The remaining 145 participants were included in the full analysis set . The total scores for respiratory and systemic symptoms during the intervention were significantly lower in the LF group than in the placebo group. The expression of CD86 and HLA-DR on pDCs was significantly higher in the LF group than in the placebo group at week 12. Adverse events were comparable between the groups, and no adverse drug reactions were observed. Conclusions: These results suggest that orally ingested LF supports the normal immune system via maintaining pDC activity, and maintains respiratory and systemic physical conditions in healthy adults. Objectives: We investigated the effects of bovine lactoferrin (LF) on the maintenance of the respiratory and systemic physical conditions. Methods: A randomized, double-blind, placebo-controlled trial was conducted. Healthy adults at Kyushu University of Health and Welfare ingested a placebo or bovine LF (200 mg/day) for 12 weeks. The primary endpoints were the total respiratory and systemic symptom scores. The secondary endpoint was the activity of plasmacytoid dendritic cells (pDCs) in peripheral blood. Results: A total of 157 subjects were randomized (placebo,  Dendritic cells (DCs) integrate the innate and adaptive immunity. Plasmacytoid dendritic cells (pDCs) express Toll-like receptor (TLR)-7 and TLR9, detect viral single-stranded RNA (ssRNA) or CpG DNA, and produce Type I interferons (IFNs). pDCs upregulate the expression of costimulatory molecules such as CD86, and major histocompatibility complex type II (MHC II) molecules such as human leukocyte antigen (HLA)-DR upon their activation. Type I IFNs stimulate downstream broad and diverse IFN-stimulated genes subsequently, and induce an antiviral state in the host cells. Type I IFNs also activate myeloid DCs, natural killer (NK) cells, CD4+ helper T cells and CD8+ killer T cells, and B cells ,2. ThereLactoferrin (LF) is a cationic iron-binding glycoprotein present in the exocrine secretions including milk, saliva, and tears, and plays an important role in the first line of host defense. Currently, LF isolated from cheese whey or skim milk produced from bovine milk is widely used as a functional ingredient in food products such as infant formula, yogurt, beverage, and supplements . LF upreThis was a randomized (1:1), double-blind, placebo-controlled trial conducted between August and November 2022 at the Kyushu University of Health and Welfare in Miyazaki, Japan. The trial was performed in accordance with the current revision of the Declaration of Helsinki  and the The eligible subjects were healthy adults between 18 and 65 years of age from Kyushu University of Health and Welfare. Subjects who met any of the following criteria were excluded from participation: subjects who regularly consume foods containing LF, subjects who plan or wish to become vaccinated or donate blood during the test period, extremely heavy drinkers , subjects who cannot sleep at night owing to irregular work or other reasons, subjects who take medicines or functional foods during the pretest period, which might affect their respiratory and systemic symptoms and/or immune function, subjects who cannot stop taking these medicines or functional foods during the test period, subjects who have serious disease in the liver, kidney, heart, lung, gastrointestinal tract, blood, endocrine system, or metabolic system, etc., or those who have a serious medical history of these, subjects having chronic disorders, subjects who are positive in the blood examination for Treponema pallidum, hepatitis B virus (HBV) antigen, hepatitis C virus (HCV) antibodies, or human immunodeficiency virus (HIV), subjects who need the application of medicine containing steroids, subjects having a milk allergy, subjects who have a medical history of drug allergy or severe food allergy, subjects who are pregnant or under lactation, or who are expected to be pregnant during the study, subjects who donated 400 mL of their blood within 12 weeks before the beginning of test-food intake, or not less than 200 mL of their blood during the pretest period, or others who have been determined as ineligible for the research subject of this study, judging from the principal researcher/doctor’s opinions on the findings of their background, physical observations, medical checkup, physical/clinical examination, and so on. For the sample size, based on an estimation of type I error (α) of 0.05, type II error (β) of 0.8, difference (δ) of 50, standard deviation (σ) of 100, and sample size ratio between groups of 1, we evaluated the sample size as 134 (67 in each group) using G*Power (version 3.1.9.7) and set the target sample size as 160 (80 in each group), considering the possible dropouts.After IRB approval, the principal researcher/doctor fully explained the details of the trial to the subjects in accordance with the informed consent form, and written informed consent was obtained from all subjects. The principal researcher/doctor enrolled 160 healthy subjects based on a background questionnaire and negative blood examination results for Treponema pallidum, HBV, HCV, and HIV. The subjects were randomly allocated to the placebo or LF group. They licked two tablets (placebo or 200 mg of LF per day) for a few tens of seconds and swallowed them afterward during the 12-week intervention. During the intervention, the subjects recorded their tablet intake, respiratory symptoms , sneezing, runny nose, and plugged nose), and systemic symptoms  in a diary. These symptoms were scored on an 8-point Likert scale from 0 (do not have this symptom) to 1 (very mild), 3 (mild), 5 (moderate), and 7 (severe), referring to the Wisconsin Upper Respiratory Symptom Survey and Jackson scale . The priThe primary endpoints were total scores for respiratory and systemic symptoms. The total scores of respiratory and systemic symptoms were calculated by summing the scores of individual symptoms obtained during the 12-week intervention. The secondary endpoints were expression of HLA-DR and CD86 on pDCs. Exploratory scores for respiratory and systemic symptoms in each 4 weeks were evaluated. Cumulative number of days of respiratory and systemic symptoms were also extracted from the adverse events record. The principal researcher/doctor discussed and evaluated all items.Any unfavorable and unintended signs, symptoms, or diseases observed during the intervention were defined as adverse events. Adverse events were evaluated using the Revised National Cancer Institute Common Toxicity Criteria Version 4.0, and events in which a causal relationship with the intake of the test foods could not be ruled out were designated as adverse drug reactions.The test food in the LF group was a round, red-orange tablet  identical to the commercial product ‘Lactoferrin Original’ . This tablet contained 100 mg of bovine LF, maltitol, indigestible dextrin, dextrin, and hydrogenated rapeseed oil. Iron content in the LF was quantified by inductively coupled plasma optical emission spectrometer . In the placebo tablet, LF was replaced with dextrin, and the tablet color was adjusted with food coloring. The tablets were similar in appearance, smell, taste, and packaging. An independent allocation manager confirmed that they were indistinguishable at the time of allocation and code breaking. The allocation manager produced a computer-generated allocation table with a block size of four (1:1 ratio), and numbered the test foods consecutively in accordance with the tables. The allocation tables were sealed in an opaque envelope and kept sealed until code breaking. The investigators numbered the enrolled subjects and assigned them to each test food item with a corresponding number. The investigators and participants were blinded until code breaking. Code breaking was performed after locking the database and the statistical analysis plan.A Vacutainer Cell Preparation Tube  was used to collect peripheral blood, and peripheral blood mononuclear cells (PBMCs) were isolated according to the manufacturer’s instructions. Isolated PBMCs were immediately used to analyze CD86 and HLA-DR expression on pDCs.6 cells/mL in RPMI-1640  supplemented with 5% human AB serum (Sigma-Aldrich), 2 mM Glutamax , and 1% penicillin/streptomycin (Thermo Fisher Scientific), and stimulated in the presence of 10 µg/mL TLR-7/8 agonist R848  for 4 h. Afterward, the cells were harvested and stained with FVS780, PE-Cy7 Mouse Anti-Human CD123, BB515 Mouse Anti-Human Neuropilin-1 (CD304), and PE Mouse Anti-Human HLA-DR  as described above. Data were collected using CytoFLEX  and analyzed using the FlowJo version 10 software (BD). Isotype controls (BD) were used to check the background caused by nonspecific antibody binding. pDCs were defined as CD123+CD304+ subsets, and CD86 and HLA-DR were used as activation markers for pDCs.Referencing a previous in vitro study , PBMCs wt-test. EZR software Version 1.40  was used for the statistical analysis [p value < 0.05 was considered statistically significant.Analyses of the efficacy were performed on the full analysis set (FAS). Consent withdrawal, eligibility violation at the start of intervention, and lost to follow-up were excluded. The total scores for respiratory and systemic symptoms were evaluated using the Mann–Whitney U test. To avoid the multiplicity issue, significant difference in both scores was required for the total significant difference. The geometric mean fluorescence intensity (MFI) of CD86 and HLA-DR on pDCs was logarithmically transformed to obtain normally distributed data, and the values at week 12 were analyzed using analysis of covariance (ANCOVA), with adjustments for the values at week 0. The values at week 0 and week 12 were also analyzed using the paired analysis , and a pn = 79; LF, n = 78). The baseline characteristics of the groups were comparable . The resulting full dataset was used for analysis   .Subjects started ingesting test foods on 8–10, 12, or 15–18 August 2022, and finished ingesting on 30–31 October, 1, 3, or 6–9 November 2022 (12 weeks). The median (interquartile range) intake rates of the test foods were 92.9% (83.3–96.4%) in the placebo group and 95.2% (86.9–100.0%) in the LF group, with no significant differences.With regard to the primary endpoints, the total scores for respiratory and systemic symptoms in 12 weeks were significantly lower in the LF group than in the placebo group . To specRegarding secondary endpoints, the expression of CD86 and HLA-DR on pDCs was significantly higher in the LF group than in the placebo group at week 12 . In bothDuring the intervention, a total of 115 subjects experienced adverse events, including the above symptoms, menstrual pain, abdominal discomfort due to excessive eating or drinking, muscle pain due to exercise, and not feeling well owing to a lack of sleep , and no significant differences were observed between the groups. No adverse drug reactions were observed. Cumulative number of days of feeling feverish, malaise, chill, cough, hoarseness, plugged nose, and head congestion during the intervention were fewer in the LF group than in the placebo group .We investigated whether the oral ingestion of LF 200 mg/day) affected respiratory and systemic symptoms, and the activity of pDCs in the peripheral blood in healthy adults. The total scores for respiratory symptoms, such as throat discomfort, hoarseness, chest congestion (sputum), sneezing, runny nose, or plugged nose, and systemic symptoms, such as feeling feverish or malaise, were significantly lower in the LF group than in the placebo group. A similar tendency was observed in the adverse events record. Alleviative effects of LF were clearer in weeks 5–8 and weeks 9–12 than in weeks 1–4 against both respiratory and systemic symptoms. Therefore, ingestion of LF for more than 4 weeks may be preferable to enjoy the benefits. Previous trials have also demonstrated that LF suppresses respiratory and systemic symptoms in healthy adults and children, which supports the results of the present trial ,20. As h0 mg/day The expression of CD86 and HLA-DR on pDCs was lower at week 12 than at week 0 in both groups, and was significantly higher in the LF group than in the placebo group at week 12. TLR7 function in PBMCs is lower in the dark season (October–March) than in the sunny season (April–September) . TherefoEndogenous LF is always present in exocrine secretions such as tears and saliva. Therefore, it is unlikely that LF itself extraordinarily stimulates the immune system. In fact, LF upregulated only CD86 expression on pDCs when there was no ssRNA present, but upregulated IFN-α, HLA-DR, and CD86 expression on pDCs in the presence of ssRNA in vitro . This suIngestion of exogenous LF may compensate for the effects of endogenous LF. Orally administered LF acts on the upper and lower digestive tracts, and also on the blood cells via the sublingual mucosa . As pDCsIn this trial, the prevalence of adverse events was comparable between groups, and no adverse drug reactions were observed. Therefore, LF was considered safe, as shown in previous studies ,19,20. FThis trial was conducted during late summer and fall in healthy men and women aged 18–38 years. In previous trials, TLR7-mediated responses in pDCs were enhanced by LF intake in elderly women , and supThe limitations of the present trial were bias in age distribution, season, lack of objective evaluation to identify the cause of respiratory and systemic symptoms, and lack of examination of proinflammatory or anti-inflammatory cytokine production. Investigations involving subjects with diverse backgrounds, in other seasons, or using objective measurements of respiratory and systemic symptoms and cytokine production are required to further improve the reliability of the results.


