the Parmotrema ELF communities. This observation was consistent with the finding that the Parmotrema ELF communities had the most diverse plant pathogen fungi, followed by the Dirinaria ELF communities and the EPF communities. These results suggest that ELF communities are similar to phyllosphere fungal communities, as previously proposed [The ELF communities tend to have a lower number of core mycobiome compared to the EPF communities. According to a previous study, the core mycobiome of ELF communities was found to be largely dominated by fungi from the Capnodiales order . In lineproposed .Our analysis compared the structure of the ELF communities with that of their neighboring environments, the EPF communities. We found that the ELF community had a less diverse, less nested, and more modular structure than the EPF community. These features suggest that the distinctive architecture of the mycobiome in lichens may be shaped by strong host-filtration processes. Additionally, we found that the ELF community exhibited similarities to phyllosphere fungal communities, with a high abundance of plant pathogen fungi. While we have provided insights into the structure of ELF communities, many questions remain unanswered, such as the origin of ELF, its ecological role, and the interactions between ELF and host lichens. Future studies are needed to explore the ecological niche of ELF.Additional file 1. Fig. S1 Community similarity ordination based on geographical distance. The ordinations are visualized in two approaches: a non-metric multidimensional scaling and b principal co-ordinates analysis based on Brat-Curtis distance. Ellipses indicate the 95% confidence interval of group variance. c PCoA plots showing ELF community variability between the two host lichens from same site. Fig. S2 Centrality indices of the fungal communities. a Betweenness and Closeness indicate vectors containing the betweenness and closeness of each node. b InDegree and OutDegree indicate vectors containing the inward and outward degree of each node. c Expected influences indicates the sums of incoming or outgoing edge weights connected to a node. Mean ± SEM, ****p < 0.001, **p < 0.01, ns not significant.


Astragalus and Danshen in IPF treatment.Idiopathic pulmonary fibrosis (IPF), a chronic progressive interstitial lung disease of unknown etiology, is characterized by continuous damage to alveolar epithelial cells, abnormal repair of alveolar tissue, and alveolar wall scar formation. Currently, the recommended treatment for IPF in Western medicine is relatively limited. In contrast, traditional Chinese medicine and compound prescriptions show advantages in the diagnosis and treatment of IPF, which can be attributed to their multi-channel and multi-target characteristics and minimal side-effects. The purpose of this study was to further corroborate the effectiveness and significance of the traditional Chinese medications We performed whole-genome methylation analysis on nine rat lung tissue samples to determine the epigenetic variation between IPF and non-fibrotic lungs using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses and quantitative reverse transcription polymerase chain reactions.Astragalus and Salvia miltiorrhiza  act on the Isl1, forkhead box O3, and Sonic hedgehog genes via regulation at transcriptional and epigenetic levels during IPF.We identified differentially methylated regions and 105 associated key functional genes in samples related to IPF and Chinese medicine treatment. Based on the methylation levels and gene expression profiles between the Chinese medicine intervention and pulmonary fibrosis model groups, we speculated that Astragalus and Danshen in treating IPF, and suggest several promising therapeutic targets for preventing and treating IPF.These findings provide novel insights into the epigenetic regulation of IPF, indicate the effectiveness of The online version contains supplementary material available at 10.1186/s12890-023-02601-6. Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive interstitial lung disease of unknown etiology. Currently, the commonly accepted pathogenesis of IPF includes continuous damage to alveolar epithelial cells, abnormal repair of alveolar tissue, and alveolar wall scar formation . The patRecent studies have found that epigenetic phenomena widely occur in pulmonary fibrosis, which opens a new avenue for its prevention and treatment. The most prevalent type of epigenetic modification in eukaryotes is deoxyribonucleic acid (DNA) methylation, which regulates gene expression by changing the activity of non-coding elements , particuAstragalus and Danshen effectively inhibited the proliferation of lung tissue fibers and matrix, intervened in myofibroblast activation, and delayed the progression of pulmonary fibrosis , Isl1, FOXO3, CCAAT enhancer binding protein beta [Cebpb], heat shock protein family A (Hsp70) member 5 [Hspa5], Shh, and BMP4) were filtered by degree value for KEGG pathway enrichment analysis. The results showed that the tumor necrosis factor (TNF), autophagy, apoptosis, cyclic adenosine monophosphate (cAMP), Hippo, phosphatidylinositol-3-kinase (PI3K)-Akt, interleukin 17 (IL-17), transforming growth factor beta (TGF-β), and FOXO signaling pathways were involved  were identified for further research. Polr2a, Isl1, Shh, and BMP4 were hypermethylated in the TRE but hypomethylated in the MOD group. FOXO3, Cebpb, and Hspa5 were hypomethylated in the TRE but hypermethylated in the MOD group.DMR-associated genes were also detected, and 105 key functional genes were identified. Several IPF treatment-related DMGs have been identified, including Isl1, an LIM homeodomain gene, plays critical roles in segmental patterning as well as cell differentiation, fate determination, and diversity production during embryogenesis [Isl1 in tumorigenesis have been extensively studied. It plays an important role in gastric cancer