Equisetum species  were used to reduce silver ions to silver nanoparticles (AgNPs). The synthesized AgNPs were characterized using UV-Vis spectrophotometry, Fourier Transform Infrared Spectroscopy (FTIR), Energy Dispersive X-ray (EDX), Transmission Electron Microscopy (TEM) and Dynamic Light Scattering (DLS) measurements. FTIR data revealed the functional groups of biomolecules involved in AgNPs synthesis, such as O-H, C-H, C=O, C-O, and C-C. EDX spectroscopy was used to highlight the presence of silver, while DLS spectroscopy provided information on the mean diameter of AgNPs, that ranged from 74.4 to 314 nm. The negative Zeta potential values  indicate the stability of the obtained colloidal solution. The study also focused on establishing the photocatalytic activity of AgNPs, which is an important aspect in terms of removing organic dyes from the environment. The best photocatalytic activity was observed for AgNPs obtained from E. telmateia, which degraded malachite green in a proportion of 97.9%. The antioxidant action of the three AgNPs samples was highlighted comparatively through four tests, with the best overall antioxidant capacity being observed for AgNPs obtained using E. sylvaticum. Moreover, the biosynthesized AgNPs showed promising cytotoxic efficacy against cancerous cell line MG63, the AgNPs obtained from E. sylvaticum L. providing the best result, with a LD50 value around 1.5 mg/mL.The ethanolic extracts of three  The potential use of nanomaterials is a continuously developing research field. Metal nanoparticles, in particular, have received special attention due to their widespread applications in areas such as medicine, electronics, cosmetics and food, optics, chemical industry and many others ,2.Among metal nanoparticles, silver nanoparticles (AgNPs) have unique properties such as chemical stability, catalytic activity, antibacterial, antiviral, antifungal, anti-inflammatory and anticancer properties, which makes them medically relevant ,4.In general, for AgNPs synthesis, a variety of chemical, physical and biological methods can be used. Biological methods currently represent the center of attention for researchers, since they provide numerous advantages, given that they involve eco-friendly processes, low costs and offer the possibility of avoiding high temperatures and pressure. Therefore, the current trend in AgNPs synthesis is the use of microorganisms, enzymes, and plants extracts. However, plants are usually preferred given their high accessibility and the presence of biological compounds that can participate in this process, as well as the feasibility of the synthesis process ,6.Acanthaceae, Amaranthaceae, Apocynaceae, Asphodelaceae, Asteraceae, Burseraceae, Dioscoreaceae, Euphorbiaceae, Fabaceae, Lamiaceae, Moraceae, Myrtaceae, Poaceae, Rutaceae, and Solanaceae [The existing literature contains numerous examples of plant extracts that can be used to synthesize AgNPs, which belong to families such as: lanaceae ,7.Compounds involved in AgNPs synthesis are distributed in different parts of the plant  and consist of primary  and secondary  plant metabolites ,9.Achillea biebersteinii flower extract [Biological actions of AgNPs depend on the size, particle morphology and composition, coating, agglomeration tendency, particle reactivity in solution, ion release efficiency, cell type, as well as on the reducing agent used for the obtaining of particles . Studies extract .Other studies have shown that nanoparticles can induce cancer cell apoptosis and oxidative stress by mitochondrial damage only in cancerous cells without affecting healthy ones, capture and adsorb cytoplasmic proteins, influence gene transcription and increase the sensitivity of cancerous cells to conventional chemotherapeutics .Cell culture studies have shown that AgNPs are able to induce cytotoxicity in human cell lines including human bronchial epithelial cells, red blood cells, macrophages, liver cells and bone cells especially for a particle size of less than 10 nm. The cytotoxicity of AgNPs has been reported to be dose, size and time-dependent .Equisetaceae family, traditionally known as Horsetails, represents a group of perennial plants that consist of a fertile stem with brown sprouts that develops in early spring and a sterile one, that grows in late spring and persists until late autumn, the latter one being of medical importance [Equisetum genus grows in different regions of Europe, as well as in North, Central and South America [The portance . The Equ America . Species America ,18. Acti America ,20.Equisetum genus is E. arvense, for which many biological activities have been confirmed, especially with applications in kidney disorders. Nonetheless, other species of the genus have been popularly used in different diseases. The few studies that focused on investigating other species of Equisetum genus emphasized their promising biochemical potential [The most known and researched species of the otential .Equisetum genus have to offer in their synthesis represent the starting point for our research. The purpose of the study is to investigate the potential use of some Horsetails species in the synthesis of AgNPs, that can offer applications in various domains, such as medicine (through the possible antioxidant and antitumor activities), as well as for environment protection through photocatalytic activity.Therefore, the numerous applications of AgNPs in different fields and the advantages that plants from the Equisetum species: E. pratense Ehrh. (Ep), E. sylvaticum L. (Es) and E. telmateia Ehrh. (Et). To the best of our knowledge, this is the first report on AgNPs obtained using either of the three Equisetum species, which emphasizes the novelty of the research.Therefore, the present study focused on structural and morphological characterization of AgNPs obtained using ethanolic extracts of three less investigated Equisetum species, the photocatalytic action was then tested. Moreover, the antioxidant and antitumor capacities of the synthesized AgNPs were also evaluated comparatively.The analyses were carried out using various techniques such as UV-Vis spectrophotometry, Fourier Transform Infrared Spectroscopy (FTIR), Energy Dispersive X-ray (EDX), Transmission Electron Microscopy (TEM) and Dynamic Light Scattering (DLS) measurements. Following the synthesis of AgNPs using extracts of E. pratense and E. sylvaticum were collected from the North-Eastern region of Romania in June–July 2017. E. telmateia was harvested in July 2018, also from North-Eastern Romania. The plants were authenticated using fresh material in the Plant Biology Laboratory of the Biology Faculty, “Al. I. Cuza” University, Iasi, Romania. After identification, the plants were dried in shade at a controlled temperature of 23 °C for 21 days, until the mass became constant. Ethanolic extracts were obtained using 10 g dry plant material mixed with 70% ethanol. The samples were placed in the ultrasonic bath at 30 °C for 30 min. The mixture was filtered and then the ethanolic extracts were evaporated using a rotavapor until the mass became constant and no residual water was left. The dried extracts were maintained in a dry, dark place at a temperature of 4 °C until further analysis [analysis ,23.3 solution under magnetic stirring. In order to study the synthesis conditions, reaction parameters such as silver nitrate solution concentrations , extract:AgNO3 ratios , pH values  and stirring intervals  were varied. After establishing the reaction conditions, the obtained colloidal solutions were centrifuged at 10,000 rpm for 30 min. In order to purify the nanoparticles, the supernatant was removed and the AgNPs were redispersed in distilled water, centrifuged and eventually separated [E. pratense, Es–AgNPs for E. sylvaticum, and Et–AgNPs for E. telmateia.For AgNPs synthesis, the resuspended extracts were added to the AgNOeparated . This opEquisetum extracts after being mixed with AgNO3. The UV-Vis spectra were recorded in the 300–600 nm range to distinguish the maximum surface plasmon resonance (SPR), using a Jasco V-530 UV-Vis double beam spectrophotometer for this determination. The FTIR spectra were obtained with a Bruker Vertex 70 spectrophotometer, by potassium bromide tableting, over a scan interval of 4000–310 cm−1. The EDX qualitative analysis was performed using a Quanta 200 Environmental Scanning Electron Microscope (ESEM) with EDX. The used EDX detector allowed rapid determination of the elementary composition of nanoparticles. The DLS measurements were performed using a Delsa Nano Submicron Particle Size Analyzer (Beckman Colter) that provided the average diameter of nanoparticles and the Zeta potential value. Additionally, TEM analysis was performed using a Hitachi High-Tech HT 7700 Transmission Electron Microscope.The formation of AgNPs was observed through the color change of Moreover, prior to the purification and characterization of AgNPs, the supernatant and the initial plant extract were analyzed taking into account the phenolic content using the modified spectrophotometric Folin-Ciocâlteu method . Gallic The photocatalytic activity of the synthesized AgNPs was studied by observing the degradation of malachite green under the influence of sunlight at room temperature and under continuous stirring. The color change was monitored over time and samples were taken at certain intervals and centrifuged. The supernatant was scanned in the 500–700 nm range ,28,29. SThe following tests were used to study the antioxidant activity: chelation of ferrous ions, LOX inhibition, the hydroxyl radical and superoxide anion radical scavenger capacities. The chelating capacity of ferrous ions was determined using ferrozine which forms a pink complex with maximum absorbance at 562 nm . The det3 cells, in Dulbecco’s Modified Eagle’s Medium (DMEM). MG63 cell line selection is based on potential further applications of the AgNPs in bone tumor treatment. The culture plates populated with cells were incubated at 37 °C. The cell lines were treated with the following concentrations: 0.5 mg/mL, 1 mg/mL and 2 mg/mL of Ep–AgNPs, Es–AgNPs and Et–AgNPs for 24, 48 and 72 h. Each fraction of the studied samples was dissolved directly in the cell culture medium with a content of 2% dimethyl sulfoxide (DMSO) [The cytocompatibility of AgNPs was evaluated using cell cultures (MG63 osteosarcoma cells) in 96-well microtiter plates, each being populated with 5 × 10e (DMSO) . The MTTe (DMSO) . The absThe absorbance determined in the experimental cultures was compared with that of the control cultures, in which the extract was not present . All experiments were performed in triplicate ,37.p-value of less than 0.01 was accepted as significant.Cell viability studies were carried out in triplicate (n = 3) for each experiment and analyzed by means of two-way ANOVA. A In the synthesis of AgNPs using plant extracts, these general steps were followed: preparation of the extract and silver salt