Blood cell count and iron biomarkers were quantified. Dietary iron intake was measured using two 24-h dietary recalls. Iron intake inadequacy was estimated using a probabilistic approach. The prevalence of anemia was 6.7%, depleted iron stores 5.1%, and IDA 1.1%. Women of all age groups, older adults, and those who were underweight or obese had the highest prevalence of anemia, and female adolescents had the highest prevalence of depleted iron stores. Female adolescents and adults were more vulnerable to depleted iron stores. Male adults and older adults had a considerable prevalence of iron overload. Except for female adolescents and adults, all groups had mild probabilities of inadequate iron intake. The main food iron contributor was wheat flour. Hemoglobin concentrations were directly associated with being an adult, having a higher income, and inversely associated with being female. Serum ferritin concentrations were directly associated with age and inversely correlated with female sex. Residents of São Paulo had a low prevalence of anemia, iron deficiency, and IDA, and sociodemographic factors interfered with these parameters.Anemia is a worldwide concern. This cross-sectional population-based study examined the prevalence of iron-deficiency anemia (IDA) among residents of São Paulo ( Anemia is a global public health problem, affecting one-quarter of the world’s population, especially children, women of childbearing age, and older people ,3,4,5,6.It is estimated that 1.62 billion people worldwide have anemia . ConsideIn Brazil, the typical process of nutritional transition with a double burden of malnutrition is currently ongoing, and following the global recommendations, a mandatory policy on the fortification of wheat and maize flours with iron has been effectively implemented since 2004. Given its safety and cost-effectiveness as a strategy to reduce IDA in the country , actionsHowever, its efficacy has been controversial due to the diverse effects observed in individuals of different ages and sexes from different areas of Brazil ,21,22,23Thus, this study aimed to estimate the prevalence of anemia associated with iron status, considering sociodemographic and lifestyle factors in a representative sample of residents from urban areas of the São Paulo city, and to evaluate the iron intake and the main food contributors to iron intake.Inquérito de Saúde de São Paulo com foco em Nutrição; ISA-Nutrition 2015), a cross-sectional population-based survey [Data were obtained from the 2015 Health Survey of São Paulo with a focus on nutrition , 2. male adults (20 to 59 years), 3. female adults (20 to 59 years), and 4. male and female older adults (60 years or older). Twenty study domains were established considering geographic and demographic domains. Adjustments were made so that the health coordinators had equal data analysis potential, and it was considered a non-response rate of 40% and that 10% of households would be empty. Further details have been published elsewhere .The individuals were randomly selected using a two-stage cluster sampling process: census tract and private households. In the first stage, 30 urban census tracts were randomly selected in each geographical area for health assistance in the São Paulo municipality. In the second stage, the households were selected in two distinct ways: for the sectors classified by the Brazilian Institute of Geography and Statistics as “common”, the households were systematically selected based on the list of households verified earlier in each area, and for the sectors classified as “special subnormal” that corresponds to sectors with favelas, the households’ segments were created considering an average size of six households .The households of each geographical area were selected based on the selection made earlier in the areas with the rarest domain: adolescents in the Midwest and older adults in the other geographical areas. Based on these rarest domains, the number of the other domains were defined and selected. There was no selection within the household. All individuals belonging to the domain for which the household was selected were included . For ISADuring the first visit to 4059 individuals in their households, trained interviewers applied a structured questionnaire encompassing sociodemographic information, lifestyle information, self-reported morbidity and deficiencies, emotional health, quality of life, use of health services, preventive health examinations, immunizations, use of medicines, health-related behaviors, characteristics of the family and the household, and expenses with health. A subsample of 890 individuals answered the first 24-h dietary recall (24HR) during the first interview, followed by a second household visit of trained nurses to collect 12-h fasting venous blood samples, anthropometric measurements, and information about dietary supplements use. The subsample also answered a second 24HR by phone after 173 days (mean) of the second household visit [Individuals who were heavy alcohol drinkers, those on enteral and/or parenteral nutrition, and pregnant or lactating women were not eligible to participate in ISA-Nutrition. During blood collection, individuals who had evidence of infectious disease had blood collection rescheduled to another date.per capita in international currency units, considering purchasing power parity, was divided into tertiles. Leisure-time physical activity was measured using the International Physical Activity Questionnaire [Sex was classified as male or female. The age groups in the ISA-Nutrition survey were grouped into adolescents (12–19 years), adults (20–59 years), and older adults (60 years and older). The start age of adolescents was defined according to the Brazilian Statute of Children and Adolescents  which coionnaire . Individionnaire ,31.2). BMI was classified as follows: for adolescents—underweight: <3rd percentile; healthy weight: >3rd percentile and <85th percentile; overweight not including obesity: >85th percentile and <97th percentile; obesity: >97th percentile [2; healthy weight: 18.5–24.9 kg/m2; overweight not including obesity: 25.0–29.9 kg/m2; obesity: ≥30 kg/m2 [2; healthy weight: 23.0–27.9 kg/m2; overweight not including obesity: 28.0–29.9 kg/m2; obesity: ≥30 kg/m2 [Weight and height were measured as described elsewhere , and thercentile . For adu30 kg/m2 . For old30 kg/m2 .Red blood cell counts were determined using an automated hematology analyzer . Serum ferritin concentrations were quantified using a chemiluminescent immunoassay , serum iron status by colorimetric assay , and serum transferrin concentrations by kinetic nephelometry . The transferrin saturation index (TS) was estimated using the ratio of serum iron (μg/dL) to the total iron-binding capacity multiplied by 100. The total iron-binding capacity was estimated using the ratio of transferrin (mg/dL) to 0.70 . An enzyY-axis concerning the variation of one unit of the X variable:Plasma usCRP concentrations were used as an indicator of acute-phase response proteins for infection/inflammation. Individuals with more than 2.0 mg/L  of us-PCAnemia was diagnosed based on hemoglobin concentrations of <13 g/dL for males aged ≥15 years or <12 g/dL for those in the other age groups .The reference levels for serum iron were 13–31 μmol/L (73–173 μg/dL) for males and 12–29 μmol/L (67–162 μg/dL) for females; for TS, it was 30–45% for both males and females, and that for transferrin was 215–365 mg/dL for males and 250–380 mg/dL for females .Depleted iron stores were identified if serum ferritin was <15 μg/L ,5,6. IDADietary data were collected using two 24HR applied on non-consecutive days using the Multiple Pass Method ,39, and The nutritional values of foods included in the survey were compared with information available in the Brazilian national food composition table , and theThe usual iron intake of each individual was estimated using the multiple-source method . The proDuring the application of the blood collection checklist, the individuals were asked if they were consuming any food supplements . If so, the supplements were listed with the respective doses and forms of use. Among those identified, the ones with iron in their composition were identified to estimate the consumption of iron by supplements.n = 1002) included the frequency of intake and the corresponding amount of dietary iron consumed. Foods and beverages were grouped according to the What We Eat in America food classification system based on the National Health and Nutrition Examination Survey, designed by the Food Surveys Research Group of the United States Department of Agriculture [Foods and beverages reported in the 24HR (iculture . The caticulture .The foods and beverages identified were distributed in the main food groups suggested by the United States Department of Agriculture: (1) dairy products, (2) protein foods, (3) mixed dishes, (4) grains, (5) snacks and sweets, (6) fruits, (7) vegetables, (8) non-alcoholic beverages, (9) alcoholic beverages, (10) water, (11) fats and oils, (12) condiments and sauces, and (13) sugars ,45.n = 13) was subdivided into 40 subgroups, which were subsequently subdivided into 118 food and beverage categories to provide a greater level of detail of the 1002 foods and beverages identified in the 24HR.Considering the similarities of foods and beverages in terms of culinary use and nutrient content, each main group , clustering, and stratification of the survey design . Categorper capita, leisure-time physical activity, and BMI classification. A 5% significance level was used for these tests.Prevalence was compared using survey-weighted chi-square tests, and medians using the Theil–Sen test for complexity sample studies  for the Skewness and kurtosis test proposed by D’Agostino, Belanger, D’Agostino  with an The final models were tested by residual analysis, and for inclusion, a 20% significance level was considered, whereas a 5% significance level was adopted for the other steps. The homoscedasticity test of the independent variables with iron intake was not successful.per capita , 16.4% (95%CI 7.7–31.5) were also diagnosed with microcytosis, hypochromia, and anisocytosis; 2.6%  with microcytosis and hypochromia; 5.4% (95%CI 1.2–21.5) with microcytosis and anisocytosis; 4.8% (95%CI 1.1–18.5) with microcytosis; and 19.2% (95%CI 10.4–32.6) with anisocytosis.Among those with anemia  was observed in the entire sample. On the other hand, 18.8% of the sample had a severe risk of iron overload. A higher prevalence of depleted iron stores was observed among adolescents, mainly among females. In contrast, a higher prevalence of severe risk of iron overload was observed among males, mainly among adults and older adults.per capita, and it was lower in the healthy weight group compared to other BMI groups , but it was considerably higher in female adolescents and adults, who had lower consumption of iron compared to males in all age groups. Dietary iron intake and the probability of inadequate intake decrease with age. Self-reported race and family income r groups .Combiron fólico”), the largest amount consumed was at a dose of 50 mg of iron per day, with ferrous sulfate being the vehicle. In general, considering the low prevalence of iron supplement use, the intake amount had no impact on the distribution of the groups , and male adults who presented severe risk (severe: ≥40%),Regarding serum ferritin concentrations, in the entire sample, a mild prevalence of depleted iron stores was also observed, concomitant with a mild risk of iron overload. In the stratification, adolescents, female adolescents, and female adults also had mildly depleted iron stores, while other groups had no problems with depleted iron stores. Conversely, it is important to mention the severe risk of iron overload observed in the study, which was more prevalent than iron depletion among adults, male older adults, 2nd and 3rd tertile of family income A higher prevalence of depleted iron stores among females, compared to males, has been observed worldwide . It can Nevertheless, although we cannot confirm, and the genetic, hormonal, and clinical issues need to be better investigated, the increased consumption of these fortified foods observed in males compared to females, over the years may be influencing the males to have this increased iron overload observed in the present study, as well as among the individuals who have hereditary hemochromatosis, as pointed out 10 years ago by Fisberg and Tosatti  and MartCançado and Chiattone  showed tOn the other hand, it can be hypothesized that the lower consumption of fortified foods by female individuals, combined with the menstrual blood loss common in women of childbearing age, may be a possible protective factor against iron overload, even when they become older adults. Genetic factors associated with anemia could also be involved and need to be studied.As depleted iron stores cannot be neglected in these women of childbearing age, the severe risk of iron overload among males is also a concern, considering the possibility of organ damage due to the pro-oxidant potential . High irFerritin is a ubiquitous and conserved iron storage protein that plays an important role in maintaining iron homeostasis, as well as serum ferritin, which is considered an acute phase reactant. In this sense, serum ferritin concentration may represent a better biomarker of inflammation than of iron status in the presence of inflammatory diseases . In thisIt is widely discussed that anemia may be associated with a condition called anemia of chronic disease ,55,79, wFurthermore, the role of other micronutrient deficiencies, such as vitamins A, B2, B6, B9, B12, C, D, and E, zinc, and copper, which are known to also lead to anemia and changes in iron metabolism ,12, shouIt is interesting to observe that a healthy lifestyle, characterized in this study by physical activity performance and healthy weight, had a positive influence on lowering the occurrence of anemia and in improving iron stores, while weight