progression and development by regulating the expression of cyclin B1 and B2  and c-MYC [Isl1 functions as a marker for well-differentiated pancreatic neuroendocrine tumors [Isl1 in the lungs, such as regulating NK2 homeobox 1 (Nkx2.1), is necessary for lung lobation and tracheoesophageal separation [Isl1 in many diseases and developmental processes are concerning. Isl1 hypermethylation is an independent predictor of disease recurrence and progression in non-muscle invasive bladder cancer [Isl1 may also help in establishing the epigenetic memory of cardiomyocyte fate commitment [Isl1 was hypermethylated in the TRE and hypomethylated in the MOD groups, indicating that methylation levels were related to IPF treatment. The expression profiles of Isl1 between the TRE and MOD groups were consistent with methylation levels. These results indicate the effectiveness of Astragalus and Danshen in IPF treatment. Although the roles and detailed mechanisms of Isl1 in IPF treatment remain unknown, Isl1 may be speculated to be a promising therapeutic target.ogenesis –39. The nd c-MYC . In addie tumors , rhabdome tumors , and neue tumors  and alsoe tumors . The rolparation . The char cancer . Isl1 mammitment . In our FOXO3, a member of the FOXO transcription factors, mediates many biological processes, such as autophagy in skeletal muscle [FOXO3 is a tumor suppressor in many cancers, including gastric [FOXO3 in IPF fibrogenesis. Its expression is downregulated, and it is hyperphosphorylated in IPF fibroblasts. FOXO3 knockout mice showed heightened susceptibility to the lung-damaging effects of bleomycin, including increased fibrosis, loss of lung function, and higher mortality. In our study, FOXO3 was hypermethylated, and its expression was downregulated in the MOD but hypomethylated and upregulated in the TRE group. Thus, to some extent, the use of TCM can act on therapeutic targets.l muscle , neural l muscle , regulatl muscle , and pril muscle . In addi gastric , colorec gastric , breast  gastric , and lun gastric . HoweverShh signaling regulates many biological processes, such as adrenocortical development [Shh during lung development. Deletion of Shh in the lung causes failure in branching and growth of the distal lung [Shh overexpression causes epithelial and mesenchymal cell proliferation to increase, leading to an abundance of lung mesenchyme but no functioning alveoli [Shh was hypermethylated and its expression was downregulated in the TRE group but upregulated and hypomethylated in the MOD group, indicating its potential regulatory role in IPF treatment. Due to the complex composition of TCM compounds, the in-depth research on the active ingredients of compound drugs and the pathological mechanism of diseases is limited. Currently, more attention is focused on single TCMs and their effective monomer components. However, TCM has synergistic effects on compatibility. Based on the research on TCM pairs, we can carry out the research on the mechanism of multi-target, multi-component, and multi-channel synergistic effects of TCM compounds. In addition, the current research on the treatment of pulmonary fibrosis with TCM mostly focuses on the prevention and treatment of target organ damage from the perspective of intervention pathway, and less on the molecular mechanism and target of TCM on the genetic level. This study deeply explores the mechanism and target of Yiqi Huoxue herbs in the intervention of pulmonary fibrosis through genome-wide methylation. It provides evidence of Chinese medicine intervention in pulmonary fibrosis on the genetic level. However, given the inherent differences between humans and other animals, the data obtained based on animal experiments also have certain limitations, and accordingly require verification in human studies.elopment , the outelopment , and medelopment . In addital lung –61. Shh  alveoli . AccordiAstragalus and Danshen act on Isl1, FOXO3, and Shh via regulation at the transcriptional and epigenetic levels during IPF treatment. Our study not only indicates the effectiveness of Astragalus and Danshen in treating IPF but also provides various promising therapeutic targets that warrant further study.In this study, we investigated whole-genome methylation patterns in the lung tissues of the CON, MOD, and TRE groups. Using this approach, DMGs in different comparison groups were identified, and 105 key functional DMGs were used for further analysis. Based on the methylation levels and gene expression profiles between the TRE and MOD groups, it can be speculated that Below is the link to the electronic supplementary material.Supplementary Material 1


The long interspersed element 1 (LINE-1 or L1) integration is affected by many cellular factors through various mechanisms. Some of these factors are required for L1 amplification, while others either suppress or enhance specific steps during L1 propagation. Previously, TRIM28 has been identified to suppress transposable elements, including L1 expression via its canonical role in chromatin remodeling. Here, we report that TRIM28 through its B box domain increases L1 retrotransposition and facilitates shorter cDNA and L1 insert generation in cultured cells. Consistent with the latter, we observe that tumor specific L1 inserts are shorter in endometrial, ovarian, and prostate tumors with higher TRIM28 mRNA expression than in those with lower TRIM28 expression. We determine that three amino acids in the B box domain that are involved in TRIM28 multimerization are critical for its effect on both L1 retrotransposition and cDNA synthesis. We provide evidence that B boxes from the other two members in the Class VI TRIM proteins, TRIM24 and TRIM33, also increase L1 retrotransposition. Our findings could lead to a better understanding of the host/L1 evolutionary arms race in the germline and their interplay during tumorigenesis. The L1 fication . Althougfication . As is tfication . Specifiin trans .Retrotransposition events were initially detected in the germline, and during early embryogenesis . Some ofTripartite motif-containing (TRIM) family proteins are crucial members in the innate immune response and have a broad effect on restricting viruses and retroelements ,49. It hTRIM28 gene, also known as KAP1 (Krüppel-Associated Box (KRAB)-Associated Protein 1) or TIF1-β , encodes for TRIM28 protein, a member of the Class VI TRIM family proteins, which share many structural characteristics . TRIM28 de novo L1 inserts in transiently transfected cultured cells and by analysis of de novo tumor-specific L1 inserts in tumors of patients with endometrial cancer, prostate cancer, and ovarian cancer. Together, our studies have identified previously unknown effects of TRIM28 on potentially distinct steps in L1 retrotransposition cycle and provided patient-based data supporting in vivo relevance of our findings.In this study using previously reported ORF2p fragments ,34,78 anPlasmids containing DNA encoding human and mouse TRIM28 were obtained from Origene  and subcloned into a PCDNA 3.1/Hygro+ vector using NheI and HindIII restriction enzymes. The coding sequences for fragments of human TRIM28 were synthesized by Genescript, including RBCCH (AA 1–509), RBB (AA 1–244), RING (AA 1–139), BB (AA 139–244), CC (AA 244–405), N-P-Br (AA 509–835), N-P AA 509–680), P-Br (AA 617–835), NHD (AA 509–617), PHD (AA 617–680), Br (AA 680–835). The break points for TRIM28 domains were used as described ,68. The 0, P-Br , includiThe ORF2 constructs were generated as previously described ,34,78. SHeLa cells were maintained as previously described . U2OS ceThe U2OS TRIM28 KO cell line was generated using CRISPR-Cas9 gene-editing using the protocol as previously described . Briefly500 000 cells were seeded 16–18 h prior to transfection in T75 flasks. 0.2 ug L1Neo or 0.6ug L1 Blast expression plasmid  and 0.8 For the L1 retrotransposition assay in Figure Five hundred thousand HeLa cells or U2OS cells were seeded in T75 flasks. Cells were transfected next day using Lipofectamine reagent (Life Technologies) following the manufacturer's protocol with 0.1 μg of pIRES2-EGFP and 0.8ug of indicated constructs. The plasmids were mixed with 12 ul of Plus reagent (Life Technologies) in 180 ul serum free media. Then 85 ul of serum free media was mixed with 15 ul Lipofectamine reagent (Life Technologies) before being added into the previous DNA mix. The transfection cocktail was added to the cells in 6 ml of serum free media for 3 h, after which it was replaced with 9 ml of Minimum Essential Media (MEM). Cells were maintained under selective medium containing 500μg/ml G418 (Life Technologies) for 14 days starting 48 h post transfection before being fixed and stained with crystal violet solution . Selective medium was changed every 3 days. The number of Neomycin-resistant colonies were counted in each flask. Statistical analysis was performed using unpaired two sample t test compared with control.Adapted from ,83, two Cell lysates were either treated (+) or not treated (−) with 5μl RNase A  and transferred to nitrocellulose membranes using the iBlot2 system (Life Technologies). The XCell Blot Module was used for wet-transfer module for ORF2p analysis. Membranes were blocked in PBS-Tween  with 5% blotting-grade blocker (BioRad) and incubated with primary antibodies overnight at 4°C. Targeted proteins were detected with corresponding primary antibodies: hORF1  antibody; abcam anti-T7 tag ab9115; cell signaling anti-FLAG #2368S; cell signaling TIF1β Antibody #4123; cell signaling GAPDH (14C10) Rabbit mAb #2118. Membranes were washed 3× in PBS–Tween for 5 min before incubation with secondary antibodies. Secondary antibody HRP-mouse anti-rabbit (Santa Cruz: sc2357) or HRP-mouse anti-goat (Santa Cruz: sc2354) was applied for 1 h at room temperature (1:5000 in 3% blotting grade blocker/PBS–Tween). Images were captured using a BioRad Gel Doc XR+ imager. GAPDH was used as loading control.2HPO4 (Sigma S3264) and 2 mM KH2PO4 (Sigma P9791), pH  7.4) and harvested using 500 μl of TLB , pH  7.2) buffer per T75 flask. The samples were centrifuged at 13 000 rpm for 15 min at 4°C. The supernatant was transferred to a new microcentrifuge tube (this is the cytoplasmic fraction). The remaining nuclear pellet in the microcentrifuge was gently washed three times with 200 ul of TLB buffer. The nuclear pellets were resuspended in TLB and the nuclear lysate samples were sonicated with a Microson XL-2000 sonicator (Misonix) 4× (6 s sonication/10s rest on ice). Samples were centrifuged at 21 130g for 15 min at 4°C. The resulting supernatant (the nuclear fraction) was then transferred to a new microcentrifuge tube. The protein concentration for each sample was determined using 595 nm wavelength OD values against a bovine serum albumin (BSA) standard.The processing of nuclear and cytoplasmic fractions was performed as previously described ,84 SpeciFor each transfection, 50 000 cells were seeded onto each glass slide. The cells were transfected with 1ug of each indicated plasmid and fixed and stained 24 h post transfection. Primary antibodies were incubated for overnight at 4°C  were incubated for 1 h at room temperature. Slides were mounted with DAPI , and confocal analysis was performed using Nikon Ti2 confocal microscope.Briefly, 4 million HeLa cells were seeded 16–18 h prior to transfection in T75 flasks and co-transfected with 5 ug of each indicated plasmid. 