Cardiovascular diseases (CVD) are a global health concern, affecting millions of patients worldwide and being the leading cause of global morbidity and mortality, thus creating a major public health concern. Sodium/glucose cotransporter 2 (SGLT2) inhibitors have emerged as a promising class of medications for managing CVD. Initially developed as antihyperglycemic agents for treating type 2 diabetes, these drugs have demonstrated significant cardiovascular benefits beyond glycemic control. In our paper, we discuss the role of empagliflozin, dapagliflozin, canagliflozin, ertugliflozin, and the relatively recently approved bexagliflozin, the class of SGLT-2 inhibitors, as potential therapeutic targets for cardiovascular diseases. All mentioned SGLT-2 inhibitors have demonstrated significant cardiovascular benefits and renal protection in clinical trials, in patients with or without type 2 diabetes. These novel therapeutic approaches aim to develop more effective treatments that improve patient outcomes and reduce the burden of these conditions. However, the major scientific achievements of recent years and the many new discoveries and mechanisms still require careful attention and additional studies. Diabetes mellitus (DM) is a metabolic disease related to chronic hyperglycemia caused by impaired insulin secretion and/or action. It occurs mainly in the older population and is often undiagnosed. More than 400 million adults worldwide suffer from it, and it is estimated that this number will increase by more than 50% in the next 20 years .Persistently increased levels of glucose in the blood result in symptoms such as polyuria, polydipsia, drowsiness, or weight loss. The consequences of uncontrolled diabetes are ketoacidosis or nonketotic hyperosmolar syndrome. Chronically elevated blood glucose levels are associated with the development of numerous complications, such as nephropathy, neuropathy, and retinopathy. However, the leading causes of morbidity and mortality in both type 1 and type 2 DM are heart failure and cardiovascular disorders [DM and heart failure (HF) are among the most widespread diseases in the adult population, and their numbers are increasing with age. It is very common for the two illnesses to co-exist in the same patient, and in people over the age of 65, as many as 22% of people with type 2 diabetes have HF simultaneously ,4. The cDiabetic cardiomyopathy is a state of ventricular dysfunction in the absence of other cardiac risk factors in diabetics . There aMost current antidiabetic drugs have an adverse effect by exacerbating cardiovascular risk factors. Some antihyperglycemic therapies, such as the use of insulin or thiazolidinediones, cause weight gain and fluid retention , while sSGLT-2 inhibitors that are nowadays available in the United States are empagliflozin, dapagliflozin, canagliflozin, and ertugliflozin. In the European Union, we also have sotagliflozin. Nevertheless, on 23 January 2023, the U.S. Food and Drug Administration (FDA) approved a new antihyperglycemic agent called bexagliflozin. It has been proven to significantly improve glycemic control with a single daily dose of 20 mg. Importantly, in addition to its hypoglycemic effect, it also shows a systolic blood pressure-lowering effect .The aim of this review is to list, discuss, and compare each individual SGLT-2 inhibitor, with a focus on the recently approved bexagliflozin. We also want to outline the most important properties and side effects of this particular group of medications.The SGLT-2 inhibitors, which include empagliflozin, are one of the more recent groups used in antihyperglycemic therapy. On the basis of numerous studies and comparative meta-analyses, it was noted that it significantly reduced HF hospitalizations in patients with both stable cardiovascular disease and acute HF. Empagliflozin reduced all-cause and cardiovascular mortality and reduced the risk of cardiovascular disease, regardless of the initial risk ,15,16,17Preclinical studies report that the use of empagliflozin reverses the effect of glucotoxicity by lowering serum methylglyoxal levels and attenuating AGE/RAGE signaling ,31. It wThe first study to evaluate the effect of SGLT-2 inhibitors on cardiovascular events was EMPA-REG OUTCOME . The stup < 0.001), respectively. The preceding effect was similar in both the diabetic and non-diabetic groups . During the course of the trial, the number of all hospitalizations for HF in patients taking empagliflozin was lower as compared to the placebo group . It should also be emphasized that patients experienced significant improvements in cardiovascular and renal outcomes regardless of their baseline diabetes status [In the randomized EMPEROR-Reduced trial, 3730 patients with chronic HF (NYHA 2–4) and a left ventricular ejection fraction (LVEF) of 40% or less were screened to assess the effect of empagliflozin on the incidence of cardiovascular death or first HF hospitalization (the first outcome), as well as the rate of all HF hospitalizations (the first-second outcome) [s status .p < 0.001), and the time to first hospitalization was prolonged. In the post-analysis of the study, improvements in parameters were observed in the form of a decrease in glycated hemoglobin levels and body weight and an increase in hemoglobin levels [A subsequent study was EMPEROR-Preserved , enrollin levels . In addip < 0.001) compared to the beginning of the study [p < 0.001). In addition, the empagliflozin group had a significant reduction in left ventricular mass  and a more pronounced increase in LVEF  in comparison with placebo. There was a notable enhancement in peak oxygen consumption (1.1 ± 2.6 mL/min/kg) and distance extension in the 6MWT (81 ± 64 m) in the empagliflozin group [The EMPA-Tropism trial examined whether empagliflozin also had a positive effect on heart failure with reduced ejection fraction (HFrEF), exercise capacity, and quality of life in non-diabetic patients . The firhe study . Similarin group ,39. All EMPULSE was a prominent trial that investigated empagliflozin’s effects in people with acute HF . The triThe individual SGLT-2 inhibitors have a similar range of action; therefore, an important factor comparing them to one another will be cardiovascular events and the occurrence of adverse events . In a me® in Europe or Farxiga® in the US, in doses of 5 or 10 mg. It was approved in 2012 by the European Medicines Agency (EMA) and in 2014 by the Food and Drug Administration (FDA) [One example of a selective SGLT2 inhibitor is dapagliflozin, used under the trade name Forxigaon (FDA) ,48. As fon (FDA) ,48,49. Don (FDA) ,51. The on (FDA) ,52.In the European Union, it is used in monotherapy or combination therapy in T2DM when patients fail to achieve normal glycemic control despite lifestyle changes, i.e., diet and adequate exercise. Dapagliflozin has been shown in numerous studies to reduce hospital admissions for heart failure and the rate of death from cardiovascular causes in both patients with and without T2DM ,53. In pThese studies indicated that HF and HFrEF, regardless of the presence or absence of T2DM, who were consuming dapagliflozin, had a lower risk of cardiovascular death . An impoAs for contraindications to the use of the drug, these are, of course, a history of hypersensitivity reactions, for example, angioedema or anaphylactic reaction, and patients on dialysis therapy . AdversePatients could experience rhinosinusitis, upper respiratory tract infections in general, headaches, back pain, or the occurrence of diarrhea . The mosCanagliflozin is one of the SGLT2 inhibitors. Primarily used to treat type 2 diabetes, it has also been studied for its potential cardiovascular benefits. Principally, canagliflozin reduced the risk of cardiovascular events in people with type 2 diabetes, regardless of the coincidence of CVD ,65,66. CCanagliflozin demonstrates cardioprotective benefits independent of a glucose-lowering effect, including preservation of cardiac function during myocardial ischemia. Canagliflozin considerably attenuates the size of myocardial infarcts ,82. Sabep < 0.05), a faster return of spontaneous circulation (p < 0.01), and increased neurological scores (p < 0.01 or p < 0.001) following resuscitation. Canagliflozin may exert its effects through the STAT-3-dependent cell-survival signaling pathway, according to this study [A remarkable cardioprotective effect against cardiac arrest and resuscitation-induced cardiac dysfunction was obtained by canagliflozin ,86. Inteis study .p = 0.002; I2 0%), but not canagliflozin , significantly reduced AF and AFL. Further studies are required to establish whether canagliflozin similarly exerts protective effects against AF/AFL development [The possible benefit of canagliflozin use in the development and progression of atrial fibrillation (AF) has been suggested . This stelopment . A summaErtugliflozin is an SGLT2 inhibitor that is used as an adjunct therapy for the treatment of DM. The cardioprotective effects of ertugliflozin among individuals with CVD have not been extensively investigated in clinical trials.In preclinical models, ertugliflozin has been found to improve cardiac energy metabolism by increasing the availability of ketone bodies as an alternative energy source for the heart. This shift in substrate utilization may help preserve cardiac function in conditions such as HF. Furthermore, the drug has demonstrated the ability to attenuate cardiac remodeling, including left ventricular hypertrophy, fibrosis, and inflammation, pathological changes commonly observed in CVD ,91.The VERTIS CV trial assessed the impact of ertugliflozin in patients with type 2 diabetes and CVD, including those with a history of HF and known a pre-trial ejection fraction. The study demonstrated that treatment with ertugliflozin reduced the occurrence and total hospitalizations for HF events. This benefit was observed in patients with and without a history of HF, as well as in those with reduced or preserved ejection fraction .2, albuminuria, and diuretic use. Additionally, ertugliflozin use was associated with decreased albuminuria and preservation of eGFR over time, indicating its potential for kidney protection in patients with type 2 diabetes and CVD [The trial also revealed that the risk reduction for the first hospitalization for HF with ertugliflozin was consistent