extremes (obesity and being underweight) were associated with a greater prevalence of anemia. The higher occurrence of anemia in individuals who were underweight can be explained by dietary inadequacy compromising iron intake; however, among individuals with obesity, dietary inadequacy and other processes related to inflammation could be the main problem.Moreover, our results indicate that individuals with more education, sufficient physical activity, and healthy weight have a better dietary iron intake, which can be related to more food awareness, while income did not interfere with dietary iron intake, as observed in Costa Rica , possiblThe probability of dietary iron inadequacies observed specifically in female adolescents and adults, indicated by the higher prevalence of depleted iron storages (associated or not with anemia), reinforces the need for policies directed at women of childbearing age worldwide ,2,5,84.An important recommendation for public policy refers to the focus on the promotion of vegetable sources of iron for women of childbearing age . ConsideTherefore, our results also highlight the importance of establishing the maximum limits of iron addition in wheat and maize flour fortification, to minimize the risk of iron overload. Kira et al.  pointed However, it is important to recognize that this is a study based on cross-sectional data, and causal inference cannot be made, providing evidence on population characteristics for identification of risk groups, and supporting health strategies to tackle anemia as a whole. Potential limitations of the study design include the selection, classification, and confounding biases, which may be minimized by using appropriate statistical strategies, such as control variables, which were adopted in the present analysis.Although the sample size may seem small, it was calculated to be representative from São Paulo, based on the most recent epidemiological data available, and considering the population demography in São Paulo city, to generate accurate estimates both at the municipal and regional levels, as described elsewhere .Furthermore, the limitations regarding potential errors in the estimation of dietary intake due to the adoption of retrospective records of food intake may be recognized; however, it is important to highlight that data collection and processing of two non-consecutive 24HR were carefully planned and applied by trained interviewers, including correction of food composition to account for iron intake considering food fortification parameters applicable in Brazil.There was a low prevalence of iron deficiency, anemia, and IDA among residents of the urban areas of São Paulo. Women of childbearing age comprised the population group with high susceptibility to depleted iron stores and presented a greater probability of dietary iron inadequacy, while male individuals were more susceptible to the severe risk of iron overload and had practically no dietary iron inadequacy. BMI, educational level, and physical activity had distinct influences on anemia, iron deficiency, and iron intake. The main foods contributing to iron intake in the study were foods with wheat flour in their composition, as well as beans and red meat, signaling the role of iron fortification programs for the Brazilian population. Future studies should focus on the risk of iron overload in certain population groups, analyzing the potential effects of diseases and genetic issues that may influence this context, in addition to factors related to nutritional transition and geographical and lifestyle factors.


An exploratory phase II, multicenter, open-label, clinical trial (NCT03687632) was conducted to evaluate the safety and effectiveness in treating persistent corneal epithelial defects (PEDs) with ST266, a proprietary novel multi-cytokine platform biologic solution secreted by cultured Amnion-derived Multipotent Progenitor (AMP) cells.Subjects with a PED were treated with ST266 eye drops 4 times daily for 28 days, then followed for 1 week. Safety was assessed by monitoring of adverse events (AEs) and serious adverse events (SAEs). Efficacy was assessed by measuring the area of the PED by slit lamp biomicroscopy. Tolerability of ST266, percentage of eyes with complete healing, reduction in area of the epithelial defect, and maintenance of a reduction in the area of the epithelial defect 7 days after treatment were recorded.P = 0.001). At follow-up, 1 week after completion of treatment, on day 35, the PED area was further reduced by 78.8% ± 37.5% (P = 0.01) compared with baseline. During 28 days of treatment, 5 eyes (41.7%) had complete wound closure. There were no AEs of concern thought to be related to the drug, and no SAEs were noted.Thirteen patients were enrolled into the trial at one of eight sites. The first patient withdrew after 5 days. The remaining 12 patients with PEDs with median duration of 39 days (range = 12 to 393 days) completed treatment. Ten of the 12 eyes had been refractory to treatment with various conventional therapies prior to enrollment. After 28 days of treatment, there was a significant decrease in mean PED area compared with baseline (66.4% ± 35.3%, In this trial, we found ST266 eye drops might promote corneal epithelization, thereby reducing the PED area, including in refractory cases in a wide range of etiologies. ST266 was well-tolerated by most patients. Treatment of a PED often begins with lubrication using preservative-free artificial tears, bandage soft contact lenses, pressure patching, debridement of the edges of the defect, scleral lenses, and/or tarsorrhaphy.–The novel secretome called ST266 (formerly known as amnion-derived cellular cytokine solution [ACCS]) was developed and produced under good manufacturing practice (GMP) conditions by Noveome Biotherapeutics, Inc. using a proprietary population of cells known as amnion-derived multipotent progenitor (AMP) cells. The AMP cells are cultured using a serum free minimal media in bioreactors and the conditioned culture media is collected over several weeks. The culture media is then further processed through filtration steps to remove certain cellular components and other debris before storage of the bulk drug product. ST266 is then quality control tested to ensure lot to lot reproducibility using a series of tests that include a panel of quantitative ELISA's, purity and cell-based potency assays. Only lots that fall within the strict specifications of these rigorous tests are used for preclinical and clinical studies. The final drug product is not concentrated and is devoid of extracellular vesicles. ST266 contains analytes at nanogram and picogram concentrations similar to levels that naturally occur in the body.–,,–In preclinical models, ST266 applied topically has been shown to have a positive impact on epithelial healing of the skin and the cornea. ST266 improved dermal epithelial healing in meshed human partial thickness xenografts in athymic “nude” rats.To date, phase I and II clinical trials have demonstrated ST266 to be safe and well tolerated in individuals with dry eye diseaseIn an exploratory phase II multicenter, open-label clinical trial, the safety and effectiveness of ST266 in healing PEDs was tested (NCT03687632). Institutional review board (IRB) approval was obtained at each investigational site, and all participating institutions (listed in the acknowledgments section) adhered to the tenets of the Declaration of Helsinki. Patients were recruited from June 2019 until July 2020. All subjects provided written informed consent prior to enrollment. Each patient received ST266 eye drops in the study eye 4 times daily and was followed weekly for 28 days, then returned for a 7 day post-treatment follow-up visit.Subjects aged 18 years or older with a PED of any size, measurable by slit-lamp and photography, were enrolled. Subjects who were actively being treated with cenegermin (Oxervate), autologous serum drops, amniotic membranes, bandage contact lens, and/or systemic steroids at greater than the equivalent of 10 mg of prednisone daily were excluded. Previous treatment with amniotic membranes or bandage contact lens was acceptable, if removed 24 hours before enrollment into the study, whereas systemic corticosteroids or immunosuppressants required a 7-day washout period to qualify for enrollment. Patients with uncontrolled eyelid or acute ocular infection, bullous keratopathy, corneal melting, recurrent corneal erosion, limbal blood vessel ischemia greater than 75% of the circumference of the limbus, or defects resulting from an alkali burn of the cornea, as determined by the investigator, were also excluded. Outside of exclusionary medications, patients were not restricted in concomitant treatment. Common ocular medications observed included topical antibiotics, steroids, and lubricating drops.2].At baseline, patients underwent standard ophthalmic assessments, including best corrected visual acuity, tonometry, slit-lamp and dilated fundus examinations, corneal fluorescein staining, and serial measurements of the size of the corneal epithelial defects: two maximum linear dimensions perpendicular to each other were used to calculate the area of the PED by approximation formula of (a * b * π/4) ). Scatter plot of the absolute measurement of CRF compared with ImageJ demonstrated a correlation coefficient of 83.22% (In addition to case report form (CRF) measurements used in the analysis, images of the PEDs were taken and analyzed by a central reader who was masked to the investigator measurements. This analysis showed a statistically significant difference in mean PED area between baseline to day 28 (t(10) = −4.1893, f 83.22% A. A Blanf 83.22% B. There Of the 13 subjects who enrolled in the study, 12 completed treatment and one withdrew early due to reasons unrelated to the study drug. Of the 12 to complete treatment, 10 exhibited no adverse events (AEs). Of the AEs reported, 11 of 16 (68.8%) were mild and included eye pain, tearing, itching, foreign body sensation, burning, photophobia, redness, lightning sensation, flashes, spots in visual field, and swelling. Five of 16 (31.3%) AEs were moderate and included pain, photophobia, tenderness, and ache. No subject experienced a serious adverse event (SAE).P = 0.958 nor between baseline and day 35 (t(9)) = 0.739, P = 0.479.Tonometry data revealed no significant difference between mean baseline and day 28 intraocular pressure (t(10) = −0.054, PEDs can be challenging to manage. Despite a better understanding of the different etiologies of PEDs and the wide range of therapeutic modalities available, some PEDs remain refractory to treatment. Aside from conventional treatments, such as patching, bandage soft contact lenses, preservative-free lubrication, and debridement of the edges of the PED, many experimental treatments have been investigated, including insulin,,Biologics, such as autologous serum eye drops and other blood-derived products, including platelets rich in growth factors, have become popular for treating PEDs, as these products contain various cytokines and growth factors, such as EGF, TGF-β1, PDGF, and FGF, that are known to promote corneal epithelial wound healing.,Recombinant human nerve growth factor  is a newer treatment for neurotrophic keratitis (NK), and has been shown to be effective for decreasing the size of PEDs in stage II NK.Amniotic membrane has been used for treating the ocular surface for over 80 years, and it was popularized in the 1990s for its wound healing and anti-inflammatory properties.In the current exploratory phase II multicenter open-label study, PEDs arising from multiple etiologies were enrolled, some previously treated with a varying array of therapies. Eight subjects had previously been treated with amniotic membraneFive eyes had complete PED closure during the 28 days of treatment. One of the 5 reopened on day 24 and then closed by day 35 during the post-treatment follow-up period. All eyes demonstrated a decrease in the size of the PED compared with baseline. Of note, in 12 of 12 subjects, the corneal wound was reduced in the area over 28 days. All 12 subjects received topical antibiotics at some time during treatment with ST266. ST266 has been shown in a preclinical rat study to improve healing despite bacterial contamination in the wounds.The data from this study suggest that a general reduction in the average PED size is present, but there are considerable fluctuations in measurements for some eyes. The descriptive pattern of PED size across time may indicate a varied response to ST266 treatment. For example, observation of PED area size versus time in patients 2, 4, 7, 9, 10, and 11 showed a strong linear trend suggesting a constant rate of re-epithelization in response to treatment see B. Notabl,The technique for measurement of PEDs has been a subject of debate.A limitation of this study is the lack of a control group. As such, it is not possible to exclude the fact that some of the improvement seen with ST266 may have happened even without the investigational drug. In addition, the heterogeneous etiologies for the PEDs also limits generalizability. However, this was intended to be an open-label study to try to detect an effect. A phase IIb randomized controlled trial is in the process of being activated in which more eyes will be enrolled, and a placebo arm will be included. In this upcoming study, the condition of the stroma (scarring and melting), as well as the endothelial cell counts (ECC) will also be assessed .Despite the small size of this trial, the results of this prospective open-label study suggest that ST266 may offer considerable benefit in the treatment of PEDs through reduction in the area of the defect. The promising reduction over just 4 weeks of treatment suggests that a longer treatment period may be needed to reach full closure, especially for patients with longstanding PEDs. To explore this, a randomized, double-masked, and placebo-controlled study is being designed to further assess the efficacy of ST266 in treating PEDs.