12 ul of Plus reagent (Life Technologies) in 180 ul serum free media was mixed with 24 ul Lipofectamine reagent and 76 ul serum free media (Life Technologies). The transfection cocktail was added to the cells in 6 ml of serum free media for 3 h, after which it was replaced with 10 ml of Minimum Essential Media (MEM). Approximately 48 h post-transfection, cells were washed as described above and harvested in 5 ml PBS. Cells were pelleted at 3000g for 5 min at 4°C and then lysed in 500 l of LEAP lysis buffer . After incubation on ice for 5 min, samples were centrifuged for 5 min at 3000g at 4°C to remove debris. Supernatant was layered on top of an 8.5% sucrose cushion that was layered on top of a 17% sucrose cushion, and then centrifuged at 36 500 rpm for 2 h at 4°C. Supernatant was removed, and pellet was resuspended in 100 ul deionized water supplemented with HALT protease inhibitor and RNAsin. Protein concentration was assessed using Bradford assay and samples were brought to equal protein concentrations. Samples were stored at –80°C in final 50% volume/volume glycerol. ORF2p-mediated LEAP reaction was performed using 1ug of each sample. The following primers were used to assess ORF2p-generated cDNA:LEAP forward primer 1: AAGGACACCTGCACCCGGATGTTCATCLEAP forward primer 2: TCCTGCGGGACCTGGAGCTGGAGATLEAP forward primer 3: CATCATCAAGAAGAGCGGCAACAACLEAP forward primer 4: CTTCAGCAAGGAGGACATCTACGCLEAP forward primer 5: AAGATTTTCGCCACCTACAGCAGCGACAAGLEAP reverse primer: AGTGGCACCTTCCAGGGTCAAGGAAReverse Transcriptase System (Promega: A3500) was used according to manufacturer's instructions. Reactions were set up at 9 ul sample. Each sample was done in duplicates with one for RT+ one for RT–. The RT+ reaction was added with 1.5 ul of AMV, the RT- reaction was added with distilled water. The final volume was brought up to 20 ul with distilled water. The following samples were incubated at 70°C for 10 min. For each sample, the same set of primers in LEAP assay were used for conventional RT-PCR.G418 resistant clones were selected as described for retrotransposition assay. Individual Neo-resistant colonies were picked and expanded in 12 well plates in 500 μg/ml G418. Genomic DNA was extracted from cells generated from individual colonies. Thirty-two clones were randomly selected from each treatment and subjected to genomic DNA extraction. GoTaq-Hot start polymerase (Promega PRM5122) was used for the PCR reactions. Each PCR reaction contained 100–200 ng of genomic DNA, 2 ul of each 10uM primer, and nuclease-free water was used to bring up the total volume of 50 μl. Typical amplification conditions were: 95°C, 3 min;  × 35 cycles; 72°C, 5 min; 4°C hold on thermocycler. Oligonucleotides used for PCR included the following: reverse primer: 5’-ATTGAACAAGATGGATTGCACGC-3’; forward primer: 5’-CAGGGATGCCCTCTCTCACCG-3’. PCR products were analyzed on 1% agarose gels.de novo insertions. In brief, the detected L1 insertions in tumor and blood/normal tissues were defined as true positive calls with the filter label ‘pass’. All the true positive L1 insertions found in tumor samples were cross-referenced with the ones detected in blood/normal tissues. Only the ones not detected in blood/normal samples but unique to tumor samples were considered as tumor specific L1 insertions. Tumor specific full-length L1 insertions were cross referenced with a list of reported polymorph L1s  on polyA-selected cytoplasmic RNA was performed by BGI as previously described . RNA-SeqP value . These dFor gene expression normalization, GraphPad Prism 9.4 was used for analysis. All genes across 12 different samples  were normalized between 0 and 100%. Zero was defined as the smallest value in each data set, and one hundred was defined as the largest value in each data set. All values in between were then normalized accordingly .Previous studies have oppositely shown that TRIM28 acts as either a potentially positive or negative regulator of L1 retrotransposition in HeLa-M2 cells and K562 cells ,58. SincTo determine whether this effect can be observed in other cell lines and to determine whether TRIM28 is required for L1 retrotransposition, we generated TRIM28 knock out U2OS cells (U2OS KO)  that do not express endogenous TRIM28 but support equal expression levels of transiently transfected human TRIM28 protein Figure . L1 retrOne possible mechanism through which TRIM28 may promote L1 retrotransposition is through interaction with L1 proteins. To test whether human and mouse TRIM28 proteins interact with human L1 ORF1 or ORF2 protein , we transiently co-expressed FLAG-tagged H-TRIM28 or M-TRIM28 with L1 ORF1p or T7-tagged L1 ORF2p and performed co-immunoprecipitation in HeLa cells using beads conjugated with anti-FLAG antibodies Figure  but not To identify the domains of the human ORF2p responsible for the interaction with TRIM28, we generated a series of T7 tagged ORF2p fragments . Plasmids containing each of these fragments were individually transiently co-transfected with a plasmid expressing either a full-length H-TRIM28-FLAG or a full-length M-TRIM28-FLAG proteins. Cellular lysates collected from HeLa cells co-transfected with these combinations of plasmids were used to perform co-immunoprecipitation followed by western blot analysis . This apTo identify whether a specific part(s) of the human TRIM28 may be responsible for increasing retrotransposition of the Neo-tagged L1, we generated plasmids containing different domains of human TRIM28 protein as previously described ,68 Figu. PlasmidTo determine whether the effect of TRIM28 fragments on L1Neo retrotransposition is dependent or independent of their ability to interact with L1 ORF2p, we performed a series of Co-IP experiments in HeLa cells using TRIM28 fragments and the ZRT fragment of human ORF2p. The ZRT construct is chosen to screen these interactions because the ZRT protein fragment contains an important enzymatic activity of the ORF2p and is easily detectable compared to other ORF2p fragments and the full-length L1 ORF2p. Western blot analysis of co-IP lysates demonstrates that all TRIM28 N- and C-terminal domains can interact with ZRT to a certain extent . In addiTo further characterize the TRIM28-ZRT interaction we evaluated subcellular localization of this interaction using western blot analysis. HeLa cells transfected with the TRIM28 and ZRT expression plasmids