solutions, obtaining of the AgNPs by mixing the two solutions in different proportions, under certain pH conditions, and for different periods of stirring time. The results are available in the + to metallic nano-silver, Ag0. The modification in color can be observed in the images found in the First of all, the obtaining of AgNPs was monitored via color change. Therefore, after completion of the reaction, a change in color from yellow to brown was observed, which was attributed to the reduction of Ag3 concentration of 1 mM; extract:AgNO3 ratio 1:9 (v:v); pH 6; stirring time of 300 min.Following all determinations, the conditions for the AgNPs synthesis were established as: AgNOE. pratense, E. sylvaticum and E. telmateia has not been previously described in literature. However, different published studies reported that the SPR value of other biosynthesized AgNPs was located between 400 and 500 nm and any shift of this value may be attributed to the metabolites involved, which serve as reducing and stabilizing agents [To the best of our knowledge, the synthesis of AgNPs obtained using g agents ,40,41,42TEM analysis provides data on the shape and size of AgNPs, while the dimensional distribution (polydispersity index) and the hydrodynamic diameter of AgNPs diameter were determined by DLS. For all three samples, TEM images  showed tThe Zeta potential values were −23.76 for Ep–AgNPs, −29.54 for Es–AgNPs and −20.72 for Et–AgNPs. These negative values show that the biomolecules found on the surface of AgNPs are negatively charged, causing rejection between nanoparticles, which means there is no aggregation and the obtained colloidal solution is stable ,44. Howe+) ions reduction and for capping and stabilizing of AgNPs were highlighted using FTIR analysis. The FTIR spectra of the extract and of the corresponding AgNPs are shown in a comparative manner in The functional groups of different biomolecules responsible for silver , which is used in various industries and can be found in aquatic and terrestrial ecosystems, therefore posing a potential danger to human health, was analyzed [In order to test the photocatalytic action of the obtained AgNPs, the degradation of a dye  and hydroxyl radicals (HO•), which destroy the structure of the dye, transforming it into smaller organic molecules, thus degrading it [The photocatalytic activity of AgNPs can be explained either through the excitation of electrons and their transition to a higher energy state due to the surface plasmon resonance effect, or through the absorption of UV radiation by AgNPs due to transitions between 4d and 5s orbitals with high energy electron generation. Furthermore, the excited electrons are accepted by oxygen molecules dissolved in the reaction medium which are transformed into superoxide anions  present an activity twice as intense compared to Ep-AgNPs (172.83 ± 0.54 µg/mL) and 1.8 times higher than that of Et-AgNPs (160.56 ± 1.72 µg/mL). EC50 represents the concentration at which nanoparticles show an inhibition/scavenger activity of 50%. The EC50 values are inversely proportional with the efficiency of the antioxidant activity.For the determination of the ferrous ion chelating capacity, when comparing the ECTherefore, it can be stated that the ethanolic extracts that were used to obtain AgNPs have the ability to chelate ferrous ions, and the intensity of the action is much higher for the obtained nanoparticles. As only some of the active constituents present in the extracts participate in the formation of AgNPs, a clear ratio comparing the efficiency of the extracts with that of the nanoparticles cannot be calculated ,10.E. sylvaticum extract are almost twice as active compared to those obtained using E. pratense and E. telmateia extracts. This could be explained through the involvement of only some of the polyphenols found in the extract in the formation of AgNPs. Correspondingly, in the case of extracts, the best antioxidant activity was also observed for E. sylvaticum [Equisetum species showed a much more intense chelating capacity of the ferrous ion than gallic acid, which was used as standard.It has been shown that nanoparticles obtained using lvaticum . As showThe active compounds present in the extracts inhibit 15-LOX (an enzyme of the oxidoreductase family) by blocking the oxidation of linoleic acid and reduce the absorbance measured at 234 nm . The abi50 value of 9.14 ± 0.53 µg/mL) present a three times more intense activity compared to that of Ep-AgNPs  and four times higher than that of Et-AgNPs .For the determination of LOX inhibition, Es-AgNPs  are 6 times more active compared to Ep-AgNPs (362.15 ± 4.29 µg/mL) and 3.3 times more active compared to Et-AgNPs (204.04 ± 24.21 µg/mL).Hydrogen donor groups are required to neutralize the hydroxyl radical. It can be seen that Es–AgNPs showed the most significant hydroxyl radical scavenging capacity, comparable to that of the chosen standard. The lowest scavenging activity of the hydroxyl radical was recorded for Ep–AgNPs. The same order was observed in the case of extracts, as well . In FiguThe superoxide anion radical is formed by reduction of molecular oxygen to accept only one electron, most often in the side reactions of the mitochondrial respiratory chain. Even if, from a chemical point of view, it is a rather weak oxidizing agent, it can generate hydroxyl radicals and singlet oxygen, which are very strong oxidants ,56.The synthesized nanoparticles capture superoxide anions and reduce the synthesis of formazan with the reduction of the absorbance measured at 560 nm. The results obtained for this determination are presented in Regarding the determination of the superoxide anion radical scavenging capacity, Es-AgNPs (178.80 ± 11.07 µg/mL) are at least 3 times more active compared to Ep-AgNPs (604.15 ± 28.00 µg/mL) and 1.6 more active compared to Et-AgNPs (298.36 ± 18.23 µg/mL).E. sylvaticum and the lowest for E. pratense. If for the neutralization of hydroxyl radicals, only the presence of hydrogen donor groups in the scavenger molecule is required, for the neutralization of the superoxide anion radical, both hydrogen donor groups and functional groups capable of neutralizing the anion charge are required instead.Nanoparticles obtained from ethanolic extracts proved to be less effective in this test compared to the first three assays. However, it can be seen that in this case, as well as in the other performed tests, Es–AgNPs showed the highest scavenging capacity, higher than that of the used standard , while the weakest results were obtained for Ep–AgNPs. It can also be noted that the antioxidant efficiency of the standard is similar as value to that of Es–AgNPs and Et–AgNPs. Correspondingly, in the case of extracts, the highest antioxidant activity was also observed for The antioxidant properties of AgNPs underline their possible use in reduction of local oxidative phenomena that occur in infectious processes.Equisetum species in terms of antitumor activity on cell lines showing osteosarcoma (MG63) [The present study aimed to evaluate the in vitro effects of AgNPs synthesized from ethanolic extracts of the three a (MG63) ,36.The presence of viable cells in the cultures incubated with the samples was assessed using the MTT method at 24, 48 and 72 h after incubation, respectively ,37. Figu50 cannot be calculated for this sample.As seen in 50 for this sample would be around 1.5 mg/mL. Consequently, the results obtained during this assay prove that silver nanoparticles obtained through green synthesis from Equisetum species could present applications in the treatment of such disorders. However, further studies focusing on the toxicity of the currently investigated samples on normal human cells must be performed in order to assess their selectivity.The cytocompatibility of Es–AgNPs at a concentration of up to 1 mg/mL can be observed in 50 for AgNPs synthesized using E. telmateia extract is slightly higher than 2 mg/mL.E. sylvaticum extract displayed the lowest cell viability, which corresponds to a high cytotoxicity. The determinations concluded that the lowest concentration that produced a change below LD50 for the analyzed cell lines is obtained for Es–AgNPs (2 mg/mL and 24 h). The highest cell viability was recorded for Ep–AgNPs, even at the highest studied concentration (2 mg/mL) and during the longest contact time (72 h), the result being slightly below 80%.After analyzing all figures, it can be easily seen that AgNPs synthesized using E. arvense and concluded that it effectively reduced human osteoclast development and function, both in osteoclast precursor cell cultures and in osteoclastic and osteoblastic cell cultures [Similarly, in 2012, Costa-Rodrigues et al. studied the in vitro effect of a methanolic extract of cultures .Equisetum species, namely E. arvense, was performed on mammalian cells, MC3T3-E1 pre-osteoblast cells. Nanoparticles prepared at a low temperature exhibited non-toxic behavior. However, for the larger AgNPs, the non-toxic behavior of silver was extended to higher concentrations, proving that decreasing the size of AgNPs increases their cytotoxicity [One of the few cytotoxicity assays done on silver nanoparticles obtained from an toxicity . One othtoxicity .Equisetum extracts showed the presence of the surface plasmonic band in the 400–500 nm range which proves the formation of nanoparticles. The DLS characterization and determination of Zeta potential showed that the biomolecules on the surface of the obtained AgNPs are negatively charged, causing a rejection between nanoparticles, which makes the obtained colloidal solution stable. The EDX characterization of AgNPs demonstrated that biomolecules found in the extracts adhered to the surface of AgNPs. Moreover, the FTIR characterization of AgNPs confirmed the attachment to their surface of functional groups such as H-O, C-H, C=O, C≡N, C≡C, COO- and -CH=CH, mostly belonging to polyphenolic compounds.The spectrum of AgNPs obtained using the three E. sylvaticum. The in vitro antitumor assay led to a low cell viability value, corresponding to a high cytotoxicity for AgNPs obtained using E. sylvaticum ethanolic extract on the MG63 osteosarcoma cell line.The photocatalytic activity assay has shown that the synthesized AgNPs can be an ecological, fast and cost-effective choice for the degradation of organic dyes from the environment. Moreover, the obtained AgNPs also possess important antioxidant action, the best overall antioxidant capacity being observed for AgNPs obtained using E. pratense, E. sylvaticum and E. telmateia. The used method is an eco-friendly alternative to classical synthesis methods and led to the obtaining of nanoparticles with important antioxidant, photocatalytic and cytotoxic activities.To the best of our knowledge, this is the first report on the synthesis of AgNPs obtained using either