Marchantia polymorpha. The protein encoded by this gene has a class III adenylyl cyclase (AC) in the C-terminal domain and class I phosphodiesterase (PDE) in the N-terminal domain; therefore, we named it CAPE (COMBINED AC with PDE). CAPE protein is likely involved in spermatogenesis and sperm motility due to its tissue-specific expression pattern in M. polymorpha and the distribution of CAPE genes in streptophytes. However, little is known about the distribution of CAPE in gymnosperms that use motile sperm for fertilization, such as cycads and ginkgo. The present study aimed to isolate CAPE genes from the cycad, Cycas revoluta, the ginkgo, Ginkgo biloba, and the hornwort, Anthoceros agerestis. Sequences with high homology to CAPE were obtained from these species. Our analyses revealed that all plant taxonomic groups reproducing via motile sperm possessed CAPE, whereas those that do not produce motile sperm did not possess CAPE, with one exception in gymnosperm Cupressales. The phylogenic distribution of CAPE almost corresponds to the evolutionary history of motile sperm production and further suggests that CAPE may be involved in sexual reproduction process using motile sperm in streptophytes.We recently isolated a novel adenylyl cyclase/cAMP phosphodiesterase gene from the liverwort,  ACs are classified according to their features into six classes (I–VI), which are evolutionarily unrelated to each other despite their common catalytic activity3. While class I, II, IV, V, and VI ACs are only present in a limited group of bacteria, most ACs belong to class III, which are widely distributed in prokaryotes and eukaryotes4. Guanylyl cyclases (GCs), which catalyze guanosine 3',5'-cyclic monophosphate (cGMP) synthesis, are homologous to class III ACs and emerged from a common ancestor6.Adenosine 3',5'-cyclic monophosphate (cAMP) is a signaling molecule that regulates many biological activities in various organisms. It is synthesized from ATP by adenylyl cyclase (AC) and hydrolyzed to AMP by cAMP phosphodiesterase (PDE) to inactivate its activity as a signaling molecule. Intracellular cAMP levels are strictly regulated by the balance of AC and PDE activities7. Class I PDEs are found in vertebrates and diverse eukaryotes. All mammalian PDEs belong to this class, which is subdivided into 11 families1. Class II PDEs are mainly found in yeasts and slime molds and class III comprises PDEs from bacteria4.PDEs are classified into three classes according to the homology in the primary structure of their catalytic sites10. In prokaryotes, the cAMP receptor protein (CRP)–cAMP complex is involved in catabolite repression in Escherichia coli11. The CRP-like cAMP-activated global transcriptional regulator, GlxR, is a global regulator that controls the expression of various genes in Corynebacterium glutamicum12. cAMP signaling is required for virulence, and is used by Bordetella pertussis that causes whooping cough13, Bacillus anthracis that causes anthrax14, Mycobacterium tuberculosis that causes tuberculosis15, and the opportunistic human pathogen, Pseudomonas aeruginosa16. In addition, in eukaryotes, cAMP is involved in various cellular functions including glycogen degradation17, sense of taste signaling18, and flagellar movement of sperm19 in mammals, fruiting body formation in Dictyostelium discoideum21, and cell division in yeast22. Furthermore, cAMP plays important roles in photosynthetic organisms, such as light signaling and cell motility in cyanobacteria24, zygote formation in Chlamydomonas reinhardtii25, and the photophobic response in Euglena gracilis26. Thus, the diverse functions of cAMP have been clarified in many organisms.The physiological roles of cAMPs have been studied extensively in various organismsArabidopsis thaliana27, Oryza sativa28, and Solanum lycopersicum29, although novel ACs, which are not classified into the existing AC classes, have been reported32. However, progress in genome and transcriptome analyses of basal land plant lineages such as bryophytes and lycophytes35 led to the identification of a protein with class III AC and class I PDE sequences, named COMBINED AC with PDE (CAPE) due to the presence of these catalytic domains in a single protein36.In land plants, no homologous sequences coding for ACs or PDEs have been identified from other organisms in the genomes of angiosperms such as Marchantia polymorpha (Mp) CAPE confirmed that both domains have each enzyme activity36. MpCAPE is specifically expressed in the spermatogenous cells of antheridia , but not in vegetative organs such as gemmalings and gametophytic plant bodies . Furthermore, CAPEs were only found in streptophytes that use motile sperm as the male gamete, such as bryophytes and ferns, but not in the genome of angiosperms36. However, the distribution of CAPE in the gymnosperms has not yet been clarified, although CAPE has not been found in the genomes of two species of Pinaceae, Pinus taeda and Picea abies38. Gymnosperms are diverse in terms of the delivery system of sperm to the egg cell. For example, Gnetales and Coniferae use non-motile sperm cells directly delivered by pollen tubes, whereas Cycadales and Ginkgo use motile sperm cells with flagella to swim to the egg cell. Thus, it is important to know whether cycads and ginkgo have CAPE.CAPE is composed of a PDE domain at the N-terminus and an AC domain at the C-terminus. Biochemical analyses using CAPE in taxa gymnosperms and the bryophyte, hornwort. Furthermore, the complete genomes from one gnetophyte, Gnetum montanum, and two zygnematophytes, Spirogloea muscicola and Mesotaenium endlicherianum, have recently been elucidated40. Thus, the present study investigated the distribution of CAPE in such streptophyte taxa in more detail.Although the complete genomes of cycads and ginkgo have not yet been elucidated, a large amount of genetic information, especially transcriptome data, is available in public databases for many streptophytes including cycads and ginkgo. In our previous study, we were unable to identify the distribution of CAPE orthologs in some species of streptophyte lineage deposited in public databases using homology searches with the CAPE amino acid sequence from M. polymorpha (MpCAPE) as a query using the BLAST program.We aimed to detect CAPE in cycad and ginkgo because we previously reported that the distribution of CAPE coincided with that of the taxonomic groups that use motile sperms as male gamete in streptophytes36. A BLAST search was performed against RNA-seq data deposited in the sequence read archive (SRA) of GenBank. A large number of short sequences (reads), which were predicted to encode CAPE, were detected from the cycad and ginkgo RNA-seq data in the SRA.We first focused on the distribution of M. polymorpha, and the moss, Physcomitrella patens, were previously reported36. We were interested in the existence of CAPE in hornwort, the remaining taxonomic group in bryophytes for which the presence of CAPE has not been investigated. A BLAST search was performed against the genome DNA sequence data of A. agrestis in the SRA of GenBank42. Many short sequences were found that were predicted to encode CAPE.The CAPE sequences in the liverwort, CAPE was not found from Zygnematophyceae and M. viridae following a search of the public RNA-seq databases36. Recently, complete genomes have been elucidated from two species in Zygnematophyceae, Spirogloea muscicola and Mesotaenium endlicherianum40, and Mesostigma viridae43. However, CAPE was not detected in these genomes.We reported that CAPE was not detected in the genomes of two species of Pinaceae, Pinus taeda and Picea abies36. The complete genome of a gnetophyte, Gnetum montanum, was recently elucidated39. However, we were unable to detect CAPE in the genome of G. montanum. On the other hand, we searched for CAPE orthologs in other taxonomic groups of gymnosperms due to the high diversity of gymnosperms. First, a homology search was performed for OneKP, which is large-scale gene sequencing data mainly based on transcriptomes of > 1000 species of plants44. CAPEs were detected from streptophyte algae and bryophytes, in which the distribution of CAPE was reported in our previous study36. Unexpectedly, scaffold sequences, of which the deduced amino acid sequences exhibit significant homology to a part of MpCAPE, were detected from gymnosperms that do not produce motile sperms, including four species each from Cupressaceae and Taxaceae  contained the sequence encoding both AC and PDE partial sequences. On the other hand, scaffolds exhibiting the homology to CAPE were not detected in any species of Pinaceae, which was consistent with a previous report that stated that CAPE was not detected in the genomes of P. taeda and P. abies36. In addition to searching the OneKP database, a BLAST search was performed against the RNA-seq data of a Cupressaceae, Cryptomeria japonica, deposited in the SRA of GenBank. Many short sequences were detected that were predicted to encode CAPE.We reported that ae Table . In partCAPE using mRNA from C. revoluta (Cr), G. biloba (Gb), A. agrestis (Aa), and C. japonica (Cj), followed by 3′, 5′- RACE PCR for C. revoluta and G. biloba. The cDNAs containing the full-length CAPE coding sequences were obtained from C. revoluta, G. biloba, and A. agrestis. The deduced amino acid sequences of CrCAPE, GbCAPE, and AaCAPE were determined and comprised 1252 (CrCAPE), 1259 (GbCAPE), and 1240 (AaCAPE) amino acids, respectively, and contained one PDE domain at the N-terminus, multiple transmembrane regions located at the middle part of the protein, and one AC domain near the C-terminus , Bos taurus type 1 AC (BtACt1C1 and BtACt1C2), and Homo sapiens type 10 AC (HsACt10C1 and HsACt10C2). The eight consensus amino acids required for catalysis and substrate binding were completely conserved in the AC domains of CrCAPE, GbCAPE, and AaCAPE , and angiosperms. Therefore, CAPE may play an important role in motile sperm function. The role of cAMP in the regulation of flagellar motility has been investigated in mammals and algae47, and it has been suggested that cAMP may also be involved in motile sperm regulation in plants. The role of cAMP in plant cells may be restricted to the function of motile sperm, and it is hypothesized that loss of the motile sperm system coincides with loss of CAPE. On the other hand, CAPE is present in gymnosperms that do not produce motile sperms such as C. japonica.The present study elucidated more precise information about the distribution of C. japonica: CjCAPE clone#1 and CjCAPE clone#2  and four species of Taxaceae   Table . This wi36; therefore, it is predicted that they will have cAMP synthesis activity. The PDE domains of CrCAPE, GbCAPE, CjCAPE, and AaCAPE also retained all eight amino acids required for metal binding and showed high match rates to MpCAPE , Ginkgo biloba , or Cryptomeria japonica . For Anthoceros agrestis, its whole genome shotgun sequence data ERX714368 and ERX71436942 were assembled with pipeline at DNA Data Bank of Japan (DDBJ) and the assembled contig data by tblastn program was searched using M. polymorpha CAPE sequence as a query. Polymerase chain reaction (PCR) primers were designed using the retrieved sequences. A BLAST search was performed at the public OneKP website using the tblastn program using the amino acid sequence of M. polymorpha CAPE to obtain the partial sequences of CAPE in gymnosperms.We used the amino acid sequence of C. revoluta or G. biloba, male strobili of C. japonica, and gametophytes