separately or together were fractionated to separate nuclear and cytoplasmic parts as previously described ,84. EachIt is reported that the higher-order assembly of TRIM28 dimers is dependent on B box interactions, which could be abolished by three characterized mutations A160D/T163A/E175R . To inveConsistent with our findings with the triple mutant B box, full-length triple mutant TRIM28 (TRIM28 3M) is unable to increase L1 retrotransposition compared to TRIM28 WT Figure , D. WestN = 4 for each condition). This time point was chosen based on previous findings that L1 retrotransposition in this assay is detectable after 36 h post-transfection (P < 0.05). We used RNA-Seq by Expectation-Maximization (RSEM) analysis and gene set enrichment analysis (GSEA) to detect cellular pathways altered in the TRIM28 WT samples compared to the Control and TRIM28 3M samples (N = 4 each). This approach also identified DNA repair as a significantly downregulated pathway when TRIM28 WT was overexpressed   Figure   Figure . Based o) Figure . These rOur results show that TRIM28 protein interacts with L1 ORF2p but not ORF1p Figure  support in vitro can be analyzed using the well-established L1 element amplification protocol (LEAP) (The RT activity of ORF2p l (LEAP) ,86,99. Il (LEAP) ,78. To al (LEAP) . This apl (LEAP) . To confl (LEAP) . This rel (LEAP) . Additiol (LEAP) . As expel (LEAP) . This prl (LEAP) . In paral (LEAP) . This fiP = 0.04). Although no significant differences were detected in TRIM28 WT versus 3M (P = 0.2), the 3M group was also not different from the Control group and there is a trend of fewer 3 kb positive clones being formed when TRIM28 WT is overexpressed compared to the 3M construct . Consistent with our findings with the LEAP assay, TRIM28 WT overexpression leads to shorter de novo L1Neo inserts, compared to the control or TRIM28 3M overexpression.To confirm that our LEAP results, which are obtained using cytoplasmic RNPs, are relevant to L1 retrotransposition products, we performed a ruler PCR assay with the reported 3 kb primers . This siin vivo, we identified patients with endometrial cancer, prostate cancer, and ovarian cancer using GDC Legacy database. Patients within each cancer group were separated in to two groups, with 8 patients demonstrating ‘High’ and 8 patients exhibiting ‘Low’ TRIM28 mRNA expression according to the reported TRIM28 mRNA expression in their tumors , and so do the patients with prostate cancer  and ovarian cancer . All patDe novo tumor specific L1 insertions were identified as present in the tumor WGS but not in blood/normal WGS and meeting MELT criteria of de novo L1 inserts as described . Based on the described inclusion criteria, we could not identify sufficient number of patients per group to conclusively determine whether TRIM28 expression levels affect the number of de novo L1 inserts per patient. However, the large number of total de novo L1 inserts identified in each group of patients has allowed us to compare their lengths. Unpaired two-sample t test analysis determined that de novo L1 insertions are significantly shorter in the ‘High’ TRIM28 expression group compared to the ‘Low’ TRIM28 expression group of patients in each cancer cohort . These values are calculated using all tumor-specific L1 inserts, which include those that may have happened before the increase in the TRIM28 expression occurred in any given tumor. The number of full-length L1 inserts in each group was too small to analyze them as a separate category. These patient-based findings agree with our findings in HeLa cells using LEAP assays  or binds to the RNA template which facilitates pausing or dissociation of the whole complex from the template RNA. Second, TRIM28 directly or indirectly binds to the mRNA template and interferes with ORF2p reverse transcription by blocking its forward progress, which ultimately leads to ORF2p pausing and/or dissociation from the RNA template. These models are consistent with the reported ability of TRIM28 to interact with both proteins and nucleic acids  and invoin vivo. TRIM28 is ubiquitously expressed throughout development and in adult tissues. In embryonic stem cells the expression level of L1 and TRIM28 are both high (de novo L1 integration is beneficial to driving cancer evolution regardless of the size of L1 inserts. The reverse order of these evolutionary steps is also possible. According to this hypothesis, L1 may have evolved to use TRIM28 to increase the number of its inserts. To counteract this the host utilized the same mechanism to reduce the number of full-length L1 inserts to halt L1 propagation.Our findings of the effect of TRIM28 on L1 retrotransposition and cDNA synthesis in different cancer models may have important biological implications for understanding evolutionary arms race between the host and L1. Most of the 500 000 L1 elements in the human genome are 5′ truncated , but theoth high ,132. It In summary, we have identified TRIM28 as a suppressor of ORF2p-mediated cDNA synthesis, but overall, a positive regulator of L1 retrotransposition. Both effects rely on the B Box facilitated oligomerization. These findings have evolutionary and disease relevant consequences that may be related to other viral and non-viral RT containing proteins. Although TRIM28 is rarely mutated in human cancers, its expression is often deregulated with overexpression being linked to poor survival. Our findings support that the ability of TRIM28 to promote tumorigenesis may include its potential to increase L1-induced genomic instability. Future mechanistic studies of TRIM and L1 proteins will be needed to provide a more comprehensive understanding of functional outcomes of their interactions on genome stability, evolution, and cancer pathogenesis.The raw data files for RNA-seq analysis in this manuscript are available under BioProject: PRJNA925462.gkad247_Supplemental_FilesClick here for additional data file.