Dscam1) gene of insects, containing four clusters of variable exons , which potentially generates tens of thousands of protein isoforms through mutually exclusive splicing, of which regulatory mechanisms are still elusive. Here, we systematically analyzed the variable exon 4, 6, and 9 clusters of Dscam1 in Coleoptera species. Through comparative genomics and RNA secondary structure prediction, we found apparent evidence that the evolutionarily conserved RNA base pairing mediates mutually exclusive splicing in the Dscam1 exon 4 cluster. In contrast to the fly exon 6, most exon 6 selector sequences in Coleoptera species are partially located in the variable exon region. Besides, bidirectional RNA–RNA interactions are predicted to regulate the mutually exclusive splicing of variable exon 9 of Dscam1. Although the docking sites in exon 4 and 9 clusters are clade specific, the docking sites-selector base pairing is conserved in secondary structure level. In short, our result provided a mechanistic framework for the application of long-range RNA base pairings in regulating the mutually exclusive splicing of Coleoptera Dscam1.Mutually exclusive splicing is an important mechanism for expanding protein diversity. An extreme example is the Down syndrome cell adhesion molecular ( Dscam1 in arthropods  protein isoforms through mutually exclusive splicing. Dscam1 protein contains 10 immunoglobulin (Ig) domains and six fibronectin type III (FNIII) domains, a transmembrane domain, and a C-terminal intracellular region. Variable exons 4 and 6 encode half Ig2 and Ig3 domains, respectively, while exons 9 and 17 encode the whole Ig7 and transmembrane domains, respectively , we found that the transmembrane domain of each species contains two variable exons (exon 17). However, the number of variable exons in exon 4, 6, and 9 clusters of the Coleoptera species vary. The number of variable exon 4s ranges from eight to 11, mostly with 9 exon variants, and does not change as much as exons 6 and 9. In the exon 4 cluster, the variable exon 4.4 was missing during evolution, resulting in only eight variants in L. decemlineata. On the contrary, 10 or 11 variable exons can be identified due to the duplication of variable exons in the S. oryzae, R. ferrugineus, D. ponderosae, and H. hampei, which all belong to the same superfamily .After annotation of erfamily , 2. Corrvariants . AlthougDscam1 exon 4 mutually exclusive splicing, we first searched the conserved intron element. Docking site-selector sequence base pairing mediating mutually exclusive splicing in Dscam1 exon 4 has been identified in Drosophila and Hymenoptera species  downstream of the last variable exon 4  of the selector sequences were completely located in intron regions, while the remaining seven selector sequences were located in the intron–exon boundary region  of the selector sequences were completely located in the exon region, and 32% (eight out of 25) of the selector sequences were located in the intron–exon boundary region. More obviously, only 7% (two out of 28) of the selector sequences of T. castaneum exon 6 completely located in intron regions, while 26 out of 28 selectors included the exon sequences  in the exon 9 cluster were found. However, 10 out of 14 chosen species shared a conserved upstream docking site . By contrast, the downstream docking sites were conserved in 14 species. Moreover, both the primary sequences of upstream or downstream docking sites were clade specific compared to that of Lepidoptera and Hymenoptera.Next, we decoded the mutually exclusive splicing mechanism of the osophila , bidirec1 exon 9 . What ising site ; the priDscam1 in Coleoptera species, it was difficult to confirm the conservativeness of evolutionarily corresponding selector sequences. Alternatively, we selected two selector sequences paired with upstream or downstream docking sites in each species. Through the alignment of so many downstream and upstream selector sequences, respectively, the core area of downstream and upstream selector sequences can form base pairing to upstream and downstream docking sites, respectively . Second, even if the minigene of the variable exon cluster was constructed, the variable exons may not be spliced normally (in vivo seems hard to carry out in practice due to the lack of model organisms in Coleoptera. However, it will be interesting if there are useful systems to solve the experimental verification problems in the future.We have used the Mfold program, which uses a minimum free energy algorithm, to perform RNA secondary structure prediction . The prenormally . Third, D. melanogaster. The experimental operation is difficult. Therefore, all the secondary structures and their effects described in this article are predicted. In the future, it is necessary to conduct systematic research on Coleoptera, explore its genetic research tools, and further experimentally verify the regulatory effect of our proposed RNA secondary structure on alternative splicing.Coleopteran insects have not been thoroughly studied, and there is no established genetic manipulation system as mature as the model organism The original contributions presented in the study are included in the article/YJ conceived the project. HD, LL, JS, and YF found the sequence. LL, YF, SZ, and JS predicted the secondary structure. YS, YF, LL, and JS made the evolutionary tree. HD, LL, and JS drew the pictures. XZ, BX, JZ, and FS analyzed the data. HD and LL wrote the manuscript. All the authors discussed the results and commented on the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Eight studies were focused on the use of PDL, six on fractional lasers (CO2 and Er:YAG), four on excimer, and two on either alexandrite or Nd:YAG.Morphea, also known as localized scleroderma (LoS), comprises a set of autoimmune sclerotic skin diseases. It is characterized by inflammation and limited thickening and induration of the skin; however, in some cases, deeper tissues might also be involved. Although morphea is not considered a life-threatening disease, the apparent cosmetic disfigurement, functional or psychosocial impairment affects multiple fields of patients’ quality of life. Therapy for LoS is often unsatisfactory with numerous treatments that have only limited effectiveness or considerable side effects. Due to the advances in the application of lasers and their possible beneficial effects, the aim of this study is to review the reported usage of laser in morphea. We present a systematic review of available literature, performed with MEDLINE, Cinahl, Central, Scopus, Web of Science, and Google Scholar databases. We identified a total of twenty relevant studies (MEDLINE  Morphea, also known as localized scleroderma (LoS), encompasses a spectrum of autoimmune cutaneous connective tissue diseases. It is characterized by inflammation, excessive collagen production, and subsequently limited sclerosis of the skin; however, in some cases, deeper tissues such as subcutaneous fat, fascia, muscles, or bones might also be involved. The incidence of morphea is estimated at 27 cases/1,000,000, with greater predominance among females (women are 2.6–6.0 times more frequently affected than men) . The etiAn active phase of morphea represents limited inflammation, presenting as red or violet patches followed by characteristic porcelain-white or wax-yellow sclerotic plaques, often accompanied by subjective symptoms such as pruritus and/or pain. After several months or years, sclerotic plaques resolve, but skin and/or deeper tissues atrophy and dyspigmentation remain as an atrophic stage. Some severe types, particularly generalized and linear, may be associated with various extracutaneous manifestations ,6. It isClinical evaluation of skin involvement in morphea should be carried out with the use of the ‘localized scleroderma cutaneous assessment tool’ (LoSCAT), that is a standard tool, together with general medical evaluation  . LoSCAT Appropriate treatment of LoS should be preceded by determining the disease type, activity, severity, and extent of lesions. In mild, limited, and superficial skin lesions, topical treatments , or UV phototherapy (UVA1 or PUVA) are sufficient, whereas widespread disease, linear, or deep types require systemic treatment . Surgical procedures are primarily dedicated to patients with linear types of LoS, but only in the inactive stage of the disease . Although morphea is not considered a life-threatening disease, the apparent cosmetic disfigurement and functional or psychosocial impairment affect the patients’ quality of life . In thisThe systematic review of the literature was performed with MEDLINE, Cinahl, Central, Scopus, Web of Science, and Google Scholar databases complementary to PRISMA  protocol. The inclusion criteria for considering studies for the review based on PICOS structure comprised the population of pediatric and adult patients diagnosed with morphea and undergoing laser treatment. The search was performed in July 2021 without a date limit. The databases were searched using the relevant MeSH terms: “morphea” OR “localized scleroderma” AND “laser”. The last day to access the databases was 15 July 2021.n = 10, Cinahl n = 1, Central n = 0, Scopus n = 2, Web of Science n = 5, Google Scholar n = 2) published between 2000 and 2021 were taken for the final analysis  up to 26. PDL was implemented in the 1980s. Its mode of action is based on the principle of selective photothermolysis, a process of selective damage to chromophores by monochromatic light. PDL targets oxyhemoglobin, resulting in coagulation, endothelial cell injury, and some photochemical effects. The most frequently used wavelengths are 585 and 595 nm, penetrating up to 1.5 mm deep into the skin ,13. It i+ cells in either the upper or lower dermis, but only the latter was statistically significant. These cells in the course of LoS are involved in fibroblast activation. Their decline indicates the diagnosis of LoS, and allows assessment of the effectiveness of applied therapy [Based on a literature search, we found eight studies on PDL in morphea. Eisen et al. were the first to report the use of 585 nm PDL in LoS in 2002 . In cont therapy . Based o therapy .However, some limitations should be highlighted. A few cases have been reported of inflammatory morphea initially treated with PDL. Kakimoto et al. , Kim et The excimer laser uses a mix of reactive and inert gases. When electrically excited, the gas mixture emits a monochromatic, coherent wavelength laser beam 308 nm), making very precise, minute changes to irradiated material. Keratinocytes and T-cells absorb the light, promoting DNA damage and reducing local inflammation and keratinocyte activity . Given t8 nm, makThe excimer laser was also successfully used to treat morphea lesions. In 2009, Nisticò et al., in a study of excimer light in skin diseases, observed that three out of five patients with LoS experienced partial clinical remission with marked improvement in skin texture after 8–12 treatment sessions; however, it was accompanied by residual hyperpigmentation . Later, The efficacy of excimer lasers in morphea mainly results from their anti-inflammatory action, since the 308 nm wavelength used in the excimer laser is the most efficient wavelength for inducing DNA breakage in lymphocytes . Thus, aThe mode of action of fractional lasers is based on fractional photothermolysis, which means generating in the skin a mesh of microthermal treatment zones surrounded by healthy, undamaged tissue ,36. This2) laser in a 27-year-old woman with linear bandlike sclerotic plaque extending from the medial thigh across the knee and ankle joint to the mediodorsal foot, complicated by a left foot contracture (plantar flexion limited to 45°) which failed to improve after several months of combination therapy with UVA-1, topical calcipotriene, 0.005%, cream, and methotrexate 20 mg/week [2 laser session resulted in an immediate subjective improvement in the range of foot motion, a 5° increase in plantar flexion as soon as after 1 week, and remarkable softening of the sclerotic band with the recovery of full plantar flexion of the involved foot at 4 months after the laser treatment, that was maintained after a 1-year follow-up. The treatment was well tolerated by the patient [2 laser, respectively [2 laser in combination with topical and injected poly-l-lactic acid (PLLA) in a 44-year-old woman [2 laser treatment every three months with subsequent applications of a filler also resulted in an excellent patient satisfaction and significant cosmetic improvement persisted at follow-up three years after the final procedure without any adverse effects, and without the need for re-treatment [In the available literature, there are reports on 24 cases of patients with LoS who have been successfully treated with fractional lasers. For the first time Kineston et al. presented a case of successful use of fractional ablative 10.6-µm carbon dioxide (CO mg/week . The add patient . Farmer ectively . In the ectively . Similarld woman . In thisreatment .2 laser treatment (3 sessions) versus low-dose (30 J/cm2) UVA-1 therapy (30 exposures) in 17 patients with plaque or linear morphea based on the clinical and histopathological assessment [2 laser that was superior to that of low-dose UVA-1 phototherapy. A very good response, i.e., ≥60% LoSCAT improvement, was obtained in 41% of morphea patients on the laser regimen versus 0% in the UVA-1 group, which showed a good response at best. Of note, according to histological variables, the fractional CO2 laser group exhibited statistically better improvement of collagen homogenization, whereas a decrease in inflammatory infiltrates was higher in the UVA-1-treated patients. Both fractional CO2 and UVA-1-treated lesions showed a significant but similar effect on a decrease in the mean profibrotic marker TGF-ß1 staining and increased MMP1 staining after treatment; however, the fractional CO2 laser group showed a statistically better decrease in ultrasonographically-assessed dermal thickness than the UVA-1 group. Additionally, patients in the CO2 laser group reported significantly higher satisfaction scores and a much lower incidence of post-inflammatory hyperpigmentation. However, compared to UVA1-treated individuals, they were more likely to experience pain during the treatment and itching within the first 24 h after the treatment [2 laser treatment over UVA-1 phototherapy may be concluded from this study. This may result from the more diverse action of fractional CO2 lasers on collagen remodeling, including: (i) an immediate mechanical effect due to formation of microthermal treatment zones (MTZ), which remove homogenized and fibrotic tissue, releasing the skin tightness, (ii) rise of multiple MMPs such as 1, 3, 9, and 13, with subsequent degradation of the thickened bundles of collagen as demonstrated by molecular studies [Recently, Shalaby et al. compared the efficacy of the fractional COsessment . The lasreatment . Nonethe studies , (iii) d studies ,56. The  studies ,41. More studies ,58. The Laser-induced coagulation creates microthermal treatment zones (MTZs) in the skin. In response to damage, there is an increase in heat shock proteins (HSPs), firstly HSP72, which participates in the activation of epidermal stem cells and cells within the dermis, promoting rapid repair and collagen remodeling (its highest expression is about 3–14 days after the laser application). During the first day, special structures are formed, i.e., microepidermal necrotic debris (MEND), containing coagulated tissue. HSP47 expression is upregulated 4–7 days after laser treatment and is maintained for 3 months, resulting in increased accumulation of procollagen and collagen I and III. An initial increase of TGF-ß1 is seen in the first 3 days after treatment, with a gradual decline thereafter over a period of 30 days. Concentrations of MMP1 and MMP3, collagen-degrading enzymes, gradually increase over 3 days, peak on day 7, and decrease during the second week after laser. Levels of MMP 9 and MMP13 remain elevated for longer periods of time, ensuring the degradation of residual collagen. The increase in bFGF, which provides the coordination of initial migration and proliferation of endothelial cells, occurs later in time. Within a month, MEND exfoliation is entirely complete, and type 3 collagen is replaced by type 1. Complete replacement of the MTZ by fresh collagen fibers occurs after approximately 3 months.There are also single case reports on the use of fractional Er:YAG laser in LoS. Kozarev presented a case of two women with morphea lesions on their lower legs who were treated with Er:YAG fractional lasers . During The fractional Er:YAG laser laser was also effective in the treatment of Parry–Romberg syndrome, a rare variant of LoS of the head. Ghorbel et al. performed four sessions every two months with the use of 2940-nm Er:YAG laser on a 50-year-old woman with stabilized skin lesions located on the left cheek. After each session, marked improvement of the texture, color, and pliability was noted. Significant cosmetic improvement was maintained for 2 years after the last treatment .Alexandrite laser is a pulsed laser system that can be short-pulsed (Q-switched) or long-pulsed. It is considered as particularly effective for the treatment of superficial pigmented lesions, but it is also beneficial in the removal of vascular lesions as well as unwanted hair or tattoos ,62. ArpeThe non-ablative 1064 nm Nd:YAG laser constitutes one of the most widely used devices in dermatological management and some of the reasons for its use include unwanted hair, vascular lesions, benign pigmented lesions, basal cell carcinoma, rhytids, cellulite, onychomycosis ,66,67. IHowever, there is only one report of the Nd:YAG laser use in LoS. Bimbi et al. described a case of a 22-year-old female with a linear morphea lesion with apparent induration and macular hyperpigmentation located on her right lower limb that was unresponsive to topical therapy . SatisfaThe studies included in the review are presented in Nowadays, the use of laser devices for the treatment of cosmetic and non-cosmetic skin lesions is increasing, due to the minimally invasive character of treatment reflected in a lower risk of side effects, rapid healing of the targeted skin, and shortened recovery time ,71,72. T2 and Er:YAG), four on excimer, and two on either alexandrite or Nd:YAG. The majority of these studies were single case reports or case series. Two studies were investigational [2 laser vs. UVA-1 therapy) [In this systematic review of the literature, we identified a total of twenty relevant studies using laser therapy for LoS. Eight studies were focused on the use of PDL, six on fractional lasers  . In all therapy) . Undoubttherapy) . Howevertherapy) ) as well2 fractional laser therapy for morphea-related contractures. On the other hand, level of evidence IB for CO2 laser treatment was specified for plaque, linear, and en coup de sabre types [2 laser versus UVA1 is a therapy with the level of evidence 2B and the strength of recommendation B [There are several previous review articles considering the use of laser treatments in patients with LoS; however, none includes as many studies as ours. According to Zwischenberger et al., 585-nm long-pulse laser therapy is supported by level ≥3 of evidence, meaning there is insufficient evidence to support its efficacy and safety . In the re types . Furtherdation B .Based on this literature review, laser therapy may be a valuable treatment in some cases of clinically evident LoS lesions or those confirmed by histopathological examination, but it should be kept in mind that this treatment does not slow down the overall course of the disease and does not prevent the formation of new lesions. Therefore, in patients with aggressively progressive lesions, general treatment should be introduced, while laser therapy may be considered as an adjuvant method to treat cutaneous complications of LoS or for cosmetic reasons to provide a better quality of life for the patients. A short recovery time that allows an early return of patients to daily activities, and a single session that enables complete or at least partial remission sustained over a long period of time, certainly form the basis for better patient compliance compared to the systematic use of topical medications . Moreove


Categorization based on quasi-joint stiffness (QJS) may help clinicians select appropriate ankle foot orthoses (AFOs). The objectives of the present study were to classify the gait pattern based on ankle joint stiffness, also called QJS, of the gait in patients after stroke and to clarify differences in the type of AFO among 72 patients after stroke. Hierarchical cluster analysis was used to classify gait patterns based on QJS at least one month before the study, which revealed three distinct subgroups . The proportion of use of AFOs, articulated AFOs, and non-articulated AFOs were significantly different among SGs 1–3. In SG1, with a higher QJS in the early and middle stance, the proportion of the patients using articulated AFOs was higher, whereas in SG3, with a lower QJS in both stances, the proportion of patients using non-articulated AFOs was higher. In SG2, with a lower QJS in the early stance and higher QJS in the middle stance, the proportion of patients using AFOs was lower. These findings indicate that classification of gait patterns based on QJS in patients after stroke may be helpful in selecting AFO. However, large sample sizes are required to confirm these results. In 2019, the global prevalence of stroke was 101.5 million people ,4. StronAn ankle foot orthosis (AFO) is a support device that can control the position and motion of the ankle by providing support for weak limbs or can position a limb into a more normal position. AFOs have been used in patients who have difficulty walking after stroke to improve gait patterns, gait speed, and energy expenditure ,11,12. AAnkle joint stiffness during gait, which is called