bearing antheridia of A. agrestis (Oxford isolate) using the cetyltrimethylammonium bromide (CTAB) method54. Frozen specimens were broken up using a mortar and pestle with quartz sand in liquid nitrogen and then incubated in CTAB solution  at 60 ˚C for 30 min. RNA was prepared from A. agrestis and C. japonica using CTAB solution supplemented with 1% polyvinylpyrrolidone-40. RNA extraction was performed three times using chloroform and precipitated with 2-propanol. The precipitates were washed with 70% ethanol and dissolved in RNase-free water. RNA was precipitated again with 2 M LiCl overnight on ice. The samples were centrifuged at 11,000 g for 30 min and the RNA was washed with 70% ethanol and dissolved in RNase-free water.RNA samples were prepared from ovules harvested at the time when sperm cells were developing in pollinated pollen grains from C. revoluta and G. biloba CAPE cDNA samples were generated by RT-PCR using total RNA samples with the following primers: CrCAPE-f, 5'-ATTTGCCAAAGATGGTGGAG-3' and CrCAPE-r, 5'-TCCTATGGGAAGCCATGAAG-3' for C. revoluta and GbCAPE-f, 5'-GCATTTCCCCTCGATAGACA-3' and GbCAPE-r, 5'-TGAATGCAGACAATCAGGGA-3' for G. biloba. Amplified PCR products were cloned into pCR-Blunt II-TOPO vectors  and the nucleotide sequences were determined. Since the 5' and 3' ends for C. revoluta and the 5' end for G. biloba were not contained in the PCR products, 5'- and 3'-RACE was performed to obtain the terminal fragments using 5'- and 3'-RACE kits (Thermo Fisher). Finally, full-length cDNA fragments were amplified by RT-PCR using primers designed using the sequences encoding the initiation and termination codons, cloned into a low-copy plasmid, pSTV29 , and sequenced to verify the full-length sequences.A. agrestis CAPE cDNA was amplified by RT-PCR using total RNA samples with the following primers: AaCAPE-f, 5'-CACCATGGCCAATTTTGATGAGGATG-3' and AaCAPE-r, 5'-TCATTTTTCTGTTAATTCACGATAC-3'. The amplified PCR products were cloned into pENTR/D-TOPO (Thermo Fisher) and sequenced. C. japonica CAPE cDNAs were generated RT-PCR using total RNA samples with the following primers: CjCAPE-f1, 5'-GGCATGCCTGTGATGTCTTA-3' for clone#1 or CjCAPE-f2, 5'-ATAGTAGATTCACCTCCATTTAG-3' for clone#2 and CjCAPE-r, 5'-TCATTTCTCTGTTAGTTCCCTGTATCC-3'. Amplified PCR products were cloned into pCR-Blunt II-TOPO vectors (Thermo Fisher) and the nucleotide sequences were determined.55. The maximum likelihood (ML) model of AC and PDE selected by the model test of MEGA 7.056. ML, the neighbor joining (NJ) method, and most-parsimonious (MP) were performed using bootstrap values.The amino acid sequences of the AC and PDE domains Tables  and S2 wSupplementary Information.


To determine the marginal bone loss and the survival, success and failure rates of narrow dental implants, a systematic literature search was carried out in the MEDLINE (Pubmed), Cochrane, Scopus, and Scielo databases for articles published between 2010 and 2021. The exclusion criteria were: systematic reviews, case reports, expert opinions; animal studies; samples of less than 10 subjects; follow-up periods of less than 36 months; smokers of minimum 10 cigarettes/day; and articles about mini-implants for orthodontic anchorage. Meta-analyses were performed to assess marginal bone loss and implant survival, success, and failure rates. Fifteen studies were included: 7 clinical trials, 3 randomized clinical trials, 3 cohort studies, and 2 case series. The total number of subjects was 773, in whom 1245 implants were placed. The survival rate for the narrow diameter implants was 97%, the success rate 96.8%, and the failure rate 3%. Marginal bone loss was 0.821 mm. All these data were evaluated at 36 months. Based on the literature, it can be considered that there is sufficient evidence to consider small diameter implants a predictable treatment option. These show favorable survival and success rates and marginal bone loss. All of them are comparable to those of standard diameter dental implants. Oral rehabilitation with dental implants provides an increase in oral health and quality of life ,2. Such Alveolar ridge resorption begins immediately after tooth extraction and this process is more intense during the first year, when about 60% of the thickness of the alveolar ridge is resorbed ,9. In thIn addition to the aforementioned, the reduction in bone width may be due to, or aggravated by, other causes such as trauma, malformation, neoplasms, use of removable prostheses and periodontal disease . This caLikewise, it is considered that in medically compromised or elderly patients, regenerative procedures carry a high risk of complications ,13. TherThe definition of NDI is not conclusive in published studies and there are several classifications according to different authors. This review has been based on that described by Klein et al. in 2014  which diThe clinical evidence comparing the NDI with SDI creates controversy in the literature; consequently, and based on the above, the objective of this systematic review is to determine the survival, success and failure rates of the NDI placed in narrow bone compared to the SDI placed in normal bone, as well as the peri-implant marginal bone loss.This systematic review has been prepared according to the PRISMA  criteria . Before Population (P): Partially or totally edentulous patients; Intervention (I): Placement of narrow dental implants in the mandible and/or maxilla; Comparison (C): Standard diameter or larger diameter implants; Outcomes (O): Implant success, survival and failure rates and marginal bone loss.Inclusion criteria: (i) articles in which the main objective was to treat partially or totally edentulous patients with narrow dental implants were considered; (ii) clinical trials, cohort studies, and case series in healthy humans; (iii) published in the last 10 years in English or Spanish; (iv) minimum 10 patients treated with NDI; (v) With a minimum follow-up period of 36 months after implant placement. Exclusion criteria: (i) systematic reviews, case reports, and expert opinions; (ii) animal studies; (iii) studies in which the sample was less than 10 subjects; (iv) follow-up period of less than 36 months after implant placement; (v) smokers of more than 10 cigarettes/day; (vi) mini-implants for orthodontic anchoring.An electronic search was carried out in the MEDLINE/PubMed, Cochrane, Scopus, and Scielo databases for articles published between 2010 and 2020. The last search was carried out on 5 March 2021.The following keywords were used: small diameter, narrow-diameter, dental implants and mini dental implants, combined through advanced search in the aforementioned databases:  OR narrow-diameter[All Fields]) AND  OR “dental implants”[All Fields])) AND  OR “dental implants”[All Fields])).All articles were initially identified by two experts Georgina González-Valls (G.G.V.) and Juan Manuel Céspedes-Sánchez (J.M.C.S.). After reading the titles, the abstracts of those that presented the potential of inclusion were read. Subsequently, the full texts of the selected articles were read to verify that the inclusion criteria were met. Any discrepancies during the selection process were resolved by one of the two authors Beatriz González-Navarro (B.G.N.) or José López-López (J.L.L.).The data were extracted by one of the authors Georgina González-Valls (G.G.V.) and entered in a data collection form (Microsoft Excel version 16.44). A second author reviewed the veracity of the collected data Elisabet Roca-Millan (E.R.M.). The following data were extracted: authors, type of study, number of patients, gender, age, diameter and length of the implants, location, type of restoration, follow-up time, number of failed implants, implants survival and success rates, and marginal bone loss.Version 2 of Cochrane Collaboration’s tool for assessing the risk of bias in randomized trials (RoB 2)  was implThe methodological index for non-randomized studies (MINORS)  was usedDifferent pooled estimates from the studies were obtained using the statistical analysis software OpenMeta [Analyst] .2 statistic and the level of significance was set at p < 0.05.Binary and continuous random-effects models were used to calculate the weighted proportions and means and the corresponding 95% confidence intervals (CI) among the studies. The evaluated variables were the survival, success, and failure rates and the marginal bone loss around dental implants, all at 36 months. Heterogeneity was assessed based on a calculation of the IA total of 971 articles were identified in the first search phase. During the second phase and after reading the title of these, the systematic reviews, case reports, and expert opinions were discarded and those that dealt with mini-implants for orthodontic anchorage were also discarded; obtaining a total of 144 articles. Subsequently, the abstracts were read and 45 articles were obtained. Finally, after reading the full text, 15 articles were included in the review that met the established inclusion–exclusion criteria ,29,30,31Of the 15 articles included, 7 are clinical trials ,22,27,31The studies were carried out in 11 different countries: 2 in Belgium ,24, 2 inThe total number of patients included in the selected studies was 773 (359 men and 370 women). All papers included both men and women. The study by Al-Shibani et al.  was the The total number of implants was 1245. In all the studies except that of Galindo Moreno et al.  the areaIn all articles the diameter and length of the implants used are detailed. Four articles ,18,19,30The main indication for NDIs in the reviewed studies was for anterior sector rehabilitation with single crowns ,25,30,31The mean follow-up time was 58 months from implant placement (range 36 to 137 months).The present systematic review meets 24 of the 27 PRISMA criteria .Of the 15 articles included in the present review, seven of them were NRCT ,22,27,31p = 0.012). Heterogeneity tests from pooled showed statistical significance . Galindo Moreno et al. [Marginal bone loss was evaluated in all the articles except those by Lambert. et al.  and Maryo et al.  obtainedo et al.  with 2.1p < 0.001) with no significant difference regarding the heterogeneity of the included studies  [p < 0.001) without significant heterogeneity  and without statistically significant heterogeneity  [Both the survival rate and the implant failure rate were described by all the studies except that of Al-Aali et al. . However= 0.773) ,27,28,31 = 0.773 ,27,28,31= 0.164) ,21,24,28NDIs are a valid treatment option that allows us to avoid more invasive procedures and higher morbidity. Patient preferences for minimally invasive treatment options, such as rehabilitation without bone augmentation, are generally high . TherefoSurvival rates for NDIs are similar to those for SDIs. We place the survival rate of those between 96.7% and 99% . Safii eWhen planning treatment with NDI, it is important to take into account factors such as its indications, the success of these, and the changes in bone remodeling that they may undergo, in addition to survival figures.Most of the articles ,21,25,30To date, there has been a lack of randomized clinical studies comparing the survival and success of NDI with that of SDI in regenerative procedures in situations of severe atrophy. Failed implants were those that presented significant mean bone loss, peri-implant radiolucency, mobility, infection, pain, discomfort, or that caused by sensorineural alteration. Forty-one implants out of a total of 1245 failed, the failure rate