This review mainly addresses applications of polymer/graphene nanocomposites in certain significant energy storage and conversion devices such as supercapacitors, Li-ion batteries, and fuel cells. Graphene has achieved an indispensable position among carbon nanomaterials owing to its inimitable structure and features. Graphene and its nanocomposites have been recognized for providing a high surface area, electron conductivity, capacitance, energy density, charge–discharge, cyclic stability, power conversion efficiency, and other advanced features in efficient energy devices. Furthermore, graphene-containing nanocomposites have superior microstructure, mechanical robustness, and heat constancy characteristics. Thus, this state-of-the-art article offers comprehensive coverage on designing, processing, and applying graphene-based nanoarchitectures in high-performance energy storage and conversion devices. Despite the essential features of graphene-derived nanocomposites, several challenges need to be overcome to attain advanced device performance. Progression in energy technologies demands the use of innovative competent nanomaterials to attain the desired high performance of those technologies . GraphenIn recent years, functionally graded graphene-reinforced composites have been focused on heavily in the literature . FunctioThis state-of-the-art review primarily covers the fundamentals of graphene and its nanocomposites. Additionally, the energy storage and conversion solicitations of graphene-derived nanocomposites are scrutinized in terms of important devices. Accordingly, high-efficiency multi-functional supercapacitors, batteries, and fuel cells are debated. To the best of our knowledge, this article is pioneering in the field of energy storage and conversion devices in terms of the review outline, the collected literature, and the wide coverage of various devices to study the effect of graphene-based nanocomposites. Few previous reviews of the literature on graphene materials have included a comprehensive survey in order to support future progress in this field . The fut2-hybridized hexagonal carbon atoms with π-orbitals [2V−1s−1 and 3000–5000 W/mK, respectively [Graphene is considered an excellent nanocarbon form ,23,24. Iorbitals ,26,27. Forbitals ,29,30. Sorbitals ,32,33,34orbitals ,36,37,38orbitals ,40. The ectively ,42. In aectively .Graphene nanosheets can be held together through van der Waals interactions ,45,46. W2-hybridized structure of graphene and styrene rings of polymer  features [−2. Jiao et al. [−1 and an efficiency of ~88% through 100 cycles. Hence, the effectiveness of using graphene in Li-ion battery anodes is established.Li-ion batteries have also been used as effective energy storage devices ,93. Prevfeatures . Li and features  designedo et al.  formed a−1. Moreover, the polyaniline-grafted graphene-oxide-derived anode had a high cycling stability and Coulombic efficiency. Guo and researchers [−1), and an extended cycling life . Chae et al. [−1 was attained. Hence, the inclusion of graphene and graphene oxide provided considerable benefits to the performance of the Li-ion battery due to dispersion and interactions with the compatible polymer structures [Conducting polymer and graphene-derived nanocomposite electrodes have been designed for Li-ion batteries . Li et aearchers  reinforce et al.  worked oructures ,107.In lithium-ion storage batteries, basically different graphene-based nanomaterials have been used for electrodes such as graphene-supported polymers, graphene-supported sulfides, graphene-supported metal oxides or alloys, etc. . The syn−2 and ~311 mA cm−2, respectively, relative to the three-dimensional graphene oxide . Moreover, the modified three-dimensional graphene oxide exhibited an elevated proton conductivity of 3.2 S cm−1 relative to the non-modified three-dimensional graphene oxide (0.7 S cm−1). The use of the three-dimensional graphene oxide with inserted sulfate ions was suggested to enhance the proton conduction capability of the nanomaterials due to their superior fuel cell performance. The fuel cell is an incipient energy conversion technological development employing nanocomposite structures for electrolyte membranes or catalysts ,112. ConLee et al.  fabricatTraditionally designed supercapacitors, batteries, or fuel cells have high price, high density, and low structural reliability problems . Before The commonly used nanocarbon nanofillers for device electrodes and components include graphene, graphite, carbon nanotube, etc. Among these, graphene has been found to be the most efficient to enhance the performance of various energy devices. Moreover, the use of conjugated polymers has been found to enhance the electron and charge transportation through these remarkable nanomaterials ,127. It More specifically, in supercapacitors, different graphene-derived nanocomposites have been utilized. Polymer/graphene nanocomposites with a high surface area and pseudocapacitance may result in a fabulous enhancement in the supercapacitor performance . ConsequAlthough promising results have been attained for numerous graphene-nanocomposite-derived Li-ion battery electrode materials, there are some prevailing challenges that need to be overcome for future developments . AccordiThe inclusion of graphene in nanocomposites may cause high performance and resilience for fuel-cell-based energy conversion devices . GraphenHenceforward, the future of graphene nanomaterials in energy devices greatly rely on the development of new innovative materials. In this context, using three-dimensional graphene-derived nanocomposites in energy systems may bring revolution in this field . The conFacile approaches need to be adopted to achieve efficient graphene-derived nanocomposites . High-peThis overview exclusively and comprehensively debates the progresses in the arena of graphene and graphene-derived nanocomposites, concentrating on the energy storage and conversion solicitations. Graphene-derived nanomaterials have been constantly researched for supercapacitance, Li-ion batteries, and fuel cell applications. These devices have been categorized among the most essential energy storage and conversion devices. The investigations exposed the stability, charge storage, charge capacity, and conversion efficiency of these energy systems while employing some efficient carbon nanomaterials. In the case of advanced supercapacitors and batteries, using graphene, graphene oxide, functional graphene, and related nanocomposite nanomaterials amended the charge storage performance as well as the power conversion and utilization capabilities of the devices. The employed polymers along with the graphene-based nanofillers were found to be important to develop the high-efficiency energy-associated devices and systems. In other words, the choice of graphene nano-reinforcements along with the selection of polymers were considered indispensable for a better performance of the energy devices. In addition, the graphene modification as well as the graphene nanocomposite processing methods have played imperative roles in the enhancement of the charge storage and energy-production-related performance of these materials, resulting in high-efficacy supercapacitors and other devices. Consequently, the indispensable features of the energy devices and systems were enhanced using graphene or its derived nanofillers, graphene synthesis methods, nanocomposite fabrication techniques, etc., leading to homogeneous microstructure, matrix–graphene