quasi-joint stiffness (QJS), in patients after stroke is an important index for decision making regarding AFO stiffness. Our previous study revealed a significant association between QJS and ankle kinetics  on the paretic side in patients with hemiparesis . HoweverMany classifications based on gait pattern  in patients after stroke have been proposed to guide clinicians when selecting the appropriate line of treatment. de Quervain et al. (1996) identified four patterns based on visual inspection of the knee joint angle on the paretic side in gait and gait speed in patients after stroke . KinsellThe objectives of this study were to clarify (i) if the gait patterns of patients after stroke could be categorized based on QJS and (ii) differences in spatial and temporal parameters, joint kinematics and kinetics, and daily-use of AFO among these groups. In our two previous studies, the average of the coefficients of variation in the QJS in the ES and MS on the paretic side were higher than that of the non-paretic side ,18. TherWe recruited 72 patients with hemiplegia due to stroke. Inclusion criteria were as follows: (1) stroke due to ischemic or hemorrhagic supratentorial lesion, (2) stroke occurring at least one month before this study, (3) ability to walk a flat path of 7 m without the use of a cane and orthosis, and (4) ability to understand instructions by physical therapists. Exclusion criteria were as follows: (1) abnormal circulatory and respiratory status, (2) history of orthopedic problems that interfered with gait, (3) brainstem or cerebellar lesions, (4) diabetic neuropathy, (5) abnormal mental status, (6) higher brain dysfunction, which would cause skewed measurements, and (7) pain in the lower or upper limb when walking, which would cause skewed measurements. The present study was approved by the local ethics committee of Tohoku University of Medicine, Japan.Information about clinical and demographic characteristics and daily-wear AFOs was obtained through interviews with the patients and from their medical records. The use of daily-wear AFOs for at least one week was divided into two types of AFO: articulated and non-articulated AFOs. All subjects who wore AFOs used passive devices, with reference to the previous study . The numThree-dimensional coordinates of 33 reflective markers attached to 12 segments  were meaThe three-dimensional coordinates and GRF data were smoothed using a bidirectional fourth-order Butterworth low-pass filter with a cutoff frequency of 6 Hz and 80 Hz, respectively. We decided the cutoff frequencies of three-dimensional coordinates based on a previous study . The cutWe calculated QJS on the paretic side from the slope of the linear regression of the ankle joint moment versus the ankle angle during the second rocker interval, which was subdivided into ES and MS following the approach of previous studies ,18. We dAll data analyses were processed using a custom MATLAB program .Z-scores; then, clustering algorithms utilizing Ward’s linkage method and squared Euclidean distance measures were applied. SGs were formed in an agglomerative manner, starting with each observation as their own SG and at every step pairing the two closest SGs together until only one group remained. The final number of SGs was chosen based on a stopping rule (a large percentage decrease in the coefficient followed by a plateau) [A hierarchical cluster analysis was used to identify subgroups (SGs) with homogeneous QJS in ES and MS. In preparation for the cluster analysis, two clustering variables  were standardized using plateau) . The numplateau) .p adjustment. We used mid-p adjustment only when performing one-sided Fisher’s exact test for multiple comparison, because Fisher’s exact test is somewhat conservative [2) was calculated to estimate the effect size. Statistical analyses were performed using a statistical software package  and JMP Pro 16 . JMP Pro 16 was used only when Fisher’s exact test was performed. In addition, one-sided Fisher’s exact test with mid-p adjustment and Bonferroni corrections was performed using a custom MATLAB program  [Differences in age, height, weight, time post stroke, and gait spatiotemporal and kinematic and kinetic parameters were compared among SGs using one-way analysis of variance followed by Bonferroni’s multiple comparison procedure. Differences in sex, paretic side, and diagnosis among these groups were tested using Pearson’s chi-squared test. The Kruskal–Wallis non-parametric test, with the post hoc Wilcoxon rank sum test with Bonferroni correction, was used to compare the SAIS among SGs. Differences in the use of AFO among these groups were tested using two-sided Fisher’s exact test followed by one-sided Fisher’s exact test with Bonferroni corrections and mid-ervative ,37. A siMA, USA) ,39.The dendrogram for the clustering results is outlined in 2,65 = 50.542, p < 0.001, η2 = 0.59) and middle stances  among the SGs.p < 0.001) and SG2 (p < 0.001).QJS in the ES in SG1 was highest, although that in subgroup 2 (SG2) was the lowest among the SGs. On the other hand, the QJS in middle stance in subgroup 3 (SG3) was lower than that in SG1 , cadence , stance time , swing time , single stance time , initial  and late double stance time , paretic step length , and non-paretic step length .p < 0.001, SG3 vs. SG1: p < 0.001) and cadence , longest stance time  and initial double stance time , and shortest paretic  and non-paretic step lengths  among the three SGs. Additionally, there were no significant differences between SG1 and SG2 for any of these parameters.SG3 had the slowest gait speed  and toe off , maximum knee flexion in swing , knee  and hip angle excursion , hip flexion at heel contact , maximum hip extension in stance , and hip flexion at toe off , but not in the kinematic parameters related to the ankle joint. Moreover, there tended to be a difference in maximum knee flexion in the ES  and knee extension in stance .p < 0.001, SG3 vs. SG1: p = 0.044) and knee  and hip angle excursion  were found in SG3 compared with the other SGs. In SG2, the maximum hip extension in stance (p = 0.001), hip flexion at push off (p = 0.037), maximum knee flexion in the ES (p = 0.050), and knee flexion at push off (p < 0.001) were significantly larger than that of SG3. However, there was no significant difference in any kinematic parameters between SG1 and SG2.A lower maximum knee flexion in the swing phase , maximum hip flexion moment in stance , first maximum knee extension moment , maximum ankle dorsiflexion moment in ES , and maximum ankle plantarflexion moment in stance .p < 0.001, SG3 vs. SG1: p < 0.001) and dorsiflexion moment  were found in SG3. In SG2, maximum hip flexion moment (p < 0.001) and the first maximum knee extension moment (p = 0.029) were larger than those in SG3. In SG1, the maximum hip extension moment in ES was larger than those in SG3 (p = 0.002).Among the three SGs, the lowest maximum ankle plantarflexion (SG3 vs. SG2: p > 0.156) in age, height, weight, time post stroke, sex, diagnosis, and paretic side among the SGs were found, as shown in No differences , as shown in In SG3, items for motor function in lower limb in the SIAS were lower than those in SG2 (ankle joint: p = 0.008). The proportion of patients with articulated AFOs in SG1 was significantly higher than that in SG2 (p = 0.040). The proportion of patients with non-articulated AFOs in SG3 was significantly higher than that in SG1 (p = 0.036).Participants who used the articulated AFOs daily wore an AFO with resistance in the plantarflexion direction due to an oil damper and without stiffness in the dorsiflexion direction  or a posterior leaf spring plastic AFO with a Tamarack flexure joint . The participants who used non-articulated AFOs daily wore an ORTOP AFO  or posterior leaf spring plastic AFO.We assessed the gait pattern based on ankle joint stiffness, also called QJS, in the gait in patients with hemiparesis using a hierarchical cluster analysis. The group was divided into three SGs, with different gait patterns based on knee kinematics, especially maximum knee extension in MS, as described in previous studies ,24,25,40The highest QJS in the ES on the paretic side was found in SG1. However, the lower limb reflex, including Achilles’ tendon reflex and passive dorsiflexion range of motion at the ankle joint, did not differ from that of the other SGs. As indicated in previous studies, QJS in the ES may contribute to other factors, such as viscous damping at the ankle joint, but not the dorsiflexion range of motion and increased muscle activity at the ankle joint in the ES due to spasticity ,41. ContSGs 1 and 2 showed a higher QJS in the MS compared to SG3, and SG2 showed the second highest QJS in the ES. Additionally, the patients had milder paresis in the lower limb on the paretic side than in SG3. Therefore, in patients with high motor function in the lower limbs, the characteristics of QJS were similar to those in the healthy controls, such as low QJS in the ES and high QJS in the MS, as shown in a previous study . Due to The lowest QJS in the ES and MS on the paretic side were observed in SG3. Additionally, evaluations of motor function in the lower limb showed severe paresis in the lower limb in SG3. Severe paresis may decrease the activity of the ankle plantar flexor muscle in the MS, thus resulting in a decrease of QJS in the MS, as shown in a previous study . BecauseThere were several limitations of the present study. First, only patients who could walk without any assistance after stroke were recruited. Patients with a slow gait speed in the previous studies used a four-point or straight cane when walking ,25. AddiFuture research should collect both kinetic/QJS and kinematic data for gait classification. However, appropriate data  for use in gait classification may depend on the purpose for which the gait classification is used. A variety of data on gait in stroke patients is needed to determine a gait classification index for appropriate treatment and orthotic selection. In our present results, the SG1 sample size was small. The use of 3D motion analysis is limited in terms of resources to measure large amounts of data, which may require the use of wearable sensors or depth sensors that are currently being developed. In addition, future research on gait classification, including patients in the subacute phase, should provide valuable information to clarify the recovery process.In summary, we classified the gait pattern based on ankle joint stiffness, called QJS, in the gait of patients after stroke and clarified the differences in the use of AFOs among the groups as follows: the proportion of use of articulated AFOs, that of non-articulated AFOs, and the non-use of AFOs were higher in SG1, with a high QJS in the ES and MS, followed by SG3, with a low QJS in the ES and MS, and SG2, with a low QJS in the ES and high QJS in the MS. However, large sample sizes are required to confirm these results.