across most baseline subgroups. However, a greater benefit was observed in three specific populations: those with an estimated glomerular filtration rate (eGFR) less than 60 mL/min/1.73 m and CVD ,94.Overall, these findings suggest that ertugliflozin may have additional cardiovascular positive effects, apart from its effects on lowering glucose levels. Thus, it might be a promising therapeutic method for those with HF and CVD. However, further research is needed to fully comprehend the underlying mechanisms responsible for these effects.2. It is available as 20 mg oral tablets, recommended to be taken once daily, regardless of the meal. Patients with diabetes and mild to moderate kidney failure have fewer treatment options compared to those with preserved kidney function. Dosage modifications have been presented in Bexagliflozin is a novel agent approved by the FDA in 2023. This highly potent and selective inhibitor of SGLT2 is indicated for adults with type 2 DM with an eGFR greater than 30 mL/min/1.73 mA 96-week phase 2 clinical study showed that bexagliflozin monotherapy led to a long-lasting, clinically relevant improvement in glycemic control, with a significant reduction in weight and blood pressure . In a clBexagliflozin has been proven to be non-inferior to other SGLT2 inhibitors. Halvorsen et al.  revealedThe summary of SGLT2 inhibitors’ effects on the kidney and heart is presented in According to recent epidemiological data , type 2 Beyond their glucose-lowering effects, all of the mentioned SGLT2 inhibitors have been proven to have additional cardiovascular benefits and could be a promising treatment option for patients with CVD and HF. Empagliflozin has been proven to be the most effective SGLT2 inhibitor in lowering the risk of HF. It is also superior in reducing death from cardiovascular causes to canagliflozin or dapagliflozin. Undeniably, bexagliflozin has been actively awaited, mainly due to the ever-increasing prevalence of T2DM as well as increased morbidity and mortality from associated cardiovascular consequences. Studies have shown its non-inferiority for hard clinical outcomes in high-risk CVD cohorts . NowadayIn conclusion, it has been proven that patients with HF and DM may benefit from SGLT2 inhibitors. The balance of profits and adverse impacts depends on the individual risk profiles.


Among the heavy smokers who also drank alcoholic beverages, the hazard ratio from the fully adjusted model was 4.3 . In the subjects with moderate alcohol consumption, the risk of smoking and alcohol combined was similar to that found in the whole population . Among the subjects with heavy alcohol consumption, the hazard ratio was 3.4 . Conclusion: These findings indicate that the detrimental cardiovascular effects of smoking can be worsened by concomitant alcohol use. This synergistic effect occurs not only for heavy alcohol consumption but also for moderate use. Smokers should be aware of the increased risk associated with concomitant alcohol consumption.Aim: To evaluate the association of alcohol and smoking combined with cardiovascular and renal events and investigate whether moderate and heavy alcohol consumption have a different impact on this association. Methods: The study was conducted in 1208 young-to-middle-age stage 1 hypertensive patients. Subjects were classified into three categories of cigarette smoking and alcohol use, and the risk of adverse outcomes was assessed over a 17.4-year follow-up. Results: In multivariable Cox models, smoking showed a different prognostic impact on alcohol drinkers and abstainers. In the former, an increase in the risk of cardiovascular and renal events was observed compared to nonsmokers (hazard ratio, 2.6, 95% CI, 1.5–4.3,  The unhealthy effects of smoking, including an increased risk for several types of cancer, chronic obstructive pulmonary disease, coronary heart disease, and cardiovascular disease in general, have been well documented since the publication of the seminal Framingham studies ,2,3,4. TAlcohol use has also been found to increase the risk of developing serious diseases, including cancer, liver cirrhosis, hypertension, and hemorrhagic stroke ,9. HowevEpidemiological data suggest that smokers are at a higher risk of greater alcohol use patterns, including greater consumption and higher binge-like alcohol use ,18. IndeHowever, little is known about the combined effect of alcohol and smoking on cardiovascular events, especially in young individuals, despite emerging concerns about the cardiovascular health of this segment of the population. Recent data indicate that treatment and control of major risk factors for cardiovascular disease among young adults are far from optimal and suggest that cardiovascular disease may now be increasing in this population ,30,31.In a previous analysis of the HARVEST population, we observed an interactive effect of alcohol and smoking on the risk of cardiovascular events in 18- to 45-year-old individuals . The aimThe HARVEST is a multicenter prospective observational study conducted in 17 hypertension units in Italy that began on 1 April 1990 ,34,35. TThe questionnaire captured data pertaining to the participants’ demographics, personal and family health, medical history, and lifestyle habits, which included information about smoking, alcohol intake, coffee consumption, and physical activity. Participants were classified into three categories according to the daily number of cigarettes smoked: nonsmokers, 1–10 cigarettes/day, and >10 cigarettes/day. They were also divided into three categories of alcohol use: 0 g/day, <50 g/day, and ≥50 g/day. Coffee consumption was defined according to the number of caffeine-containing coffees drunk per day. For the present analysis, participants were divided into two categories: coffee drinkers and nondrinkers. Information on physical activity was collected using a previously published classification ,34. A faDuring the follow-up, visits were scheduled every 6 months. Antihypertensive treatment was started following the recommendations of international guidelines available at the time of the visit see . Then, t2). We ascertained vital status and the incidence of fatal and nonfatal events from medical records and interviews with attending physicians and patients’ families.Cardiovascular events included fatal and nonfatal strokes, fatal and nonfatal ST-elevated acute myocardial infarction, non ST-elevated acute coronary syndromes, any myocardial revascularization procedure, heart failure needing hospitalization, any aortic or lower limb revascularization procedure, and development of permanent atrial fibrillation. Renal events were defined as chronic kidney disease at stage 3 or higher , physical activity (yes/no), parental history of cardiovascular disease, BMI, total cholesterol, average 24 h systolic and diastolic BPs, and incident hypertension needing antihypertensive treatment diagnosed during the follow-up were also included. No violations to the proportional hazards assumption were detected by inspection of survival curves. Hazard ratios and corresponding two-sided 95% confidence intervals were derived from the regression coefficients in the Cox models. Tests for interaction effects were conducted in multivariate regression analyses by adding the product of smoking and alcohol in the models. A two-tailed probability value ≤0.05 was considered significant. Analyses were performed using Systat version 12  and MedCalc version 20.218 .Quantitative variables were reported as mean ± SD unless specified. For follow-up duration, the median and interquartile range (IQR) were calculated. Categorical variables were reported as percentages and differences in the distribution were tested by χClinical characteristics of the subjects stratified by smoking and alcohol use are reported in Smokers were more frequently alcohol drinkers than nonsmokers. Alcohol consumption was proportional to the number of cigarettes smoked per day . Coffee 2 4.5; p = 0.033). The most common events were acute coronary syndromes , with 4.9% among men and 2.1% among women .During a median follow-up of 17.4 (IQR 8.9–23.0) years, there were 108 adverse events (8.9%). Of these, 96 were fatal and nonfatal cardiovascular events (7.9%), and 12 were renal events (1.0%). The rate of total events was higher among the 879 men  than the 329 women  (chip < 0.001). In the latter, the risk did not achieve the level of statistical significance  with a significant interaction between smoking and alcohol use (p < 0.001) (p < 0.0001).The risk of cardiovascular and renal events from a multivariable Cox model including age, gender, BMI, parental history for cardiovascular disease, coffee consumption (yes/no), physical activity (yes/no), total cholesterol, mean 24 h systolic and diastolic BPs, and incident hypertension in the subjects stratified by smoking and alcohol drinking is reported in < 0.001) . Among tThe survival curves for the participants stratified according to smoking and alcohol combined are reported in p < 0.001) (p = 0.011) , and 22.2% in the smokers with heavy alcohol consumption (≥50 g/day). In a sensitivity Cox analysis, the risk of smoking and alcohol combined in the subjects with moderate alcohol consumption  was similar to that found in the whole population  . Among t= 0.011) .In this prospective cohort study of young-to-middle-aged subjects with stage 1 hypertension followed for over 17 years, we observed that the detrimental effects of smoking on cardiovascular health were worsened by concomitant alcohol use confirming our previous findings that smoking and drinking can act synergistically to increase cardiovascular risk . In addiTobacco consumption is widely recognized as an important risk factor for cardiovascular disease and multiple chronic noncommunicable diseases ,3,4,5,6 According to the American Heart Association, ideal cardiovascular health should include the combination of the 7 factors that compose the so-called “Life’s Simple 7”: not smoking, having a healthy diet pattern, adequate physical activity, healthy body weight, and healthy BP, cholesterol, and blood glucose in the absence of pharmacological treatment . AlcoholA strong synergistic effect between smoking and alcohol has been found in several clinical conditions, including some types of cancer ,23,24, lA J-shaped relationship between alcohol use and