interactions, electron conduction, specific capacitance, charge capacity, high charge storing capability, charge–discharge, life cycle, charge density, power density, cyclic concert, reliability, and numerous related features of these systems.Briefly speaking, in this article, we debated the topical research advancements in the graphene and graphene-derived nanocomposites for important energy storage and conversion systems. Although substantial developments have been made so far, the marvelous potential for real-world applications in cutting-edge energy systems still requires further research. Importantly, the fabrication of graphene and its related nanocomposites for diverse energy applications are still in premature stages, where gaps exist in understanding the atomic/molecular level functioning, essential limits, and failure of devices. Moreover, the high surface area of graphene-derived nanomaterials plays a significant part in supercapacitors and batteries for charge storage. The overall nanocomposite design supporting the combination of a high surface area and pseudocapacitance of graphene may result in a tremendous improvement in the charge-storing potential of devices. To overcome the challenges of energy storage systems, it is essential to develop and understand the multidisciplinary tactics and connections between materials science, engineering, microstructures, electrochemistry, physical features, and interactions.Research has revealed the potential of graphene nanocomposites towards the electrodes of rechargeable Li-ion batteries. The high-energy densities of Li-based materials were found to be favorable for electronic energy storage. Mostly, the anode systems in Li-ion batteries benefited from using graphene nanocomposites due to their high surface area and conductivity values. All of these factors caused the high power density and rate performance of the Li-ion battery anode. In this context, the high surface area of graphene can accommodate more Li-ions to improve the energy density and capacity of the batteries. Carefully designed graphene-nanocomposite-based electrodes have been applied for commercial-level applications. On the other hand, the high surface area, aggregation trends, and porosity of graphene-derived nanomaterials can also decrease the performance of the electrode materials due to the formation of the solid electrolyte interphase layer causing a low first cycle Coulombic efficiency. Hence, the overall practical applications of graphene nanocomposites in energy storage systems have been limited. Moreover, using porous graphene-based materials may absorb large amounts of electrolytes, leading to battery swelling during electrochemical processes and also cost issues.Moreover, unusually high physical/chemical properties of graphene nanocomposites have been used for fuel cell applications due to their efficient proton conduction. Graphene-nanocomposite-derived fuel cell catalysts have cost effectiveness, high durability, and insensitivity to carbon monoxide during electrocatalysis relative to platinum-based catalysts. In proton-conducting electrolytes, graphene derivatives have oxygen functionalities to hold water and develop the proton transportation channels. However, due to blending with polymers, some challenges may arise like aggregation, decreased surface area, and homogeneity of the electrolyte membranes. Hence, graphene has been blended with different polymers in order to fabricate nanocomposites for enhancing the processing, mechanical stability, chemical and electrochemical features, and other functions for applications in supercapacitors, batteries, and fuel cells. Although great evolutions have already been attained, the research in this field still faces several unresolved glitches.Hence, this overview portrays the developments in the field of graphene nanomaterials for energy storage and conversion. In this context, several graphene nanofillers and derived nanomaterials along with polymers such as conjugated polymers, thermoplastics, and thermosets have been used for nanocomposites in order to attain advanced features of the energy devices. Appropriately adopting graphene-derived nanomaterials offers some notable future opportunities towards important energy storage and conversion systems. Moreover, constant research efforts may resolve the challenges related to the design, properties, and performance of applying graphene materials in energy devices. Forthcoming research may extend the use of the above conversed graphene-based nanomaterials towards efficient photovoltaics, light-emitting diodes, nanogenerators, microelectronics, and numerous other devices.


Due to its outstanding properties, graphene has emerged as one of the most promising 2D materials in a large variety of research fields. Among the available fabrication protocols, chemical vapor deposition (CVD) enables the production of high quality single-layered large area graphene. To better understand the kinetics of CVD graphene growth, multiscale modeling approaches are sought after. Although a variety of models have been developed to study the growth mechanism, prior studies are either limited to very small systems, are forced to simplify the model to eliminate the fast process, or they simplify reactions. While it is possible to rationalize these approximations, it is important to note that they have non-trivial consequences on the overall growth of graphene. Therefore, a comprehensive understanding of the kinetics of graphene growth in CVD remains a challenge. Here, we introduce a kinetic Monte Carlo protocol that permits, for the first time, the representation of relevant reactions on the atomic scale, without additional approximations, while still reaching very long time and length scales of the simulation of graphene growth. The quantum-mechanics-based multiscale model, which links kinetic Monte Carlo growth processes with the rates of occurring chemical reactions, calculated from first principles makes it possible to investigate the contributions of the most important species in graphene growth. It permits the proper investigation of the role of carbon and its dimer in the growth process, thus indicating the carbon dimer to be the dominant species. The consideration of hydrogenation and dehydrogenation reactions enables us to correlate the quality of the material grown within the CVD control parameters and to demonstrate an important role of these reactions in the quality of the grown graphene in terms of its surface roughness, hydrogenation sites, and vacancy defects. The model developed is capable of providing additional insights to control the graphene growth mechanism on Cu(111), which may guide further experimental and theoretical developments. Graphene, as the first isolated two-dimensional 2D) material , has attD materiaThe CVD process is highly dependent on the synthetic parameters, such as the partial pressure of precursors, temperature, substrate surface, and carbon solubility in the metal substrate ,36,37. A domains ,41,42,43 domains . Moreove domains ,46, therThe controllable synthesis of large-sized graphene using CVD is still a challenge due to the complex growth process and its sensitivity to growth conditions . A detaiThe mechanism of graphene growth in CVD has a multiscale character, i.e., it requires multiscale modeling approaches to properly complement experimental investigations. Numerous theoretical studies, employing ab initio calculations , moleculWhen combined with first principle