Background: Our study aimed to determine the correlation between the clinical staging of Fuchs' endothelial corneal dystrophy (FECD), rate of endothelial cell loss, and corneal biomechanical properties.Methods: This study combined a longitudinal retrospective/prospective analysis of corneal endothelial cell loss and a prospective cross-sectional analysis of corneal biomechanics of Fuchs' endothelial dystrophy. The trial was registered at the Thai Clinical Trials Registry as TCTR 20160927004. FECD was diagnosed by the presence of corneal guttata detected by slit lamp microscopy; the disease severity was classified into four stages using the modified Stocker's classification. In vivo confocal microscopy, Scheimpflug imaging, and Corneal Visualization Scheimpflug Technology were performed to evaluate endothelial cell count, central corneal thickness, and corneal biomechanical properties. Linear mixed modeling analyses were used to estimate the endothelial cell densities in a 4-year period. The corneal biomechanics were compared among the stages using Corvis ST parameters.Results: Eighty eyes from eighty subjects were enrolled . The mean endothelial cell density was 1228.35 cells/mm2. The year-by-year reduction rate was 94.3 cells/mm2 . Corneal endothelial cell losses in Fuchs' endothelial dystrophy were estimated to be 7.7, 7.8, and 8.4% per year for stages 1, 2, and 3, respectively. The mean corneal thicknesses of stages 1, 2, and 3 were 556 ± 32, 623 ± 33, and 648 ± 50 mm, respectively. For the corneal biomechanical parameters, the A1-length and A1-time were significantly different between stages 1 and 3 .Conclusions: In the advanced stage, corneas significantly changed their biomechanical viscoelastic behavior by decreasing resistance, as measured by a longer A1-length and shorter A1-time. Fuchs' endothelial corneal dystrophy (FECD) is characterized by thickened Descemet's membrane, guttae formation, and progressive loss of corneal endothelial cells. This causes corneal swelling, and the cornea becomes opaque in the advanced stages of the disease. The standard treatment for significant visual disturbance in FECD is endothelial transplantation . The central corneal endothelial cell density (ECD) declines at a rate of 0.6% per year in normal physiology . Cross-sCorneal biomechanics include corneal elasticity and stiffness. Most researchers focus on this property in ectatic corneal diseases, which have abnormal collagen and extracellular matrix. Their corneas are usually thinner and have weaker biomechanical properties than those with normal corneal tissue . The patThis study aimed to determine the rate of endothelial cell loss using longitudinal data, which provides information on disease progression, and investigate corneal biomechanical properties at different stages of severity in patients with FECD.This study was a longitudinal retrospective/prospective analysis of ECD and a prospective cross-sectional analysis of corneal biomechanical properties in FECD. This study followed the tenets of the Declaration of Helsinki and was approved by the Institutional Review Board of the Faculty of Medicine, Chulalongkorn University (COA No. 594/2016) and registered at the Thai Clinical Trials Registry (TCTR 20160927004). Informed consent was obtained from all participants.The corneal dystrophy database at King Chulalongkorn Memorial Hospital was searched to identify patients who had FECD diagnosed by cornea specialists between 2008 and 2017, and their medical records were reviewed. All reviewed patients were invited to participate in the study by phone. Only the patients who agreed to participate and provided informed consent were enrolled in the study. Patients with a history of intraocular trauma, laser peripheral iridotomy, corneal infection, previous corneal transplantation, contact lens wearers, or patients who could not undergo investigations were excluded from the study. In cases in which both eyes of a patient qualified for inclusion, only the right eye was included. The collected data included age, sex, family history of FECD, underlying systemic diseases, presence of corneal edema, presence of corneal endothelial pigment deposits, history of previous cataract surgery, ECD, and central corneal thickness (CCT). On the visit date, non-contact computerized tonometer and slit-lamp examinations were performed to determine the extent of corneal guttae, presence of corneal edema, and subepithelial scarring to grade the disease severity according to an established 4-scale severity grading system (modified Stocker's classification) , Corneal Visualization Scheimpflug Technology , and Scheimpflug imaging  were used to measure ECD, biomechanical properties, and CCT, respectively.Three non-invasive instruments were employed by an experienced technician: 2 were chosen. Automated analysis by the manufacturer's software (NAVIS) was traced. If the automated software failed to correctly identify the cell borders, the blinded technician will perform manual cell identification at the center of each cell. Subsequently, the manufacturer's software calculated and reported ECD. If no endothelial cells were identified, the sample was defined as uncountable.A Confoscan 4 microscope with a 40x objective lens was used to image corneal endothelial cells; the best quality images with a region of interest > 0.05 mmScheimpflug technology was used to automatically record corneal dynamics at 4,300 frames per second after deformation by a collimated air puff in the sitting position. To assess the corneal biomechanics, the Scheimpflug images were classified into three states: applanation 1 (A1) or the flattened state, while the cornea rebounded inward; highest concavity (HC); and applanation 2 (A2) or the flattened state, while the cornea rebounded outward. In each state, Corvis ST measured the length of the flattened cornea (A1-length and A2-length), elapsed time , and velocity (A1-velocity and A2-velocity). In the HC state, the deformation amplitude , peak distance (the distance between two apexes at HC), and HC radius (the radius of curvature of the cornea at the time of maximum concavity) were calculated. These 10 Corvis ST parameters are critical for the evaluation of corneal biomechanical properties.Pentacam scans automatically when patients directly fixate on a target light. Within a couple of minutes, the CCT was obtained using apical corneal pachymetry.All patient characteristics, including age, sex, underlying systemic diseases, family history of FECD, time of EC counts, extent of guttae area, presence of corneal endothelial pigment deposits, history of previous cataract surgery, intraocular pressure, and CCT were analyzed using means and standard deviations for continuous variables and counts and percentages for categorical variables. The data were analyzed for normal distribution using the Shapiro–Wilk test.p < 0.25 in the bivariate models were considered sufficiently important for consideration in the multivariable model. The exception to this rule was ECD, the study effect, which was forced into the multivariable model regardless.Linear mixed modeling analysis was used to estimate the corneal ECD over time and between each stage of FECD. This model was chosen because the ECD of each patient's eye was repeatedly measured over time. Both bivariate and multivariate analyses were performed to estimate unadjusted and adjusted effects. All predictors with p < 0.05. All analyses were performed using the R program version 3.4.1 (2017) .To analyze the corneal biomechanical parameters, general linear modeling was used to fit both the continuous and categorical predictors of each corneal biomechanical parameter throughout all analyses. Statistical significance was set at in vivo confocal microscopy was not performed at every visit. The mean ECD at baseline was 1228.35 cells/mm2, and 11 subjects had uncountable cells at the first presentation.There were 164 patients enrolled; however, only 80 patients met the inclusion criteria. There were 80 eyes from 80 patients; 63 (79%) were female and 17 (21%) were male, with a mean age of 61.9 ± 11.1 years (range: 21–84 years). In the 4-stage clinical severity grading system, 42 (52%), 26 (33%), and 12 (15%) patients were in stages 1, 2, and 3, respectively. No patients were categorized as stage 4. The demographic data, corneal characteristics, and CCT are summarized in 2  for each year following diagnosis. Although there were some differences in the reduction in ECD with stage levels , the differences were not statistically significant. Of the factors  that were included in the multivariable model, the presence of family history tended to have an effect on ECD: on average, 237.39 cell/mm2 per year fewer than those without a family history ; however, this was not statistically significant  for each stage are shown in F = 18.24, df = 3, df = 76, p < 0.001). The post hoc comparisons revealed that only stage 3 had a significantly longer A1-length than stage 1 .The 10 corneal biomechanical parameters recorded by Corvis ST are outlined in F = 3.50, df = 4, df = 75, p = 0.01). The analysis of the individual differences showed that stage 3 differed from stage 1 .There was a statistically significant difference in A1-time between stages 1 and 3. When the adjusted models (age and lens) were applied, at least two stages differed . Stage 2 had an average of 0.21 mm longer A2-length than stage 1 . Stage 3 had an ~0.37 mm longer A2-length than stage 1 . After accounting for potential confounders , no statistically significant difference was noted .The A2-length in clinical stages 1, 2, and 3 increased with statistical significance  for each year since diagnosis, the rates of ECD loss in our study  were greater than those in a previous study by Hatou et al. (2 zone) was altered, the remaining endothelial cells were still able to adequately function. This suggests that corneal thickness may be a better indirect indicator of corneal endothelial cell function. Following this concept, Sun et al. , increased A2-velocity, and greater deformity amplitude . The difThis study had some limitations. First, some necessary information may not be recorded owing to the retrospective data for endothelial cell loss analysis. Second, there was no control group to avoid any bias.In conclusion, this was the first longitudinal study to demonstrate ECD loss at each stage of FECD severity. Furthermore, it measured corneal biomechanical transformation in FECD by comparing corneal viscoelastic properties at each stage of corneal severity. Our findings provide a better understanding of the natural history, pathophysiology, and prognosis of FECD and can inform more specific and appropriate treatment plans.