cardiovascular mortality has been described in several studies and meta-analyses, suggesting an increase in risk among heavy drinkers and a protective effect among light users ,11,12,13Tobacco and alcohol use have reciprocal influences on potentiating cravings and interact with each other leading to more frequent use and higher consumption levels . A recenThe mechanisms underlying the interactive cardiovascular effects of smoking and alcohol remain elusive. In a previous study, we observed an interactive effect of these two factors on 24 h catecholamine output, suggesting that increased sympatho-adrenergic activity may be a contributing factor to the synergistic cardiovascular effect of tobacco and alcohol use . In addiWe acknowledge that the present study has several limitations. First, in our study, the status of lifestyle factors was self-reported by the participants and thus subject to misclassification. However, health behaviors were checked frequently during the follow-up, showing that lifestyle habits were constant and well-reported ,33. SecoThe present findings indicate that there is an important synergistic effect between tobacco and alcohol consumption on cardiovascular and renal outcomes in young hypertensive subjects. The multiplicative risk associated with smoking and alcohol drinking also occurred for moderate alcohol consumption, emphasizing the importance of controlling the concurrence of unhealthy lifestyle factors. Strategies for improving lifestyle behaviors are well recognized and should be applied and reinforced, particularly in young patients with hypertension. Indeed, increases in heart disease and cardiovascular mortality has been reported in recent years in younger adults, which reflect recent unfavorable trends in cardiovascular risk determinants and events ,46. Toba


Background: Intertrochanteric fractures are a global health concern, especially in aging populations like the Republic of Korea. Surgical treatments like intramedullary nailing are preferred for their benefit. Various hip nails are used worldwide, each with unique features and challenges. This study aims to compare the GS hip nail with the Affixus hip fracture nail for the treatment of intertrochanteric fractures. Material and Methods: This retrospective study, conducted at a single center, included 179 patients who underwent intramedullary nailing for intertrochanteric fractures using the GS hip nail or the Affixus hip fracture nail. Excluding specific cases, 43 patients in the GS group and 46 in the Affixus group met the minimum 6-month follow-up criteria. Result: The GS group exhibited a significantly shorter mean operation time (43.26 min) compared to the Affixus group (51.11 min). Radiographically, both groups showed no significant differences in their reduction quality, tip, and apex distance (TAD), or Cleveland index in the immediate postoperative window. However, the GS group achieved a greater valgus reduction based on the contralateral femoral neck shaft angle (NSA). At 6 months post-operation, there were no significant differences in TAD or advancement and sliding distances. Complication rates were similar between the two groups, with no implant breakages. Clinical outcomes, as measured via mHHS and EQ-5D-5L, showed no significant differences. Despite a slightly higher implant cost, the GS group had a lower total hospital cost than the Affixus group, but this was not statistically significant. Conclusions: This study highlights the efficiency of the GS hip nail in reducing the operation time compared to the Affixus hip fracture nail with comparable radiologic and clinical outcomes. Further research with long-term follow-up and larger patient studies are needed to fully assess its effectiveness in improving patient outcomes in hip fracture treatment. The annual incidence of hip fractures worldwide is predicted to increase, and this is attributed to the growing elderly population globally. It is known that the increase in the elderly population is set to become more prominent in Asia and Latin America compared to Europe and North America . The RepThere have been numerous clinical and biomechanical studies analyzing the outcomes of various hip nails available worldwide, including the Gamma nail , the Proximal Femoral Nail Antirotation , and the Affixus hip fracture nail . However, these devices have also encountered issues such as cut-out, implant breakage, femoral shaft fracture, and subsequent loss and reduction in clinical practice ,12,13,14In the Republic of Korea, the GS hip nail  has also been developed for the treatment of proximal femoral fractures. Lag screws come in the following two forms: screw-shaped and blade-shaped. The screw-shaped design allows for easy insertion and simple length adjustment, making it easier to achieve fixation in firm bone with the tip located just beneath the articular surface. However, it can lead to fixation failure due to the rotation of the femoral head. On the other hand, the blade-shaped design requires significant force to counteract femoral head rotation, thus preventing varus deformity. However, it has the disadvantage of having a sharp blade that can potentially lead to a cut through . The GS The primary objective of this study is to compare a reduction in the risk of postoperative complications, such as fracture fixation failure, cut-out, and back-out, when treating intertrochanteric fracture patients using the lag screw type of the GS hip nail, known for its advantages, with those treated using the Affixus hip fracture nail.This study was conducted after receiving the approval of the Hallym University Kangnam Sacred Heart Hospital Institutional Review Board (IRB) (2023-08-019). This study is a retrospective study conducted at a single university hospital  on a total of 179 patients who underwent surgical treatment for intertrochanteric fractures with the GS hip nail and the Affixus hip fracture nail devices via intramedullary nailing from September 2019 to February 2023. Patients who underwent revision surgery due to the failure of a previous operation, those with pathologic fractures, individuals who underwent bilateral surgery, and patients who were lost to follow-up due to death or other reasons were excluded from the study. Ultimately, 43 patients in the GS hip nail group (GS group) and 46 patients in the Affixus fracture nail group (Affixus group) met the minimum 6-month follow-up requirements .The demographic data of patients included age, gender, body mass index (BMI), bone mineral density (BMD), time to operation, length of hospital stay, preoperative hemoglobin levels, smoking status, American Society of Anesthesiologists (ASA) score, Koval classification, and AO/OTA classification.Patients were encouraged to use wheelchairs and initiate training to walk as soon as their general condition permitted. Depending on each patient’s general function and preference, dual crutches or a walker were allowed to support permissible weight-bearing.All surgeries were performed by a single surgeon, with patients placed in a supine position on a radiolucent fracture table under either general anesthesia or spinal anesthesia. Closed reduction was conducted using the C-arm device present in the operating room. After the traction of the fractured limb, a reduction was achieved by internal or external rotation and adduction or abduction, depending on the fracture pattern, followed by a confirmation of the reduction status with an image amplification device. In cases where anatomical reduction was not satisfactory for the anteroposterior (AP) and lateral view, percutaneous techniques were employed using long Kelly-–Rankin hemostatic forceps to assist in fragment reduction. In the AP view, efforts were made to achieve maximal anatomical alignment or a slight valgus alignment with positive medial apposition. In the lateral radiographic view , efforts were made to ensure a smooth anterior cortex. After achieving reduction, a guide pin was inserted just medial to the greater trochanter as an entry point. Following this, a reamer was used to create a canal in the proximal femur, and a metal sleeve was inserted. The guide pin for the lag screw was inserted to target the inferior at 1/3 of the head in the AP view, which was aimed centrally in the lateral view. Efforts were made to ensure that the length of the lag screw was 25 mm or less from the tip-apex distance (TAD). In all cases, a 125-degree cephalomedullary nail was inserted, taking into account the neck–shaft angle (NSA) of the femur. A set screw was then inserted in the proximal portion of the metal sleeve to enhance the stability of the lag screw fixation.First, we measured the operation time (minutes) and blood loss through intraoperative evaluation. To assess the patient’s overall tendency to bleed, we measured the difference between the preoperative hemoglobin levels and the hemoglobin levels on the first day after surgery (POD 1) (Difference = PostOP Hb − PreOP Hb), which was obtained through preoperative blood tests.postOP) (mm), which is the distance between the attachment of the lag screw and the cortical bone of the femoral head [postOP) and investigated the difference between these two values .For radiologic evaluation, AP and lateral view imaging was performed immediately post-operation, and reduction quality was assessed according to the classification by Chang et al. , categorral head . Additioral head . We meas6m) and the difference between this value and the immediate postoperative TAD (TAD difference = TAD6m − TADpostOP) were assessed. Additionally, the 6 month NSA (NSA6m) and the difference between this value and the immediate postoperative NSA (NSApostOP) were evaluated (NSA difference = NSA6m − NSApostOP).Patients were regularly followed up even after discharge , and at each visit, radiographic evaluations were performed. Bone union was confirmed when callus or cortical bone continuity was observed in at least three directions along the anterior, posterior, medial, and lateral cortex following the fracture line. The 6 months TAD  and sliding distance (mm) ,24. For All measurements obtained through radiographic examinations, whether in AP or lateral views, were corrected for magnification using the distal diameter of the nail as a reference.In the final follow-up of radiographic examinations, postoperative complications