calculations, the KMC method emerges as a promising approach that is capable of characterizing graphene growth with atomistic resolution and for significantly larger time and length scales compared to other models. Such multiscale models were developed by, e.g., Li et al. , who usen et al.  proposedu et al. , who shoan 10 nm ,60,61,62an 10 nm  developeDespite all these outstanding efforts to model the growth of graphene for isolated flakes, important questions, concerning the impact of the kinetic pathways on the atomistic scale of reactions involved, remain open. Developing KMC methods that are capable of providing insight into the atomistic scale requires consideration of a complete list of reactions, which was previously limited to the size of the simulated graphene flakes ,60,61. FIn this paper, we report a DFT-based KMC model capable of dealing with a comprehensive list of reactions, ranging from the dissociative adsorption of CHIn order to analyze the growth mechanism of graphene, we utilized the density functional theory (DFT) method to determine the activation energy barriers for relevant possible reactions involving the species of interest, i.e., H, C, CH, CHSince Li et al. reported activation energy barriers of the set of reactions happening during the CVD of graphene on Cu(111) using CHecursors , we firsecursors . In parti et al. . TherefoThe BEEF-vdW functional makes it possible to determine the barrier height for reactions on surfaces very accurately, since it was especially designed for the processes on surfaces . At the We found a reasonable agreement between the results obtained by the PBE-D3 and PBE-D2 , but a mi et al. . This wai et al. ). TherefUsing the activation energy barriers and reaction energies obtained, we calculated the linear scaling relations for the detachment of “small species” from the graphene edges . We alsoFinally, we have to mention that we also calculated the hydrogenation and dehydrogenation reactions, which were missing in the previously reported approach. We have added these reactions to the list of reactions used in the KMC method see . They arIn order to study the mechanism of graphene growth on a Cu(111) surface, we utilized the KMC protocol with reaction rates obtained from the DFT as parameters. Eight different simulations were carried out to examine the impact of partial pressures on the rates of chemical reactions occurring during the growth process see .Each simulation resulted in a non-equilibrium steady-state being reached at approximately on plots . In the ions see . Conversions see .ver time . After atate see . This petate see b. Since tate see c,d.We conducted a comprehensive analysis of the spatio-temporal evolution of all the samples refer to . Our fin quality ,39,42,72time see . It is cFinally, we observed a high concentration of H adatoms  in detail. The precursor used in this study is CHtion see  and the tion see . Therefocess see .Moving on to the next set of reactions, CThe contribution of edge attachments of carbon and its dimer were found to be the most stable, while other attached species were more likely to be hydrogenated and detached, as indicated by the arrows in To successfully grow graphene, it is crucial to allow ring closure reactions, which are highlighted by the brown region in d growth . The incTo evaluate the impact of (de)hydrogenation on graphene growth, we conducted KMC simulations with and without the set of (de)hydrogenation reactions for samples S1 hydrogenation on the quality of the grown graphene is evident from the results. The surface roughness of the samples was substantially lower in the presence of (de)hydrogenation reactions, with values of 1.64 nm and 1.85 nm for S1 and S2, respectively, compared to 1.98 nm and 2.12 nm, respectively, when the reactions were absent. Additionally, the inclusion of (de)hydrogenation reactions led to a decrease in root-mean-square roughness, whose values were reduced by 0.27 nm and 0.36 nm for S1 and S2, respectively. To further analyze the roughness of the samples, we used the radius as a measure and calculated the roughness profile, which shows the deviation of the radius from the mean radius hydrogenation reactions, as demonstrated by the surface roughness analysis and the observed reduction in the number of hydrogenated edges and defects (vacancies). The results suggest that incorporating (de)hydrogenation reactions on the edges leads to higher quality graphene material.For the development of the multiscale model, we considered the CVD conditions depicted in reaction ,75; therreaction , we neglAll quantum mechanical calculations were performed using density functional theory with the BEEF-vdW  and PBE To calculate reactions between “small species”, e.g., the dehydrogenation of methane, the Cu(111) surface was modeled by a four-layer 3 × 3 slab (with 2 bottom layers being frozen), see  in Ref. .In the present study, we have not considered subsurface processes. However, we included five reaction barriers for the formation and reconstruction of the hexagon at the graphene edge, which were proposed by Chen et al. . TransitCollection scheme, in which the algorithm traverses the lattice points to collect species and, after considering their neighboring sites and the reference reactions list, collects all possible reactions from current configuration as a list; (ii) a Selection scheme, where the algorithm calculates the cumulative  rate of all possible reactions collected in the previous step and generates two random numbers ; and (iii) an Update scheme with three stages, which include executing the selected reaction based on its type, advancing the simulation time using the second random number generated before, and updating the lattice configuration accordingly.To efficiently simulate CVD growth processes with elementary steps characterized by diverse rates, we developed an algorithm based on the rejection-free KMC approach, known as the BKL algorithm ,88. We aWe calculated all reaction rates, except for the CHry (TST)  using acximation  considerOne of the main aims of this study was to determine how the growth of graphene using CVD is affected by the partial pressures of CHTo study the CVD growth of graphene on a Cu(111) surface at 1300 K, we developed a multiscale model by combining first principle calculations and the KMC method. By implementing a kinetic Monte Carlo program, we were able to accurately represent relevant atomic scale reactions without any additional approximations, thereby allowing us to study long time and length scales. Specifically, we were able to observe graphene flakes of up to 38 nm that formed in 0.157 s under different CHrefer to . SpecifiWe also identified the role of individual reactions that occurred during graphene growth and studied the reaction pathways in detail. Data analysis suggested that the carbon monomer (C) and dimer , thus providing insights into how controlling parameters affect the quality of the material. This method is particularly helpful for understanding the kinetics of CVD graphene growth and other experimental observations at the relevant temperatures and pressures used in the production of graphene. However, it is important to note that our model assumed a hexagonal lattice, which limited the growth mechanism to hexagonal symmetry. This means that we cannot model topological defects such as rings of atoms with five or seven atoms. Additionally, our simulations began with an initial flake as a ribbon (measuring 0.284 × 100 nmgraphene . We did graphene ,92. Ther