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Institutional Review Board of Faculty of Medicine, Chulalongkorn University (COA No. 594/2016). The patients/participants provided their written informed consent to participate in this study.UR, MJ, and NK devised the idea of the methodology, drafted the protocol, and proofread the manuscript. MJ conducted the literature review and drafted the manuscript. UR and NK provided verification and editing. All authors contributed to the article and approved the submitted version.This work was supported by the Ratchadapiseksompotch Fund, Faculty of Medicine, Chulalongkorn University (grant number RA 59/084).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Few studies assessed the association between major adverse cardiovascular events and adherence to warfarin and direct oral anticoagulants (DOACs) in patients with atrial fibrillation (AF). Therefore, we aimed to evaluate the effects of adherence to oral anticoagulants (OACs) in patients with AF using claims data (July 2014–April 2019). Using the initial 3-month medication possession rate (MPR), patients were categorized into adherent (MPR ≥ 0.8) or non-adherent (MPR < 0.8) groups. Propensity score matching of non-adherent group to adherent group was conducted for warfarin (1:1) and DOAC (1:3), respectively. Incidence of ischemic stroke, myocardial infarction (MI), intracranial hemorrhage, and all-cause death was assessed in the matched cohort . The hazard ratio (HR) for adherence to OAC was estimated using the Cox proportional hazard model with adjusting covariate including age and sex. The risk for ischemic stroke, MI, and all-cause death was lower in the DOAC adherent group than in the DOAC non-adherent group . Adherence to OAC was not associated with the risk of intracranial hemorrhage . Commitment programs to improve adherence in patients with AF could maximize drug effectiveness and safety. AF is known to increase the risk of stroke and can lead to thromboembolism and heart failure5. In AF, complications such as stroke and myocardial infarction (MI) are reportedly associated with high mortality. Comorbidities such as hypertension, history of a previous stroke, and coronary heart disease are not only associated with the development of AF, but also elevate the risk of stroke6. According to clinical guidelines, managing the risk of fatal AF complications such as stroke and myocardial infarction is important7. Patients should be treated with oral anticoagulants (OACs) to prevent the occurrence of these complications. A risk-factor-based assessment should precede initiating medication treatment to manage the risk of stroke. Using the CHA2DS2-VASc clinical stroke risk score, AF patients who are eligible for OACs are identified, and those with “low stroke risk” are not recommended for antithrombotic treatment. For selecting OACs, direct oral anticoagulants (DOACs) are preferred over vitamin K antagonists, mostly warfarin, as DOACs result in a lower risk of bleeding and are less affected by time in the therapeutic range (TTR) while securing similar effectiveness when compared with warfarin8. Nevertheless, patients with AF taking anticoagulants need to be carefully observed owing to the increased risk of bleeding11.In an ever-aging era, the prevalence of atrial fibrillation (AF), characterized by irregular and rapid heart rate, is expected to grow by 2.5 times over the next 50 years7. In previous studies, adherent use of DOACs revealed superior clinical outcomes without bleeding risk. In a retrospective observational study, the adherent DOAC users reportedly showed lower risks of ischemic stroke and systemic embolism than the non-adherent ones12. Conversely, the risk of stroke increased 3–4 times when patients discontinued oral anticoagulant (OAC) administration15. Increasing age, presence of comorbidities, and frequent dosing schedules have been reported as risk factors for poor adherence to OACs17. Although adherence to OACs is important in preventing stroke, studies on clinical outcomes other than stroke are limited. Besides stroke, AF can also lead to MI and even death; however, studies assessing clinical outcomes other than stroke remain limited. Considering these complications, it is crucial to evaluate the benefits of OAC adherence. Moreover, intracranial hemorrhage must be assessed to verify the safety of adherence7. Therefore, we aimed to evaluate the risk of ischemic stroke, MI, intracranial hemorrhage, and death following adherence to DOACs or warfarin.Along with regular monitoring of warfarin use, guidelines emphasize the importance of actively promoting adherence to and persistence of DOAC treatment18. Disease diagnoses were coded according to the International Classification of Disease-10th revision (ICD-10).Herein, we employed the claims data from July 1, 2014 to April 30, 2019, provided by the Health Insurance Review and Assessment Service (HIRA). The HIRA data are medical claims data covering approximately 98% of the total Korean population. The database contains information about demographic characteristics, including sex, age, insurance type, and healthcare resource utilization data, such as the type of medical procedure, diagnosis of disease, costs, and medication use. Information on medication use includes the generic name, prescription date, daily dosage, quantity, duration, and general codes19. OACs included warfarin and DOACs . The first day of OAC prescription was set as the cohort entry date. The index date was defined as 90 days after the cohort entry date. We measured adherence during this 90-day period, according to a previous study20. We included only those patients who did not switch drugs or did not die within 90 days of the cohort entry date.The target population in this retrospective cohort study Fig.  included2DS2-VASc score; this score predicts the risk of stroke and is calculated based on patient characteristics21. As described previously14, the CHA2DS2-VASc score was calculated based on the diagnosis code within a year before the cohort entry date  were included; patients who received OACs within 1 year before the cohort entry date were excluded, and only newly treated patients were selected. Patients diagnosed with valvular diseases  were excluded; only patients with non-valvular AF were included22. Patients diagnosed with ischemic stroke, MI, and intracranial hemorrhage within 90 days of adherence determination were also excluded.OAC initiation is recommended based on the CHA24 and is calculated by dividing the total number of prescription days for the initial 90 days by 90 days. Based on the MPR, the population was categorized into the adherent (MPR ≥ 0.8) or non-adherent (MPR < 0.8) groups.The medication possession rate (MPR) is used to evaluate medication adherencep-value was less than 0.05, the MPR of the initial 3 months was considered to be positively correlated with the MPR in the last 3 months. The linearity was determined using a scatter plot. Odds ratios (ORs) and 95% confidence intervals (CIs) were estimated by performing univariable logistic regression analysis to assess causation between the two variables. Initial adherence was considered to affect later adherence when the p-value was less than 0.05.As patients were categorized by early adherence, we established another cohort to determine whether early adherence to OACs continued for the later adherence. Newly treated patients with AF taking OACs were included using the same criteria as mentioned above. Among them, patients censored due to switching or death within 365 days were excluded  ischemic stroke, (2) MI, (3) intracranial hemorrhage, and (4) death. The diagnostic codes were confirmed based on a previous study that validated the codes for clinical outcomes27. Concomitant medications, such as aspirin, antiplatelets, beta-blockers, and calcium channel blockers, were considered potential confounders. The operation definitions of pre-clinical history and concomitant medications are presented in Supplementary Tables Demographic characteristics, including sex, age group, and type of insurance on the cohort entry date, were assessed. Within 1 year before the cohort entry date, pre-clinical history of diabetes mellitus, hypertension, dyslipidemia, MI, prior percutaneous coronary intervention, prior coronary artery bypass graft, chronic renal failure, chronic obstructive pulmonary disease, unstable angina, cognitive disease, heart failure, stroke, vascular disease, intracranial hemorrhage, and cancer, which could affect the adherence to OACs or outcomes, was evaluated2DS2-VASc and HAS-BLED scores, representative risk scores for stroke and bleeding, respectively, were calculated based on diagnosis codes within a year before the cohort entry date (Supplementary Table 14. The Charlson comorbidity index (CCI) was used to estimate the burden of underlying diseases that could affect OAC adherence28. Additionally, the number of emergency room (ER) visits and outpatient visits was included as a covariate for each patient.The CHA2DS2-VASc, HAS-BLED, CCI, and the number of ER and outpatient visits33. Matching was performed using a greedy algorithm34. Non-adherent warfarin users were matched 1:1 with adherent warfarin users, owing to the insufficient number of patients in the adherent group. For rivaroxaban, apixaban, dabigatran, and edoxaban, we performed 1:3 matching for each DOAC . A standardized mean difference (SMD) between the adherent and non-adherent groups was estimated to compare the distribution of variables used for matching. Covariates with SMD ˃ 0.1, which can provide evidence of an imbalance between matched groups, were included in the survival analysis.Propensity score (PS) matching was performed to minimize the potential impact of confounders on outcomes. Multivariable logistic regression estimated the PS for the adherent group using the following variables within a year before the cohort entry date or at the cohort entry date: age group, sex, type of insurance, pre-clinical history, co-medication, CHA2DS2-VASc score ≥ 4, and (3) a group of patients with CCI ≥ 4. All statistical analyses were conducted using SAS version 9.4 .The incidence of outcomes was calculated by dividing the number of individual events by the total follow-up period and presented as 100 person-years (PY). The Cox proportional hazards model was used to estimate the adjusted