such as cut-out, cut through, back-out, non-union, delayed union, osteolysis, and implant breakage were investigated. Clinical outcomes were assessed using the modified Harris Hip Score (mHHS) ,26 at 6 ® 9.4 64-bit program . Continuous variables were presented as the mean ± standard deviation. To assess whether data followed a normal distribution, the Shapiro–Wilk test was utilized. If data satisfied the criteria for a normal distribution, a two-sample t-test was employed to compare the two groups. If data did not meet the assumptions of normality, Wilcoxon’s rank sum test was applied. Categorical variables were presented as frequencies and percentages. Pearson’s Chi-square test was used to analyze categorical data, and if the expected cell count was less than 5 and exceeded 20%, Fisher’s exact test was utilized. All tests were two-tailed with a significance level of 5%. Decimal places for all values were presented for up to two decimal points. p-values were reported for up to four decimal places, and if the p-value was less than 0.0001, it was indicated as <0.0001. Statistical significance was considered for p-values less than 0.05.The statistical analysis of these results was conducted using the SAS2) was 22.73 for the GS group and 23.97 for the Affixus group, and BMD averaged −3.03 and −2.73, respectively. The time to operation was 3.93 days for the GS group and 2.46 days for the Affixus group, although this difference was not statistically significant. Hospital stay was significantly shorter in the GS group at 13.42 days compared to 17.80 days in the Affixus group. Preoperative hemoglobin levels (g/dL) were similar between these two groups, measuring 12.08 and 12.05, with no statistical difference  were 73.02 and 73.20, respectively. Gender distribution in the GS group included 16 males and 27 females, while the Affixus group had 22 males and 24 females. The average BMI  post-operation was 23.97 and 25.22 for the GS and Affixus groups, respectively, but this difference was not statistically significant. In terms of the Cleveland index, the GS group had 1 case in zone 2, 29 cases in zone 5, 1 case in zone 6, 8 cases in zone 8, and 4 cases in zone 9. In the Affixus group, there was 1 case in zone 1, 1 case in zone 2, 4 cases in zone 4, 26 cases in zone 5, 3 cases in zone 6, 10 cases in zone 8, and 1 case in zone 9. However, this difference was also not statistically significant. The NSA on the contralateral side measured 123.06 for the GS group, which was statistically and significantly smaller compared to 132.25 for the Affixus group. However, the NSA on the operative side measured 130.39 and 130.82 for the GS and Affixus groups, respectively, with no statistically significant difference. As a result, the difference between these two values was 7.33 for the GS group and −1.44 for the Affixus group, indicating that the GS group achieved a greater valgus reduction  was 23.18 for the GS group and 22.94 for the Affixus group. The difference between immediate postoperative and 6-month postoperative examinations (TAD difference = TAD6m − TADpostOP) was −0.79 and −2.28 in the GS and Affixus groups, respectively, but this difference was not statistically significant. Regarding the NSA at 6 months (NSA6m), the GS group had a value of 120.31, whereas the Affixus group had 126.20, showing a statistically significant difference. The difference between the NSA both immediately post-operation and at 6 months (NSA difference = NSA6m − NSApostOP) was −10.08 for the GS group and −4.61 for the Affixus group, indicating a statistically significant varus change in the GS group compared to the Affixus group (At 6 months post-operation, the TAD (TADus group .In the case of advancement distance, the GS group had a value of 1.89, which was slightly less than the Affixus group at 2.05, but this difference was not statistically significant. Conversely, the sliding distance was higher in the GS group with a value of 3.87 compared to 3.73 in the Affixus group, but again, this difference did not reach statistical significance .In terms of complications, in the GS group, there were 38 cases with no complications, 3 cases of cut-out , 1 case In the Affixus group, there were 34 cases with no complications, 5 cases of cut-out , 1 case There was no statistically significant difference in the occurrence of complications between the two groups, and neither group experienced implant breakage .In terms of clinical outcomes, the GS group had an mHHS of 50.00, while the Affixus group had a higher mHHS of 54.98. In terms of EQ-5D-5L, the GS group had a score of 0.47, while the Affixus group had a higher score of 0.60. However, there was no statistically significant difference between these two groups for both mHHS and EQ-5D-5L scores .In terms of financial outcomes, the GS group had a higher implant cost of USD 611.78 compared to USD 590.53 in the Affixus group. However, the total hospital cost for the GS group was lower at USD 2471.92 compared to USD 2529.12 in the Affixus group, and these figures showed no statistically significant difference .Intertrochanteric fractures have their limitations in conservative treatment, and surgical intervention is the primary and often the only effective treatment method. While there has been considerable debate in the past, there has been a growing trend toward surgical treatment using intramedullary nailing in recent years . This isIn this study, there were no statistically significant differences between the two groups in demographic data, excluding hospital stay. These demographic factors included age, gender, BMI, BMD, time to operation, preoperative Hb level, smoking status, ASA score, Koval grade, and fracture classification. In a study conducted by Lefaivre et al. , it was The difference in postoperative Hb levels measured on the first day postoperatively and their respective preoperative Hb values did not show a statistically significant difference between the two groups. However, the operation time was significantly shorter in the GS group, with a mean of 43.26 min compared to 51.11 min in the Affixus group. It is noteworthy that the surgery was performed by a single surgeon, and even considering the insertion of the anti-rotation screw with the Affixus hip fracture nail, the GS group’s operation time was approximately 8 min shorter, which is a significant finding.In the postoperative radiologic evaluation, there was no significant difference between the two groups in terms of reduction quality, the TAD, and the Cleveland index. However, it was observed that the contralateral side’s NSA in the GS group had a significantly greater varus angle. Considering that the operative side’s NSA showed no significant difference between the groups immediately after the operation, it suggests that the GS group underwent a reduction at a significantly greater valgus angle compared to the native anatomy. This became more evident in the 6-month follow-up radiologic evaluation, where a pronounced difference was observed. The GS group, which had a greater valgus reduction, actually experienced a varus collapse of approximately 10 degrees, from 130.39 degrees to 120.31 degrees, after 6 months. By contrast, the Affixus group showed a lower varus collapse of about 4 degrees, decreasing from 130.82 degrees to 126.20 degrees during the same period. Many previous studies have reported that if varus collapse occurs postoperatively, it eventually leads to one of the most common and critical mechanical complications of intramedullary nailing, known as cut-out ,44,45,46At the 6-month follow-up radiologic evaluation, there was no significant difference between the two groups in terms of advancement distance and sliding distance. According to Shin et al. , a lag sAlthough there were slight differences favoring the Affixus group in clinical outcomes, as mentioned earlier, this can likely be attributed to the longer time to operation in the GS group. However, it is important to note that these differences did not reach statistical significance, so they may not be considered noteworthy results. In terms of implant cost, it was observed that the GS group incurred slightly higher costs compared to the Affixus group. On the other hand, from the perspective of the total hospital cost for patients, the GS group was found to be less expensive. This is likely due to the shorter hospital stay in the GS group, as mentioned earlier, and since no statistically significant differences were observed, these findings may not be considered particularly significant.This study has several limitations. First, it is a retrospective study, not a prospective one. While demographic data did not statistically differ between the two groups, patient matching was not performed. Additionally, as this study focused on fracture patients, native femoral NSA had to be inferred from the NSA of the contralateral side, introducing potential bias due to asymmetry between both femurs. It should be noted that this study only provides short-term outcomes, and long-term follow-up is necessary in the future. Furthermore, the sample size of patients was relatively small, warranting larger-scale research. However, despite these limitations, this study holds value as it is the first unique research conducted on the GS hip nail after its introduction into clinical practice. Moreover, it benefits from being a single-center study performed by a single surgeon, which adds strength to its findings.This study serves as an initial exploration into the clinical outcomes of the GS hip nail, highlighting its unique attributes. Notably, it demonstrates that the GS hip nail procedure, performed by a single surgeon, significantly reduces operation time compared to the Affixus hip fracture nail, indicating its efficiency. Moreover, both radiologic and clinical outcomes appear to be comparable when compared with the Affixus hip fracture nail. However, it is important to recognize that this study provides short-term outcomes, and future research should include long-term follow-up to fully evaluate the performance of the GS hip nail. Additionally, a larger patient study should be considered for a more comprehensive understanding of its effectiveness. This research lays the foundation for further investigations into the GS hip nail’s role in improving patient outcomes in the treatment of hip fractures.