is 3% at 3 years ,28,31. VMarginal bone loss over time is another important factor influencing the predictability of treatment. Assaf et al. , suggestThe NDIs were mostly used for restorations in the anterior sector, mainly upper lateral incisors or lower incisors. These locations usually have limited interdental space and a thin alveolar ridge. Placing an implant too close to an adjacent tooth can lead to interproximal bone loss, a factor that can negatively affect the final position of the papilla and the soft tissues at the supracrestal level . TherefoOverdentures as rehabilitation in edentulous jaws and with severe atrophy are another of the most frequent indications before the NDI. These are indicated in those cases in which we do not have enough bone to place an SDI and we do not want to perform any bone regeneration procedure. Therefore, we must be aware that NDIs are normally inserted in very atrophic edentulous jaws, which represents very challenging surgical situations . OcclusiSurvival, success, and marginal peri-implant bone loss rates obtained in our review are similar to those of other reviews by other authors. The 97% survival obtained in our review is slightly higher than that obtained by other authors: 95.6% . The sucThe main limitation of this systematic review is the heterogeneity of the different articles included in terms of study design, follow-up period, sample size, and type of prosthetic rehabilitation. Some articles do not provide data on marginal bone loss, and others do not evaluate the success rate, or the data they provide is not sufficient to include them in the meta-analysis.Based on the results obtained in this review, it can be stated that NDIs are a predictable treatment option. This is due to their favorable survival, success, and average bone loss rates. All of them are comparable to those of the SDI or those of regenerative bone treatments at a horizontal level and bone expansion techniques. Therefore, it can be concluded that NDIs are a valid therapeutic option in cases in which there is not enough bone volume in the horizontal direction to place an SDI. NDIs are a viable option in medically compromised or elderly patients to avoid more invasive procedures, thus reducing morbidity and treatment time.


To construct and validate a radiomics nomogram for preoperative prediction of survival stratification in glioblastoma (GBM) patients with standard treatment according to radiomics features extracted from multiparameter magnetic resonance imaging (MRI), which could facilitate clinical decision-making.A total of 125 eligible GBM patients  were randomly divided into a training cohort (n = 87) and a validation cohort (n = 38). Radiomics features were extracted from the MRI of each patient. The T-test and the least absolute shrinkage and selection operator algorithm (LASSO) were used for feature selection. Next, three feature classifier models were established based on the selected features and evaluated by the area under curve (AUC). A radiomics score (Radscore) was then constructed by these features for each patient. Combined with clinical features, a radiomics nomogram was constructed with independent risk factors selected by the logistic regression model. The performance of the nomogram was assessed by AUC, calibration, discrimination, and clinical usefulness.There were 5,216 radiomics features extracted from each patient, and 5,060 of them were stable features judged by the intraclass correlation coefficients (ICCs). 21 features were included in the construction of the radiomics score. Of three feature classifier models, support vector machines (SVM) had the best classification effect. The radiomics nomogram was constructed in the training cohort and exhibited promising calibration and discrimination with AUCs of 0.877 and 0.919 in the training and validation cohorts, respectively. The favorable decision curve analysis (DCA) indicated the clinical usefulness of the radiomics nomogram.The presented radiomics nomogram, as a non-invasive tool, achieved satisfactory preoperative prediction of the individualized survival stratification of GBM patients. Glioblastoma (GBM) is the most frequent primary malignant tumor of the central nervous system with the characteristics of highly aggressive growth, high recurrence rate, and poor prognosis, defined as Grade IV glioma or glioblastoma multiforme according to the 2016 World Health Organization classification of brain tumors . DespiteRecent investigative studies have identified several prognostic factors commonly used to predict the prognoses of GBM patients, such as the age of diagnosis, laterality, radiotherapy, chemotherapy , KarnofsIn this research, the main purpose was to develop and then independently validate a nomogram based on radiomics score (Radscore) for preoperative prediction of individual OS stratification probabilities for GBM patients who would receive standard treatment. Moreover, three feature classifiers were established to evaluate the value of the selected radiomics features for differentiating survival stratification of GBM patients.This retrospective study included 125 patients with newly diagnosed GBM undergoing open craniotomy at the First Affiliated Hospital of Zhengzhou University from January 2018 to January 2020 according to the following criteria. The inclusion criteria were as follows: 1) patients with GBM confirmed by pathological report; 2) patients with complete data of medical and imaging records before surgery; and 3) patients with standard treatment, i.e., maximal safe surgical resection followed by radiotherapy plus adjuvant chemotherapy with temozolomide, then maintenance with 6–12 months of temozolomide as single-agent therapy. The exclusion criteria were as follows: 1) patients with biopsy only; 2) patients without complete medical records; 3) patients with incomplete image data and image artifacts; and 4) patients with radiotherapy or chemotherapy alone after surgery. The medical ethics committee of the First Affiliated Hospital of Zhengzhou University approved this retrospective study.There were 4 clinical features and 2 conventional imaging features collected for each patient, including age at diagnosis, gender, preoperative Karnofsky performance status (pKPS), preoperative epilepsy status (pEPI), located lobe , and hemisphere . Regular follow-up was applied to every patient until death or June 2021 through the clinic or phone, once every month for the first 6 months after surgery, and every 3–6 months thereafter. Each patient was separated into short or long survival group according to the OS of 12 months. Then, the patients were divided randomly into training cohort  used for model construction and validation cohort  used for model evaluation.Four imaging sequences were selected from the head MRI undergone before surgery of each patient, i.e., T1-weighted contrast-enhanced imaging (T1C), T1-weighted imaging (T1), T2-weighted imaging (T2), and T2-weighted fluid-attenuated inversion recovery imaging (T2F). The imaging was performed on five models of MRI scanners from two manufacturers, i.e., Verio, Prisma, TrioTim, and Skyra of Siemens and Discovery MR750 of GE Medical Systems. The T1 sequence was acquired with the following range of parameters: repetition time (TR)/echo time (TE), 163–1,750.03 ms/2.46–25.176 ms; slice thickness, 5 mm; spacing between slices, 6.5–6.75 mm. The T1C sequence was acquired with the following range of parameters: TR/TE, 21–3,900 ms/2.32–92 ms; slice thickness, 0.9–5 mm; spacing between slices, 6.50–6.75 mm. The T2 sequence was acquired with the following range of parameters: TR/TE, 3,800–5,673.8213 ms/92–117 ms; slice thickness, 5 mm; spacing between slices, 6.5–6.75 mm. The T2F sequence was acquired with the following range of parameters: TR/TE, 5,000–8,400 ms/81–157.732 ms; slice thickness, 5 mm; spacing between slices, 6.5–6.75 mm.Image preprocessing was performed by 3D Slicer software (v4.11.0). First, skull-stripping was executed by the Swiss Skull Stripper module, and T1, T2, and T2F sequence images were registered to T1C sequence images. Next, N4 bias field correction was applied to correct the intensity unevenness of each sequence. Ultimately, image normalization  and image resampling  were performed in Python environment by the PyRadiomics package.The region of interest (ROI) was delineated on T1C using 3D Slicer software separately by two neurosurgeons, who were blinded to the clinical data and had over 5 years of clinical experience.The radiomics feature extraction was executed in the Python environment with the PyRadiomics package, which was an open-source python package for the extraction of Radiomics features from medical imaging. The Three feature selection steps were performed to avoid overfitting before establishing the model. First, the stable features, which were defined by intraclass correlation coefficients (ICCs) >0.8, were selected between the two groups of ROIs drawn by the two neurosurgeons . Next, tTo understand the ability of these OS status-related radiomics features to discriminate the OS state, three supervised machine learning algorithms were applied in the training cohort, including random forest (RF) algorithm, support vector machine (SVM) algorithm, and logistic regression (LR) algorithm, and tested in the validation cohort. The performance of training and validation cohorts was evaluated by AUC. Then, these features with non-zero coefficients were used to construct the Radscore for each patient. Finally, the radiomics nomogram based on Radscore from the training cohort was established by logistic regression and assessed by the validation cohort. For the two cohorts, the discriminative ability of the radiomics nomogram was quantitatively measured using AUC, and the calibration curves were plotted based on observed probabilities and the nomogram-estimated probabilities . To evalThe continuous variables were analyzed by Student’s t-test or Mann–Whitney U-test, and chi-square test, Yates’ correction, or Fisher’s exact probabilities were performed in the categorical characteristics. Generally, a two-sided p-value < 0.05 was considered statistically significant. All data analyses were performed in Python (v3.7.6) and R software (v4.0.5).A total of 320 newly diagnosed GBM patients were collected. According to the inclusion and exclusion criteria, we included 177 patients whose image quality met the criteria and 52 patients without standard treatment were excluded. 