hazard ratio (aHR). Age, OAC type, sex, CCI, and CHA2DS2-VASc score were included as confounders in the model. These models are presented in the Supplementary Table The study was approved by the Institutional Review Board of Sungkyunkwan University (SKKU-201910031-UE001), South Korea. As patient claims data from the Health Insurance Review and Assessment Service (HIRA) had been anonymized and de-identified, the Institutional Review Board of Sungkyunkwan University waived the requirement for informed consent. The study was conducted in accordance with the Declaration of Helsinki.As patient claims data which support the findings of this study had been anonymized and de-identified publicly available information, the institutional review board waived the requirement for informed consent.In total, 67,147 patients were included after PS matching Fig. . Table 1The incidence rates per 100PY and aHR for events according to the adherent and non-adherent groups are shown in Fig. 2DS2-VASc score, and CCI. In all subgroups, the risk of ischemic stroke and death was lower in the adherent group than in the non-adherent group. The aHR for death was 0.46 (95% CI 0.41–0.51) in the younger group and 0.57 (0.53–0.60) in the elderly group. Adherence to OACs improved efficacy against MI in the lower CCI group , but not in the higher CCI group . As the type of OAC was included as a covariate in the subgroup analysis, the hazard ratio for each OAC is presented in Supplementary Table Figure p < 0.0001), revealing a significantly positive correlation; this has been depicted through a scatter plot presented in Supplementary Fig. Furthermore, we assessed the association between the initial 3-month adherence and subsequent 3-month adherence  and MI  without the risk of bleeding , when compared with non-adherent use12. A previous retrospective study, using Medicare claims data, reported a lower HR for ischemic stroke in the adherent group (0.62) than in the non-adherent group (0.74), compared with the non-use group14 although it was difficult to directly compare our findings with these results. Beyond previous studies, we observed the benefit of adherence with stratifying the type of DOACs, which were on not only ischemic stroke , MI  but also all-cause death .Although the OACs themselves have suboptimal protective effects for AF patients from stroke risk, adherence was associated with better benefit of DOACs. The protective effect of adherent DOAC usage in our study was similar to that observed in previous studies. In the United States, the risk of ischemic stroke was found to be comparatively high following non-adherent DOAC usage AF complications still occurred in patients with good adherence in this study, even if the incidence rates of the adherent group were lower than those of the non-adherent group. These may also be affected by factors other than adherence, such as age or disease burden. In the subgroup analysis, the protective effect of OACs, which reduced the risk of ischemic stroke and MI, was found to be considerably high in younger patients and patients with a lower disease burden. As shown in our study, the benefit of adherent use of OACs was smaller in the older patient group than in the younger patient group when comparing point estimations. Additionally, the group with a high disease burden showed less reduction in the risk of adverse events than the low disease burden group.35. Kachroo et al. have reported that elderly patients with AF are more likely to discontinue treatment36. The presence of comorbidities affects not only the rate of OAC prescription, but also adherence, as previously reported that patients with high CCI are likely to demonstrate low adherence to OACs38. Previous studies also showed that stroke patients with AF who took DOACs were to be more likely to have non-modifiable factors such as being old and being female, and factors that favor thrombotic effects such as hypertension, diabetes mellitus, and dyslipidemia compared to stroke patients without AF40. Therefore, monitoring adherence in these high-risk patients is important, as recommended in clinical guidelines. Achieving optimal adherence in this patient group is essential to avoid any additional cardiovascular events, as the aforementioned patients who took DOACs were vulnerable to thrombotic factors. As reported in a recent study41, adding a statin to anticoagulant therapies would be worthwhile.Consistent with our results, Rutherford et al. have reported that the risk of stroke or bleeding is affected by age and comorbidities, as elderly patients or those with substantial comorbidities receive a reduced dose42. This may be explored in further study.Interestingly, the statistical significance of aHR on adherence differed depending on the type of DOACs. Rivaroxaban and edoxaban seemed to lower the risk of ischemic stroke but apixaban and dabigatran didn’t. The difference between two types is dosing schedule. Apixaban and dabigatran are common on taking twice daily whereas rivaroxaban and edoxaban take once daily. Adherence itself can be affected by frequency of dosing14. The deviation in incidence between studies could be attributed to patient inclusion criteria, as our study only included intermediate to high-risk patients: male patients with a CHA2DS2-VASc score ≥ 2 only and female patients with a CHA2DS2-VASc score ≥ 3 only. The study by Kim et al., which included patients with a mean CHA2DS2-VASc score of approximately 5, showed that the incidence rates of ischemic stroke were 4.21 and 5.84 per 100PY in the adherent and non-adherent groups, respectively12. Furthermore, racial differences may affect the incidence of stroke43.The incidence rates of ischemic stroke attributed to DOAC adherence deviated from those previously reported. In our study, the incidence rates of ischemic stroke were 3.51 and 4.07 per 100PY in the adherent and non-adherent users, respectively, while a previous study has revealed that these rates were 1.20 and 1.92 per 100PY in the adherent and non-adherent users, respectivelyOur study had several strengths. The risk of adverse health outcomes due to blood clots and the risk of bleeding, implying suboptimal control, were traced within the same dataset; therefore, the benefit of adherent OAC usage was examined from various angles. By applying the simplified study design, the findings can be interpreted easily in a cause-and-effect manner to deliver a straightforward message that adherence is related to adverse health outcomes in patients. Additionally, the results were representative of the Korean population with AF, as we used a population-level database covering the entire Korean population.44, prescription days can be used to assess adherence to OACs, and patients with AF should be assumed to be under optimal control. In addition, as this study aimed to elucidate the clinical effect of adherence and not the therapeutic effect of OACs, factors other than adherence would have little effect on the results. Second, a misclassification bias may be present, as we used diagnostic codes of claims data to define outcomes and covariates operationally. To verify that the procedure was executed for disease remission, we limited the diagnostic codes. Furthermore, all claims of procedure codes are reviewed by HIRA to assess the appropriateness of the executing procedure before reimbursement; therefore, the procedure codes have their own accuracy and completeness. Third, the protective effect of adherence to OACs might have been overestimated, as the adherent group was older and had a slightly greater disease burden than the non-adherent group. However, the SMD revealed a balance between the adherent and non-adherent groups. Moreover, to derive the HRs of health outcomes related to adherence, we used the Cox proportional hazard model to adjust for patient characteristics, including age, sex, CCI, and the CHA2DS2-VASc score. Furthermore, we successfully verified the robustness of the study results, as the protective effect of adherent OAC use was uniform, regardless of patient characteristics, such as age group, the CHA2DS2-VASc score, and CCI, in various one-way sensitivity analyses. Comedication such as a statin may affect cardiovascular events through blood-thinning effects41. Although we did not observe statin use, we included dyslipidemia as one of the matching variables, and the adherent and non-adherent groups were well balanced, as shown by the SMD.A few limitations should be considered when interpreting the present findings. First, we could not capture the data of patients who failed to administer prescribed medications despite the dispensed prescription, as adherence was estimated by prescription days using claims data. Especially for warfarin, the MPR could not serve as an alternative indicator to the period in the therapeutic range, which could be verified by the international normalized ratio (INR). However, as guidelines for patients with AF recommend that patients taking OACs should be regularly monitored by the clinician17. In our study, we simplified the study design and simultaneously performed Pearson’s correlation test to support the assumption that adherence persisted within the first 3 months of the follow-up period. Patients with high initial adherence were shown to be more likely to adhere in the later period than those with low initial adherence. Similarly, a previous study using medical records from the outpatient clinic of a cardiology department in Korea reported that adherence to OACs was similar, regardless of the treatment period16. Based on the assumption of adherence persistence, we demonstrated the differences in protective effects of OACs between the adherent and non-adherent groups.It is challenging to illustrate the impact of adherence on health outcomes because adherence is a time-varying covariate. To overcome this limitation, Hernandez et al. previously conducted a Cox proportional hazard analysis using time-dependent exposure, while Brown et al. performed the same analysis for diverse study periods, such as 3, 6, and 9 monthsAdherent use of DOACs in patients with AF could be beneficial for reducing the risk of ischemic stroke, MI, and death, without increasing the risk of bleeding. Warfarin adherence presented a lower risk of death. Adherence to OACs could be more effective in younger patients and patients with lower CCI, particularly in preventing MI. Efforts to improve adherence in patients with AF taking OACs may help reduce the burden of cardiovascular diseases.Supplementary Information.