The reduced hydrodynamics inside the mesocosms favored the deposition of organic matter and the sinking of cells from the water column towards the sediment. Consequently, MPB abundances increased over the course of both experiments and significant changes in the taxonomic composition of the community were recorded. Species richness increased in summer while it slightly decreased in autumn, this latter due to the increase in relative abundances of taxa favored by high organic loads and fine grain size. By coupling classical taxonomy with 18S rRNA gene metabarcoding we were able to obtain a comprehensive view of the whole community potential, highlighting the complementarity of these two approaches in ecological studies. Changes in the structure of MPB could affect sediment biostabilization, water turbidity and lagoon primary production.MOSE is a system of mobile gates engineered to temporarily isolate the Venice Lagoon from the Adriatic Sea and to protect the city from flooding during extreme high tides. Within the framework of the Venezia2021 program, we conducted two  According to [With the climate change effects intensifying, extreme events are becoming more and more frequent, severe, and unpredictable, and sea level is predicted to rise between +0.6 and +1.2 m within the end of this century . The Ven the sea ,6. MOSE rding to ,8, it isrding to , and itsrding to , the lenrding to , and therding to ,13 and wrding to . Moreoverding to , with serding to , the autThe microphytobenthos (MPB) constitute an important component of the benthic ecosystem. Indeed, this community contributes to the total production, plays a crucial role in the benthic food web and in sediment stabilization through the production of extracellular polymeric substances (EPS), and regulates nutrient fluxes and other biogeochemical processes at the sediment interface ,19,20,21In the last two decades, DNA metabarcoding has been more and more widely used in addition to traditional taxonomic studies, especially on microbial communities. Nowadays, the available sequencing techniques and databases are extremely advanced and constantly upgraded. Nevertheless, it is clear to taxonomists that classical taxonomy still cannot be fully replaced by molecular tools; instead, the two approaches are complementary and provide different but equally important information ,33,34.Assessing the impacts that new infrastructures might have on the ecosystems of interest is of pivotal importance in order to establish effective and sustainable protection strategies. Within the framework of the “Venezia2021’’ project, the present study was part of the enclosure experiments aimed at monitoring the MOSE impact on the Venice Lagoon ecosystem. A great effort was made in order to set up and run such challenging in situ experiments in an unstable and sensitive environment such as the Venice Lagoon. By simulating the MOSE closure, these experiments aimed at identifying structural and functional alterations in the planktonic and benthic populations. In this work, we focused on the effects of the altered hydrodynamics induced by the operational phase of the MOSE system on the MPB community. In particular, we investigated the modifications in MPB abundance and composition, the latter through both operator-driven classical microscopy  and NGS sequencing. This way, we were able to obtain a more comprehensive and detailed overview than would have been possible by using either approach alone, and to highlight pros and cons of both techniques.We hypothesized that the reduced hydrodynamics and oxygenation of the water column and sediment portions enclosed by the barriers induce modifications in the MPB total abundance and structure. Our guiding questions were: (1) Does the particular matter settling from the water column towards the sediment have a stimulatory effect, leading to an increase in MPB absolute abundances? (2) Do these modified hydrodynamic conditions affect the MPB structure, resulting in an increase of opportunistic species? Furthermore, to investigate the MPB community structure, we compared two methodologies, i.e., classical taxonomy and the more innovative 18S rRNA gene metabarcoding. Therefore, our third question was: (3) Are the higher resolution and efficiency of the molecular tools preferable over the time-consuming but strictly operator-driven classical microscopy?In this comparison, Cyanobacteria were not included since we applied primers to target only microalgae, but they were kept in the presentation of the taxonomic results because prokaryotic photosynthetic organisms are considered part of the MPB community .We expected to obtain a wider taxonomic characterization from the 18S rRNA gene sequencing, but to obtain a more reliable quantification of the living and photosynthetically active taxa of the MPB community from the classical taxonomy. Finally, we briefly discussed the ecological implications of the modified MPB community structure on sediment biostabilization and lagoon ecosystem functioning.2, a mean width of 15 km and an average depth of 1.5 m [The Venice Lagoon is located at the north of the Adriatic Sea A and is of 1.5 m . It is cof 1.5 m . The Venof 1.5 m .In this study, a shallow (<75 cm) non-anthropized area, Palude di Cona, with organic enrichment and low hydrodynamics, and subjected to hypoxic events, in the northernmost part of the basin, close to the bayhead estuaries of some plain rivers , was chosen for the experiments. This area is a typical brackish area, surrounded by salt marshes with an average depth of 0.8 m during the mean tidal conditions , and preThe mesocosm experiments were carried out in Palude di Cona, near the Dese River mouth A. The fi3 each) consisting of a cylindrical zinc-galvanized iron frame (∅ ca. 100 cm) coated by a transparent nylon cylinder (∅ ca. 106 cm)  D,E, were 106 cm) B,C. Sinc 106 cm) E. In thi 106 cm) F.At each sampling, water temperature, salinity and dissolved oxygen concentration were recorded by a multiparameter probe EXO2, YSI, Xylem Inc., USA, prior to sediment sampling, inside and outside the mesocosms.2, sample depth = 31 cm). At the first sampling time (T0), sediments were sampled outside the three replicate mesocosms, in close proximity to them. Then, the cylindrical nylon bags of the 18 mesocosms, initially lowered to allow water exchange, were pulled up, and the experiment began. At each following sampling time (T1–T4), sediments were collected from inside the three replicate mesocosms, one from each row, according to a chronology aimed at identifying any responses of the induced closure on the benthic microalgal community in the short term (after 4 h from the positioning of the structures = T1) and in the long term , starting from an initial state (T0). Once in the laboratory, the sediment cores were extruded and the top sediment layer (0–1 cm) was collected for chemical  and microbiological analyses (microphytobenthic abundance and community composition). The field experiment lasted 48 h in July 2019, and 28 h in October 2020 due to the high tide and the consequent actual activation of the MOSE in the second experimental day, which forced us to stop the second experiment in advance. In the first experiment, one of the three replicates at T4 was lost due to a logistic issue. Notwithstanding, each mesocosm enclosed a sediment area of 0.785 m2, quite a representative portion of the lagoon; therefore, two replicates were still depictive enough.Virtually undisturbed sediment samples were taken by a hand-operated sediment corer using polycarbonate sample tubes  collected from each mesocosm and sampling time, sieved at 2 mm and pretreated with 10% hydrogen peroxide before being analyzed with an LS 13 320 Laser Diffraction Particle Size Analyzer, Beckman Coulter, USA. Data are expressed as sand, silt, and clay percentages following the Udden-Wentworth grain-size classification .For TOC, TN and BPC analyses, sediment was freeze-dried, ground in a ceramic mortar and then sieved through a 250 μm iron steel sieve .For TOC and TN determination, triplicate subsamples of about 8–12 mg were weighed directly into silver and tin capsules using a microultrabalance with an accuracy of 0.1 μg. Before TOC analysis, subsamples were treated directly into capsules with increasing concentrations of HCl (0.1 N and 1 N) to remove the carbonate fraction . C and N2O and C-CHO-EDTA) were analyzed following the method described by [−1 for C-CHO, 0.50 g C g−1 for C-PRT and 0.75 g C g−1 for C-LIP, and the sum of carbohydrate, lipid and protein carbon was referred to as BPC sensu [Subsamples were processed in triplicates for the determination of carbohydrates, lipids and proteins. Colloidal and EDTA extractable carbohydrates  were withdrawn using a syringe and directly fixed with 10 mL of formaldehyde -buffered solution CaMg(CO3)2, in pre-filtered bottom seawater (0.2 μm filters). After manual stirring, 20 μL aliquots of the sediment suspension were drawn off from the slurries and placed into a counting chamber. Only cells containing pigments and not empty frustules were counted under a Leitz inverted light microscope  using a 32X or 40X objective  [In this study, by the term microphytobenthos (MPB), we refer to the microscopic eukaryotic algae , and prokaryotic photosynthetic organisms, such as filamentous cyanobacteria. For MPB analyses, from each mesocosm replicate, three aliquots of homogenized sediment (2 cmication) . For eacication)  or threeication)  and WoRMication)  websitesication) ,58,59,60ication) .For 18S rRNA gene metabarcoding analysis, sediment from each mesocosm replicate of T0 and T final (T4 for Summer 2019 and T3 for Autumn 2020) was sampled with a sterile spatula and stored at −80 °C until further processing.DNA was extracted from each sample using the DNeasy PowerSoil Pro Kit (Qiagen) following the manufacturer’s instructions and quantified with a Qubit Fluorimeter . The samples were then amplified with degenerated primers covering the V9 region of the 18S rRNA gene from position 1389 to 1510  . PCR mixThe