125 patients were finally included in our study . The clinical features, conventional imaging features, and Radscores of patients in the training cohort, validation cohort and total cohort, are summarized in In this study, a total of 1,304 radiomics features for each sequence were extracted, including 14 shape features, 18 first-order statistics features, 68 texture features, 172 LoG features, 688 wavelet features, 86 square features, 86 square-root features, 86 logarithm features, and 86 exponential features. A total of 5,216 radiomics features were calculated from four imaging sequences for each patient. Among them, 5,080 radiomics features were stable after being screened by ICCs. After that, 777 radiomics features were selected by t-test. Ultimately, the optimal regulation weight λ (λ = 0.029470517025518096) was determined for the LASSO algorithm, and 21 features with non-zero coefficients were selected for OS stratification of GBM patients. The detailed names and weights of the 25 radiomics features are shown in There were three supervised machine learning algorithms models constructed to determine the ability of these OS status-related radiomics features to discriminate the OS stratification. The detail performances of the three models are shown in . In the training cohort, the average values of Radscore were significant differences in the short OS and long OS groups . Similarly, the mean Radscore of long OS was 0.165, which was significantly higher than that of short OS  in the validation cohort.Then, the Radscore for each patient in training and validation cohorts was constructed for further analysis, which was calculated by multiplying each feature coefficient by the corresponding feature value and summing. The corresponding fitting formula is listed in To establish the radiomics nomogram, the logistic regression based on Radscore, clinical features, and conventional imaging features was applied to find independent predictors of OS stratification by univariate and multivariable logistic regression. The results of logistic regression are presented in . The ROC curve, which was based on the probability of long OS according to the Radscore, was used to evaluate the sensitivity and specificity of the nomogram. The AUCs of the nomogram were 0.877 and 0.919 in the training and validation cohorts, respectively, which indicated favorable sensitivity and specificity . The calibration curve of the proposed nomogram based on the training cohort was constructed, and a favorable calibration was confirmed in the validation cohort . Moreover, the result of DCA showed that the nomogram to stratify the OS of GBM patients could yield clinical net benefits .Then, the radiomics nomogram was constructed according to the multivariable logistic regression A total of 125 newly diagnosed GBM patients with standard treatment were included in this study, and the objective was to develop and validate a preoperative prediction model for OS stratification. Only one independent predictive factor was found to be associated with OS stratification. The Radscore factor was adopted in a clinically relevant nomogram model that can predict the probabilities of OS stratification for the GBM patients. The radiomics nomogram demonstrated that a patient was more likely to have long OS if he had a higher Radscore before operation and would receive standard treatment. This may help neurosurgeons with preoperative planning and allow for better education for these patients or those family members who were extremely concerned about the postoperative survival or hesitated to continue the treatment for various reasons before the operation.The nomogram, as a tool of prediction, integrates a variety of independent predictive factors and visualizes the overall impact of these factors on survival in each patient to help clinicians develop intervention plans . Owing tAt present, there have been some studies that make efforts to predict the OS of GBM. These studies have selected some independent clinical risk factors related to OS, such as age at diagnosis, gender, KPS, MGMT, IDH, radiotherapy, chemotherapy, and radiotherapy combined with chemotherapy , 20, 21.Recently, several studies about the nomogram for predicting survival of GBM based on radiomics were published. Zhang et al.  developeHowever, our study still has some limitations. Firstly, this was not a multicenter study, although we had independently validated the model. More datasets from multimedical centers are needed to independently validate the robustness and repeatability of the radiomics nomogram. Second, although with high efficiency and sparsity, the combination method of the t-test and LASSO regression may be less stable when a large number of features were involved in the model. Other feature selection methods should be investigated in future work. Finally, these MRI images come from different imaging scanners and models, which may cause heterogeneity bias. In order to avoid this situation, all MRI images involved in the study were normalized and resampled before feature extraction. The same scanner and model are expected for MRI images in future researches for the convenience of image processing.In conclusion, to help neurosurgeons make better preoperative planning and patient education, our research developed and validated a radiomics nomogram based on multiparameter MRI imaging. The presented radiomics nomogram, as a noninvasive tool, achieved satisfactory preoperative prediction of the individualized survival stratification of GBM patients.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 Ethics Committee of the First Affiliated Hospital of Zhengzhou University. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.XJ and XW designed the study. XJ, DS, YW, and SW collected the clinical and radiomics data. XJ and FY preprocessed patients’ MR imaging and drew the ROI. XJ, DS, and YZ analyzed the data and developed the prediction model. XJ wrote the manuscript. All authors contributed to the article and approved the submitted version.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.


CLOCK rs11932595 and CRY1 rs3741892 had a higher abdominal obesity risk than those in the OFC. The GG genotype of PER2 rs2304672 in the VLFC showed greater risks for obesity and abdominal obesity. In conclusion, these findings suggest that macronutrient intake patterns were associated with obesity susceptibility, and the associations were different depending on the circadian clock genotypes of the CLOCK, PER2, and CRY1 loci.Although the impacts of macronutrients and the circadian clock on obesity have been reported, the interactions between macronutrient distribution and circadian genes are unclear. The aim of this study was to explore macronutrient intake patterns in the Korean population and associations between the patterns and circadian gene variants and obesity. After applying the criteria, 5343 subjects  from the Korean Genome and Epidemiology Study data and nine variants in seven circadian genes were analyzed. We defined macronutrient intake patterns by tertiles of the fat to carbohydrate ratio (FC). The very low FC (VLFC) was associated with a higher risk of obesity than the optimal FC (OFC). After stratification by the genotypes of nine variants, the obesity risk according to the patterns differed by the variants. In the female VLFC, the major homozygous allele of  CLOCK), aryl hydrocarbon receptor nuclear translocator-like , period homolog , and cryptochrome  regulate the circadian rhythm mechanism [PER and CRY genes by binding to enhancer elements. Increased proteins of PER and CRY inhibit ARNTL-CLOCK-mediated transcription. This transcription–translation negative feedback loop leads the circadian rhythm, which takes 24 h [The circadian clock governs 24 h rhythms and regulates the sleep–wake cycle. In mammals, circadian rhythms influence metabolism and physiological processes . Furtherechanism . The ARNkes 24 h ,4.CLOCK and ARNTL influence body weight control, the development of obesity, and susceptibility to metabolic diseases [Multiple evidence from mouse models and human studies have reported a link between the risk of disease and clock genes ,12,13,14diseases ,18,19,20diseases ,23,24,25The master clock located in the hypothalamic suprachiasmatic nucleus can be regulated by the light–dark cycle ,26,27, wDietary macronutrients are important to maintain health and physiological functions. In previous nutritional intervention studies, the results mainly focused on the effects of low-fat or low-carbohydrate diets on obesity-related features such as weight control ,39,40,41Several studies that investigated the effects of nutritional challenges on the circadian system reported that altered feeding cycles under an obesogenic diet were related to metabolic disorder ,44. MacrThis study used the Korean population data from the Korean Genome and Epidemiology Study (KoGES), provided by the Center for Genome Science, National Institute of Health, Korean Centers for Disease Control (KCDC) and Prevention, Chungcheongbuk-do, Korea . A localCLOCK, ARNTL, PER1, PER2, PER3, CRY1, and CRY2  were excluded. We conducted linkage disequilibrium (LD)-based pruning (r2 > 0.2); one SNP which had the highest MAF was selected from each LD block using PLINK software version 1.09 [Genomic DNA derived from blood samples was genotyped with the Affymetrix Genome-Wide Human SNP Array 5.0 kit  , and 100and CRY2 . SNPs wiion 1.09  and Haplion 1.09 . Utiliziion 1.09 ,54, we sA validated semi-quantitative food frequency questionnaire with 103 food items was used for assessing dietary data . The con2 according to Asia–Pacific BMI cut-off from the World Health Organization Report [Anthropometric measurements were obtained  by trained staff in cohort study . In the n Report . The abdn Report .p-value of <0.05 was considered to be statistically significant. Bonferroni correction was applied to correct for multiple testing (Bonferroni corrected p < 0.011).Data were presented as the mean ± standard deviation, number, and percentage. ANOVA analysis with Tukey post hoc comparison test was used to identify group differences, and Welch’s ANOVA with Games–Howell test was used to adjust for unequal variances. The Chi-square test was used to analyze categorical variables. Multiple logistic regression analysis was used for exploring the associations between genotypes and disease after adjustment for covariates, such as age, body mass index (BMI), sleep duration, alcohol intake, tobacco consumption, physical activity, energy intake, and number of regular meals. Statistical analyses were performed using SAS software version 9.4  and RStudio ver.1.2.1335 . A 2), whereas female VLFC had a higher BMI (25.3 ± 3.4 kg/m2). In the VLFC group, the portion of rural subjects was greater than other groups . The proportion of urban subjects was highest in the OFC group . The VLFC had a lower lean body mass and body fat than other groups in males . In contrast, female VLFC had a lower lean body mass (39.8 ± 4.6 kg) and higher body fat (15.7 ± 4.9 kg). Furthermore, the female VLFC showed a higher waist to hip ratio (0.91 ± 0.05) compared to other groups.The main characteristics of all the included participants are shown in The nutritional intake including total energy, carbohydrate, protein, and fat was highest in the OFC group and lowest in the VLFC group. However, carbohydrate intake did not differ by FC group in females. The VLFC group had a significantly higher % of energy from carbohydrate intake  and consequently a lower % of energy from protein and fat  than in other groups . ConsideIn contrast, the VLFC and LFC had an inadequate composition of macronutrients, which fell outside the AMDR with a higher carbohydrate and lower fat intake. Because the OFC had a macronutrionally balanced diet with optimal proportions, we designated the OFC as the reference group in our further analysis. The FC ratio was 0.14 ± 0.03, 0.23 ± 0.02, and 0.34 ± 0.08 for male VLFC, LFC, and OFC respectively; and 0.11 ± 0.03, 0.19 ± 0.02, and 0.31 ± 0.09 for female VLFC, LFC, and OFC respectively.The prevalence of disease according to the tertiles of the FC ratio is shown in To investigate the association of macronutrient composition and genetic variations of circadian clock genes, we stratified subjects by the genotypes of nine SNPs and analyzed the risk of obesity . The homCLOCK rs9312661, CRY2 rs7951225, and the GG genotype of CRY1 rs11113192 showed increased risks of obesity; however, significances were diminished after the Bonferroni correction (CRY1 rs11113192 and the FC on obesity was observed (p-interaction = 0.009); however, the significance disappeared after multiple corrections. No statistically significant differences were found for abdominal obesity.The risk of disease was increased in the VLFC group, particularly in females . In the rrection . An inteCLOCK rs9312661 in the VLFC showed an increased incidence of abdominal obesity compared with the reference group . In addition, under the VLFC condition, CRY1 rs3741892 had a significantly greater obesity risk than the reference regardless of genotype . Intriguingly, the associations between macronutrient intake patterns and obesity risks were different depending on the genotypes of CLOCK rs11932595, PER2 2304672, and CRY1 rs3741892. The major allele homozygous, AA genotype, of rs11932595 in the VLFC had a higher risk of abdominal obesity than the reference group , but not in subjects carrying the minor G allele. Regarding CRY1 rs3741892, which showed a higher obesity risk in both genotypes, the GG genotype, but not the CG/CC genotype, had a greater incidence of abdominal obesity . Moreover, females with the GG genotype of PER2 rs2304672 in the VLFC had significantly higher risks of obesity and abdominal obesity compared with the references , whereas no differences were detected in minor C allele carriers.In females, both genotypes of CLOCK gene expression in the skeletal muscle, small intestine, colon, pancreas, and subcutaneous adipose tissue . These findings indicate that genetic variants might influence circadian gene expression levels in important metabolic tissues.To explore the potential role of genetic variants on circadian gene regulation, we conducted an eQTL analysis at the SNP selection step. The four SNPs , which had association with macronutrient intake patterns and obesity risk, contributed to gene expression in various tissues involved in metabolism . For inse tissue . Moreove results , showed CLOCK, PER2, and CRY1. In the VLFC pattern, the major allele homozygous genotype of rs11932595, rs3741892, and rs2304672 had greater risks of obesity and abdominal obesity than the reference group, whereas minor allele carriers had no difference in risk. These findings indicate that macronutrient intake patterns were associated with obesity susceptibility, and the associations were dependent on circadian clock genetic variants, particularly in females. To the best of our knowledge, this is the first study to investigate the roles of dietary macronutrient distribution and circadian clock genes in disease risk in the Korean population.In the present study, we explored macronutrient intake patterns in a Korean midlife population and observed associations between patterns and circadian clock gene variants and obesity. A categorization of the three patterns by the FC ratio revealed the high carbohydrate and relatively low-fat intake of subjects. The prevalence of obesity and abdominal obesity increased in the VLFC compared to the OFC in females. After stratification by the genotypes of nine SNPs, the obesity risk according to the patterns was different according to the genetic variants of PEPCK) and glucose-6-phosphatase (G6Pase) in liver and kidney [CLOCK, PER2, and CRY1. Thus, these results suggest that the identification of patterns of dietary macronutrient distribution and understanding the effects of interactions between patterns and circadian genes are essential for the prevention of obesity.Dietary macronutrients induced alterations of circadian clock gene expression and phase shift in tissues ,33,34,35d kidney . TherefoCLOCK polymorphism, has a role in the development of obesity, diabetes, and metabolic syndrome [CLOCK rs1801260 affected the incidence of metabolic syndrome, and the association was more apparent after the stratification of monounsaturated fatty acid intake [CLOCK in various tissues, these results imply that circadian genetic variants might regulate circadian genes as well as clock-controlled genes, resulting in different metabolic phenotypes. Having investigated the effects of genetic variants and macronutrient patterns on obesity risk, we found four significant SNPs. According to the eQTL analysis, the four SNPs influenced gene expression in various tissues  for adjustment in a statistical analysis process, the possibility of effects induced by potential confounding factors, such as residential area, socioeconomic position, and health-related behaviors, should be considered.CLOCK, PER2, and CRY1. These findings suggest that the low dietary FC ratio influences obesity susceptibility and the association depends on circadian clock genetic variations. Our findings highlight an important role of the association of macronutrient distribution and circadian clock on obesity.In conclusion, we investigated Korean macronutrient intake patterns and found associations between the patterns and circadian clock gene variants, and obesity risk. The VLFC pattern was related to higher incidences of obesity and abdominal obesity in females. After the genotype stratification of nine SNPs of circadian genes, the association between the FC ratio and obesity risk differed by the genetic variants of


F2, 60 = 0.88, p = 0.421]. This non-confirmatory pilot trial suggests that low-threshold, viable access to mental healthcare can be offered to multiple burdened refugees by culturally adapting an intervention, providing it in a scalable format, and addressing a transdiagnostic symptom.Refugees are exposed to multiple stressors affecting their mental health. Given various barriers to mental healthcare in the arrival countries, innovative healthcare solutions are needed. One such solution could be to offer low-threshold treatments, for example by culturally adapting treatments, providing them in a scalable format, and addressing transdiagnostic symptoms. This pilot trial examined the feasibility, acceptance, and preliminary effectiveness of a culturally adapted digital sleep intervention for refugees. Sixty-six refugees participated, with 68.2% of them seeking psychological help for the first time. Only three participants did not show clinically significant insomnia severity, 93.9% reported past traumatic experiences. Participants were randomly assigned to the intervention group (IG) or the waitlist control group (CG). Insomnia severity, measured by the Insomnia Severity Index, and secondary outcomes  were assessed at baseline, 1 and 3 months after randomization. The self-help intervention included four modules on sleep hygiene, rumination, and information on mental health conditions associated with sleep disturbances. 66.7% of the IG completed all modules. Satisfaction with the intervention and its perceived cultural appropriateness were high. Linear multilevel analyses revealed a small, non-significant intervention effect on insomnia severity of Hedge's g = 0.28 at 3-months follow-up, comparing the IG to the CG [ The United High Commissioner for Refugees (UNHCR) reports on 82.4 million forcibly displaced people in the end of 2020, including 34.4 million people displaced abroadConcerning (a) the use of scalable and low-threshold treatment formats –24, digiBarriers could be further reduced by (c) culturally adapting treatments, that is by considering refugees' contexts and strains in their treatment , 57. TheCombining the three illustrated approaches to overcome mental healthcare barriers among refugees, we culturally adapted a digital sleep intervention. A randomized controlled pilot trial was conducted to evaluate the feasibility, acceptance, and preliminary effectiveness of the resulting eSano Sleep-e intervention among refugees, which should allow drawing conclusions on conducting a final randomized controlled trial. Thereby, we aimed to investigatewhether refugees adhered to the intervention;whether refugees were satisfied with the intervention and perceived it as culturally appropriate; andwhether preliminary evaluations indicate that the intervention improved insomnia severity and other sleep associated and mental health conditions in refugees.http://www.consort-statement.org)  receiving access to the eSano Sleep-e intervention and a waitlist control group (CG). The study was registered in the German Clinical Trial Register (identifier: DRKS00018949). Deviating from the registration protocol, more than the 40 initially planned participants were included [the planned number based on the Consolidated Standards of Reporting Trials (CONSORT) guidelines for pilot and feasibility studies  and an aent.org) , 74.https://esano.klips-ulm.de/de/trainings/schlafprobleme-sleep-problems/sleep-e/), interested participants could get detailed information on the study procedure, and they were provided a link to study registration.The study was conducted online in German and English language. Recruitment was carried out from March 2020 to February 2021 and took place mainly in Germany, with a small part also in Austria, Switzerland, and Liechtenstein. The last follow-up assessment was completed in July 2021. Emails, posts, and flyers referring to the study homepage were distributed through diverse recruitment channels. Among the channels were institutions working with refugees ,  media , personal contacts and on-site advertisement . On the study homepage , applying permuted block randomization with randomly varying block sizes of two and four. Randomized participants were emailed information on the further study procedure. Participants of the IG received immediate access to the intervention after randomization; participants of the CG received access to the same intervention after completing the final assessment, following a waiting period with no intervention provided. Follow-up assessments took place 1 month (T2) and 3 months after randomization (T3). Participants received reminders to complete pending assessments. After T3, participants had the chance to win a 50€ Amazon voucher.After registering and signing the digital informed consent, participants were emailed a link to the baseline assessment (T1). Here, inclusion criteria for study participation were assessed, which were (1) age ≥ 18 years, (2) sufficient English or German language skills, (3) access to an internet device, (4) flight background. The only exclusion criterion was suicidality, unless the participant was in secured primary care. No cut-off for the severity of sleep disturbances was used as inclusion criterion so to obtain information on who might be interested in using the intervention, regardless of meeting clinical symptoms. This should also allow participation of refugees with subclinical symptoms to facilitate an initial and transdiagnostic access to mental healthcare. Eligible participants were randomized 1:1 to the IG or the waitlist CG after completing the baseline assessment. Randomization was conducted by an independent researcher not further involved in the study, using an automatic randomization software  in both a browser and mobile-based format. It consisted of four modules, which were recommended to be completed within 4 weeks. It took 30–45 min to complete all contents of one module; a module could be completed as a whole or broken down into different units. Contents aimed at normalizing mental health problems, promoting health behavior, and facilitating access to the health system. They included psychoeducation on sleep difficulties, rumination, and information on associated mental health conditions; sleep hygiene rules; exercises to deal with rumination; links to further mental healthcare options; and relaxation exercises  of completed modules among the participants of the IG, as well as the rates of non-starters and completers. Data was derived from the intervention platform.Acceptance outcomes included intervention satisfaction and its perceived cultural appropriateness. They were measured at T3 within those participants of the IG who had started the intervention at that time. Satisfaction with the intervention was assessed with the Client Satisfaction Questionnaire adapted for Internet Interventions  CSQ-I;   . Seven iSeveral secondary outcomes were measured. Sleep quality was assessed with the Pittsburgh Sleep Quality Index  PSQI; 1 FOSI-SF MFI; 20 PHQ-9;  RHS-15;  on level 1 and individual participants on level 2 . This analytical procedure was chosen as it allows for the simultaneous analysis of time and group effects . MissingWe conducted stepwise multiple regression analyses to examine whether acceptance of and adherence to the digital intervention predicted changes in the primary or secondary outcomes from T1 to T3. Perceived cultural appropriateness, client satisfaction, and number of completed modules at the time of T3 were used as predictors.n = 33) and CG (n = 33). Their sociodemographic characteristics are illustrated in n = 39, 59.1%). In total, 58 (87.9%) of the participants were from countries in the orient, the remaining 8 (12.1%) from sub-Saharan African countries. Participants spoke 15 different native languages, with 43 (65.2%) having Arabic as (one of) their native language. 36.4% of the participants (n = 24) were currently employed, 37.9% (n = 25) were enrolled at a university. Thirteen participants (19.7%) were in current psychotherapeutic treatment, 45 (68.2%) had never sought psychological help before. Regarding baseline symptomatic, three participants did not show clinically significant insomnia severity; 30 (45.5%) showed subclinical insomnia severity, and 33 (50.0%) showed clinical insomnia severity. Sixty-two participants (93.9%) reported past traumatic experiences, 14 (21.2%) reported alcohol or drug abuse.n = 57) and those who dropped out of the study (n = 9), or between participants of the IG who completed the training (n = 22) and those who did not (n = 11) were revealed concerning language abilities, level of education, and student status. Rates of lower language skills and lower educational level were higher among participants who did not complete T3 or the intervention, whereas rates of higher language skills and higher educational level were enhanced among participants who completed T3 and the intervention. Similarly, the number of university students was higher among study and intervention completers than among non-completers. Detailed information on the revealed group differences are presented in Sociodemographic data  and baseParticipants of the IG completed a mean of 2.9 (SD = 1.7) modules, which equals 72.0% of the intervention. Five participants (15.2%) did not start the intervention, 22 participants (66.7%) completed all modules see . At the Intervention satisfaction among the participants of the IG who started the intervention was found to be high with a mean CSQ-I score of M = 24.0 (SD = 6.6). The lowest rate of agreement was revealed in the statement “The training has met my needs,” which 15 participants (57.7%) partly or totally agreed with. The highest rate of agreement was shown in the statements “The training helped me deal with my problems more effectively,” “In an overall, general sense, I am satisfied with the training,” and “I would come back to such a training if I were to seek help again,” which 19 participants (71.4%) partly or totally agreed with, respectively. Details on the ratings are shown in Perceived cultural appropriateness was shown to be high with a mean CAQ global score of M = 79.3 (SD = 10.3). All four subscales yielded high mean scores, with language being the scale with the lowest ratings  and structure being the scale with the highest ratings (77.4%). The lowest perceived appropriateness was shown on the item “It would have been better for me to do the training in my native language,” which 23.1% (strongly) supported. The highest appropriateness was shown on the item “I think the training looks good,” which 88.5% (strongly) supported. Details on the ratings are displayed in the p = 0.112) and an effect size of g = 0.40. At T3, a mean of M = 11.5 (SD = 5.0) was observed in the IG and a mean of M = 12.9 (SD = 5.0) in the CG, with a non-significant estimated mean difference of -1.6  and an effect size of g = 0.28.Multilevel analyses of the ISI are illustrated in detail in Per protocol analyses on the primary outcome were conducted including only those 22 participants of the IG that had completed at least three modules of the intervention at the time of T3. Analyses revealed a non-significant group x time interaction effect. Between-group mean differences of insomnia severity at T2 with -2.4  and T3 with -1.9  were non-significant, revealing effect sizes of g = 0.49 and g = 0.35, respectively. Detailed results are illustrated in F2, 54= 4.97, p = 0.010, revealing a significant estimated mean difference at T3 between the IG and CG of -2.1 .Regression analyses investigating the influence of the CSQ-I, the CAQ, and the number of completed modules at T3 on the primary outcome did not yield significant results. Among the secondary outcomes, scores of the PSQI, the FOSI, the PHQ, the RHS, and some subscales of the MFI were significantly predicted by CSQ-I or CAQ scores. None of the outcomes was significantly predicted by the adherence to the intervention. Results are illustrated in All in all, 98 negative effects were reported that occurred during study participation , of which 12 (12.2%) were reported to be caused by the intervention, and 86 (87.8%) were reported to be caused by other circumstances. Concerning the negative effects caused by the intervention, three related to the factor symptoms, with slightly, moderately, or strongly worsened symptoms; five related to the factor deficiencies, revealing slightly or moderately perceived deficiencies of the intervention; one related to the factor dependency, with a moderate dependency on the intervention; two related to the factor stigma, with a moderate or strong stigma in relation to the intervention; and one related to the factor hopelessness, with a moderate hopelessness due to the intervention. Details on negative effects are displayed in This is the first trial investigating a digital sleep intervention in refugees. Thereby, three low-threshold approaches were combined to engage refugees: using a scalable digital format for the intervention, addressing somatically perceived and transdiagnostic sleep disturbances, and culturally adapting the intervention. The findings of this non-confirmatory trial indicate that the self-help intervention holds the potential to provide multiple burdened refugees with initial access to mental health treatment. So, on the one hand, although there were no inclusion criteria to the study regarding a cut-off for insomnia severity, only three of 66 participants showed no clinically significant insomnia severity based on the ISI . MoreoveThe particular target group of burdened and underserved refugees showed high satisfaction with, perceived appropriateness of, and adherence to the digital sleep intervention, with rates comparable to the original intervention , 81. ImpWhen interpreting the results, two aspects should be taken into account. First, a large proportion of participants had high levels of education and language skills, and the intervention dropout rate seemed higher among participants with lower levels of education and language skills. Second, almost three-quarters (72.7%) of the IG participants needed to be motivated to start or continue with the intervention at least three times, and intervention completion took long . This may suggest that the intervention completion was difficult and required skills that could mainly be fulfilled among highly educated refugees, which may be an easier-to-reach subgroup of refugees.To increase adherence, acceptance, and effectiveness in refugees with a lower level of education, it might be necessary to further adapt the intervention. (1) As suggested by the results of the CAQ, the intervention should be translated into native languages of refugees. (2) As access to the intervention was shown to be difficult by the frequent reminders on starting the intervention, the intervention should be provided on an easy-to-access platform with lower technical demands. In addition to reminders, persuasive design principles aiming at user engagement may be promising in this regard . (3) MorThe conducted pilot trial had several limitations. First, due to the pilot character of the trial, the sample size and, thus, the power to detect clinical significant effects was limited. Second, the digital sleep intervention and study participation material was only available in English or German. This posed high demands on (possible) participants, potentially hindering interested refugees with higher language barriers from participating in the study and the intervention. Third, along with this, a large part of the participants had a high level of education, which does not adequately reflect the situation of refugees, herewith indicating a selection bias and, thus, a limited generalisability. Fourth, a large proportion of Syrian refugees participated in the study. Although this roughly maps the distribution of refugees in Germany, the generalisability for refugees from other countries of origin is limited. Fifth, the used self-report questionnaires to assess the effectiveness and acceptability of the intervention were largely not evaluated for the target population, with exception of the RHS-15 . This laIn conclusion, this pilot trial indicated that providing a treatment in a digital format, targeting transdiagnostic sleep disturbances, and conducting a cultural adaptation resulted in an intervention that seemed feasible to establish initial healthcare access for hard-to-reach, burdened refugees. Acceptance and adherence levels were high, and no serious negative effects due to the intervention were reported. However, this non-confirmatory pilot trial indicated only a small, non-significant intervention effect on improving insomnia severity. Evaluating a refined cultural adaptation focusing on language, intervention complexity, and engagement strategies may be a promising next step toward bridging the mental health treatment gap for refugees.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 Ethics Committee at the University of Freiburg, Germany. The patients/participants provided their written informed consent to participate in this study.KSpa, JB, and LS conceived the study conceptualization and design. DL provided the intervention template GET.ON recovery, KSpa and LS adapted the intervention content, HB provided access to the intervention platform. KSpi provided expertise in sleep problems and treatment. KSpa and EH collected data, conducted the statistical analyses, and wrote the draft of the manuscript. HB, JB, and LS supervised the work. All authors contributed to the manuscript revision, read, and approved the submitted version.KSpa is supported by the German Academic Scholarship Foundation. The article processing charge was funded by the University of Freiburg in the funding programme Open Access Publishing.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.