pooled PCR products were then indexed and subsequently normalized according to the “16S Metagenomic Sequencing Library Preparation’’ protocol , with one main modification: the PCR amplicons were normalized using the SequalPrep Normalization Plate kit . Finally, amplicon libraries were equally pooled, purified using 0.7X volumes of AMPure XP beads  and sequenced in two separate runs (for 2019 and 2020 samples) of a MiSeq Illumina (2 × 300 bp). Primers were removed using cutadapt, v.2.1 . The anaNavicula rhombica, i.e., a tube-dwelling diatom whose abundance in samples might be random or often uncountable. An additional matrix (e) [An independent t-test was performed between the water temperature, salinity and dissolved oxygen concentration recorded inside the mesocosms at the beginning and at the end of the experiments. A preliminary Spearman’s correlation analysis was performed through Past 4  in orderl matrix  was constrix (e) ) and domtrix (e) ).Before multivariate analyses, the biotic matrices were square-root transformed and Bray–Curtis similarity matrices were generated. To visualize differences in taxa assemblages among the different sampling points, a non-metric multidimensional scaling ordination (nMDS)  was perfComparison among experimental times was complemented by a visual representation of diversity, using k-dominance curves : speciest-test: R = −2.81, p < 0.05).Prior to sediment sampling, water temperature, salinity and dissolved oxygen were recorded by a multiparameter probe, inside and outside the mesocosms  . No sign−3, observed in one replicate of T0, and 137,600 ± 4064 cells cm−3 obtained in one replicate of T1 (−3), while the highest was recorded at T4 .In July 2019, the total MPB abundance ranged from 39,000 ± 1742 cells cmte of T1 A. The avThe MPB community was dominated by diatoms, whose abundance varied between 82.07% at T2-R3 and 98.03% at T4-R1.−3 observed at T2-R1, and 252,600 ± 30,547 cells cm−3 at T3-R1. The lowest average abundance  of the whole experiment was observed at T1, with the highest  at T3  at T3 B.As of July 2019, the MPB community was dominated by diatoms, which represented a variable fraction between 87.14%, obtained at T2-R1, and 98.86% obtained at T2-R2. When diatoms were the least abundant (T2-R1), all other groups, except phytoflagellates, showed their Relative Abundance (RA) maxima.Tryblionella and Thalassiosira, with RAs of 39.6% and 17.6%, respectively. We identified three genera belonging to Cyanobacteria: Anabaena, Oscillatoria and Spirulina, with the first two being the most abundant at T0 (average RA = 0.7% and 0.6% respectively). We were able to identify only one genus of Chlorophyta, namely Scenedesmus, recorded at T0.Considering July 2019, within the Bacillariophyceae class, we identified 26 genera, among which the most abundant were Mastogloia, Gyrosigma and Surirella, with RAs of 20.26%, 16.40% and 11.72%, respectively. Three genera were identified within the Cyanobacteria: Anabaena, Oscillatoria and Merismopedia, although with relatively low mean abundances of 0.5%, 0.4% and 0.3%, respectively. In the Chlorophyta group, as in July 2019, only the genus Scenedesmus was identified, present solely at T2-R1 (average RA = 1.12%).In October 2020, we identified 25 genera within the Bacillariophyceae class, among which the most abundant were Tryblionella cf. acuminata  and equitability of the MPB community  was cuminata . The comMastogloia braunii , considering both the experiments, and consequently the lowest dominance .In October 2020, the most abundant diatom species (RA ≥ 0.5%) in all experimental time points was  braunii . The genTryblionella. On the contrary, at the beginning of the experiment, two replicates of T0 and one replicate of T1 showed the typical cut-off form of a community dominated by a few species  and the end of the experiment (T4). The taxa ncreased A.Surirella cf. robusta, G. fasciola, Nitzschia fasciculata, G. macrum and Mastogloia braunii. The LefSe analysis at T3 determined Surirella spp., G. macrum and G. fasciola together with Entomoneis sp. and Amphora spp. as the most discriminating taxa. In contrast, Tryblionella cf. apiculata, Mastogloia cf. acutiscula, Biddulphia spp., Entomoneis cf. alata and Gyrosigma cf. balticum displayed significantly higher abundances at T0 than at T3  and phytoflagellates. LEfSe detected 21 and 44 ASVs overrepresented at T0 compared to T4 and T3 in July 2019 and October 2020, respectively. On the other hand, 8 and 12 ASVs, respectively, were overrepresented at T4 and T3 compared with T0 of the two experiments  was described as a nutrient-loving species that thrives under high-organic matter conditions [Nitzschia panduriformis  was also reported to be part of a phosphate-loving assemblage [Psammodyction cf. constrictum and Tryblionella cf. hungarica, Nitzschia cf commutata was the third most discriminating species between the beginning and the end of the summer experiment. This species was previously reported to have some affinity for TOC-enriched sediments since it reached up to 27% of the MPB relative abundance in a harbor area [The microalgal biodiversity inside the mesocosms varied during the experiment. Interestingly, in summer, from an initial state dominated by a few species, the community species richness increased , particuodictyon . Nitzschnditions . This spnditions . Similarsemblage . Furtherbor area .Paralia sulcata, greatly reduced their abundance during the experiment. This is a tychopelagic diatom  that lives loosely associated with sediments [In contrast, other taxa, particularly ediments  and usuaediments . NeverthGyrosigma, namely G. macrum and G. fasciola , testifying that the induced lower hydrodynamics favored the reproduction of this taxon.From the LEfSe analysis, In the sediments, together with typically benthic taxa, we detected some planktonic forms. This was observed especially in July 2019 and was due to cells sinking from the water column towards the sediments following the reduced hydrodynamics. This result was also an anticipated consequence derived from the reduced horizontal water fluxes in the mesocosms, similarly to what we could expect in case of MOSE closure. From both the indices and the LEfSe analysis, an overall loss of richness (d) and diversity (H’) in the sediments was evident, from the beginning toward the end of both the experiments, with many taxa disappearing or becoming significantly less abundant , and few others appearing or becoming significantly more abundant .It is interesting to note that the taxa whose abundance significantly increased at T3 in October 2020 belonged almost entirely to the Naviculaceae family, which is known for its wide ecological valence. However, under various types of stress , tolerant taxa take over, and become dominant components of the MPB community ,80,81.Thalassiosira was identified by both techniques due to the size and the state of the still-intact cells in the surface sediments. In contrast, other genera, such as Tryblionella and Psammodictyon, were not detected through metabarcoding, almost certainly due to the fact that these genera are still considered part of the large genus Nitzschia  in the used database. Similarly, the very abundant genus Mastogloia was likely listed as undetermined Pennales because the databases are not yet implemented with many pennate benthic diatom forms. In fact, it is possible that the benthic organisms are less characterized from a genomic point of view than the planktonic ones. Nevertheless, as a rule, the 18S sequencing was able to reach a much higher resolution within samples compared to classical taxonomy, and to detect many taxa non-detectable through microscopy. For instance, some diatoms were probably not identified by classical taxonomy due to their small size, e.g., the genus Minutocellus that belongs to the picophytoplankton (size < 3 um) [Plagiostriata [Chaetoceros. However, since 18S rRNA gene metabarcoding can also quantify DNA residues from dead organisms, e.g., it cannot discriminate between living photosynthetically active cells and dead organism/DNA residues, it leads to an overestimation of community biodiversity. In general, molecular tools may provide a more accurate picture of what is present in the community, but in some cases, and especially for larger cells such as diatoms, the classical taxonomy is more informative from a quantitative point of view, i.e., much more reliable in terms of the absolute abundances used in ecological studies. Moreover, we observed that databases on microalgae from surface sediments obtained from metabarcoding are still poorly implemented and often give an assignment only at a higher hierarchical level .As already evidenced in previous studies ,33,34, b < 3 um) , or the ostriata . FurtherThese results confirmed that both these techniques provide important but complementary information that should be integrated in studies aimed at investigating natural communities from both taxonomic and functional perspectives. In summary, molecular tools based on total DNA provide a picture of everything present in the community, dead or alive, while classical taxonomy can quantify what is alive at a given moment, which is of utmost importance in ecological studies.Navicula, Nitzschia, Amphora and Thalassiosira, among others, are known to produce EPS, whereas Paralia is not an EPS-producer   and are roducer ( and refeall ones . Recentlbilizer” . Therefobilizer”  and overTo the best of our knowledge, this is the first study that investigated the effect of a storm-surge infrastructure by simulating in situ its impacts on biological communities through mesocosm experiments, by employing both classical taxonomy and molecular tools.The MOSE is a monumental engineering project with enormous resources devoted to its realization. Its utility in attempting to protect Venice City from flooding deserves to be recognized. Nevertheless, Venice, its economy and its biological communities cannot be preserved unless the health and functioning of its lagoon is also preserved.


The 95% and 63% Bivariate Contour Ellipse Area (BCEA), axial length, Foveal Avascular Zone (FAZ) area, center macular thickness and volume were assessed. The relationship between these parameters was explored. (3) Results: Statistically significant differences were found among the four groups in best corrected distance visual acuity (BCVA) (p < 0.001), BCEA 95% (p = 0.002) and BCEA 63% (p = 0.002), but not in the FAZ area, central macular thickness, central macular volume and axial length. Eyes with amblyopia had poorer BCVA and larger fixation instability than controls. Inter-ocular differences were more significant in patients with strabismic amblyopia, particularly in BCVA (p = 0.003), central macular thickness (p < 0.001) and central macular volume (p = 0.002). In amblyopic eyes, BCEA 95% and 63% were correlated with BCVA, but not with the FAZ area. (4) Conclusion: Amblyopia is associated with a reduction in fixation stability and BCVA, although there is a general lack of correlation with structural changes, suggesting a complex interaction between anatomy and function in amblyopia.(1) Background: Amblyopia is an ocular condition leading to structural and functional changes. The relationship between these changes is complex and remains poorly understood. (2) Methods: Participants included 31 children aged 5 to 9 years with strabismic ( Amblyopia is a neurodevelopmental disorder that results from an abnormal visual experience during a critical period of visual development ,2. Risk Current developments in microperimetry have allowed researchers to explore fixation stability in amblyopia ,19,20,21Previous researchers have explored the association between retinal structural parameters, such as the FAZ area, and visual function parameters, including visual acuity, visual fields and fixation stability, in retinal vein occlusion, diabetic retinopathy and other pathologies ,27,28. HThus, it was the aim of the present study to assess the possible correlation between fixation stability, defined by BCEA, and structural parameters, such as the FAZ area, in a sample of children with unilateral strabismic, anisometropic and mixed amblyopia. Additional study variables were axial length (AL), best corrected distance visual acuity (BCVA) and stereoacuity, as well as central macular thickness and volume. Results from the amblyopic eyes were compared with those of the contralateral eye, and with those of a non-amblyopic control group.A cross-sectional study was conducted at the Institut Català de la Retina  from September 2021 to May 2022. The study was approved by the Ethics Committee of the Grupo Hospitalario Quirón Salud, (ICR-13/21-2022/10-OFT-ICR) and was compliant with the principles of the Declaration of Helsinki. Informed consent was obtained from legal guardians after a detailed explanation of the nature and possible consequences of the study.Children aged 5 to 9 years were recruited for this investigation. Participants in the study had either strabismic, anisometropic or mixed unilateral amblyopia. Amblyopia was defined as BCVA of ≥0.2 logMAR for the worse eye and an interocular difference of ≥0.15 logMAR. In addition, for anisometropic amblyopia, interocular differences in refractive error needed to be of 2.00 diopters (D) or more in sphere and/or 1.50 D in cylinder. No anisohyperopic participants were included in the study. In addition, strabismic amblyopia required a minimum angle of manifest deviation of 5 prism diopters. Patients diagnosed with neurological pathologies, retinopathies and/or maculopathies, glaucoma, nystagmus, media opacities, systemic diseases, cardiovascular or renal diseases, retardation and/or prematurity were excluded from the study, as were those with a manifest deviation of 30 prism diopters or more. In addition, a control age and gender-matched group of healthy non-amblyopic subjects was included.Monocular BCVA was evaluated with the Early Treatment Diabetic Retinopathy Study (ETDRS) test at 4 m and recorded letter by letter in logMAR notation. Stereoacuity was evaluated with the TNO stereo test, with the aid of red–green filters (a score of 800 arc seconds was assigned to patients failing to identify any of the pictures). For both BCVA and stereoacuity, patients received instructions on the procedure and an initial trial was conducted to ensure they fully understood what was expected of them. For this trial, the largest letter of the ETDRS test was used for BCVA measurements, and the butterfly plate of the TNO test for stereoacuity, which corresponds to 1500 arc seconds. Exactly the same procedure was employed for all patients and all measurements were conducted by an examiner unaware of the patient group allocation, although in patients with strabismic amblyopia this single-blind experimental condition could not be maintained. All patients included in the study were able to follow the BCVA and stereoacuity measurement procedures without difficulties.Refractive error was measured using the retinoscopy technique, with and without cycloplegia. Visual alignment was measured with the cover test in both near and far vision, and the angle of deviation of strabismic patients was determined with the cover test and a prismatic bar, both in conditions of best corrected visual acuity.Fixation stability was measured using the Macular Analyzer Integrity Assessment (MAIA) microperimeter  and followed the BCEA methodology described by Crossland and co-workers, with a fixation strategy of 30 s . Briefly2, as previously described  was measured with the OCT-A Spectralis  with angiography software. To determine the FAZ area, a scan pattern of 10 × 10 degrees (consisting of 512 scanned sections separated by 6 µm) was centered on the fovea. Internal fixation was used to ensure proper alignment of the eye. It may be noted that in certain patients with large fixation instability, internal fixation may be eccentric instead of central. However, if the fixation moved outside of the initial internal fixation zone, the OCT stopped the test until the patient correctly fixed the stimulus again. Each scan was automatically segmented. Finally, the FAZ area was calculated manually employing the tools provided by the Spectralis software: the boundaries of the FAZ area were delineated by the freehand tool and calculated in mmescribed  35]. Ax. Ax2, asMacular thickness and volume were measured using the OCT-A Spectralis. A macular cube scan pattern of 20 × 15 degrees  centered on the fovea was acquired. Internal fixation was used for proper alignment of the eye. Each scan was automatically segmented and an optometrist examined all macular cubes to ensure that the analyzed macular thickness and volume corresponded to the foveal center.10 values. The Shapiro–Wilk test was employed to explore data normality, whereupon results are accordingly described as mean and standard deviation (SD), or median and range. Inferential analysis was conducted with the ANOVA or Kruskal–Wallis tests, with the corresponding post-hoc Bonferroni or Dunn–Bonferroni pair-wise analysis and correction to account for multiple comparisons, or with the paired Student’s t-test or Wilcoxon tests. In addition, possible associations between variables were analyzed with the Spearman coefficient of correlation test, and the Chi-Squared test was employed for frequency distribution analysis. A p-value less than 0.05 was considered to denote statistical significance.SPSS version 27  for Windows was used for data analysis. Values of BCEA were transformed to their corresponding log2 and a minimum expected difference between amblyopic and fellow eyes of 0.36 log deg2, and considering a 95% statistical power. The required minimum sample size was 18 participants per group for pair-wise comparisons.Sample size calculation was performed using G*Power version 3.1.9.2 software . The study of Subramanian and co-workers  was usedn = 9), anisometropic (n = 16) and mixed (n = 6) amblyopia. The control group included 14 non-amblyopic children , aged 6 to 9 years (7.6 ± 1.0 years). No statistically significant difference was found in neither age nor sex distribution between the three amblyopic and control groups (p > 0.05).A total of 31 children were included in the amblyopic group  aged 5 to 9 years (mean ± SD of 6.6 ± 1.1 years), with strabismic (p < 0.001), stereoacuity (p < 0.001), BCEA 95% (p = 0.002) and BCEA 63% (p = 0.002), but not in the FAZ area, central macular thickness, central macular volume and axial length . A pair-wise analysis with the post-hoc Dunn–Bonferroni test revealed statistically significant differences in BCVA and stereoacuity between control and strabismic, anisometric and mixed amblyopic groups , with superior values of both visual function parameters in the control group. Regarding BCEA 95% and BCEA 63%, the Dunn–Bonferroni or Bonferroni tests revealed statistically significant differences between control eyes and strabismic and anisometropic eyes , but not between mixed amblyopic and control eyes. Strabismic eyes tended to have the largest BCEA 95% and BCEA 63% results. By pooling the data from all amblyopic groups, statistically significant differences were found between BCEA 95% and BCEA 63% (p < 0.001).For the analysis, first eyes with different types of amblyopia were compared amongst them and with one of the eyes of subjects from the control group. p = 0.003; central macular thickness, p < 0.001; central macular volume, p = 0.002). No statistically significant interocular difference was found in control subjects.For the second part of the analysis, inter-ocular differences between the amblyopic eye (or non-dominant eye in controls) and the dominant eyes were calculated for amblyopic patients and control subjects. p = 0.007, p = 0.043, p = 0.026 and p < 0.001, respectively). Post-hoc pair-wise analysis revealed statistically significant inter-ocular differences in BCVA between the anisometropic and control groups (p = 0.007) and between the mixed and control groups (p = 0.018); in BCEA 95% between anisometropic and control groups (p = 0.033); in BCEA 63% between the strabismic and control groups (p = 0.042), and between the anisometropic and control groups (p = 0.045); and in central macular thickness between all three amblyopic groups and the control group .Inter-ocular differences amongst groups in BCVA, BCEA 95%, BCEA 63% and central macular thickness were statistically significant (p < 0.001) and BCEA 63% . BCEA 95% and 63% showed a very strong correlation . Statistically significant moderate to strong correlations were found in the non-dominant eyes of the control group between the FAZ area and both central macular thickness  and central macular volume . However, in the amblyopic groups, only a weak correlation was evidenced between the FAZ area and central macular thickness . Upon examining each amblyopic group independently, the only significant correlation was between BCVA and central macular thickness in the strabismic group , as well as between both BCEA outcomes. No statistically significant correlation was evidenced in any group between BCEA 95% or BCEA 63% and the FAZ area. In particular, in strabismic and mixed amblyopia, there was no correlation between angle of deviation and fixation stability. In addition, inter-ocular differences in BVCA in amblyopic eyes were weakly correlated with inter-ocular differences in BCEA 95%  and BCEA 63%  and moderately correlated with inter-ocular differences in central macular thickness .By pooling the data from all amblyopic groups, and considering only the amblyopic eyes, statistically significant moderate correlations were found between BCVA and both BCEA 95% , as well as AL, in children with different types of amblyopia and in normal controls. The relationship between structural and functional parameters was investigated.Fixation stability, determined with the BCEA 95% and 63% was found to be particularly compromised in strabismic and, to a less extend, in anisometropic amblyopia, as compared with control eyes. No difference between control eyes and those with mixed amblyopia was found, although the reduced sample size for this type of amblyopia led to a slightly underpowered analysis, thus increasing the probability of type I error. These findings are in agreement with previous reports ,19. For To determine fixation stability, a fixation strategy of 30 s was implemented, which is the current minimum available interval for the MAIA microperimeter to provide reliable measurements. Given the age of some of the participants, they needed encouragement to maintain fixation on the target stimulus . Previous researchers have employed longer fixation strategies, of 45 s or even 1 min intervals . It mustNo statistically significant differences were found in the FAZ area between amblyopic and control eyes, albeit a trend was observed in which anisometropic and mixed amblyopic eyes had larger FAZ areas than strabismic and control eyes. These findings would be in agreement with previous literature documenting larger FAZ areas in the SCP layer in anisometropic amblyopia ,30, albeUpon examining inter-ocular differences in functional and structural parameters, that is, amblyopic eye versus the contralateral eye, or both eyes of the control group, statistically significant differences were predominant in patients with anisometropic amblyopia, particularly in BCVA, central macular thickness and central macular volume. Inter-ocular differences in BCEA 95% and BCEA 63% were larger in strabismic and anisometropic patients than in the control group. Similarly, inter-ocular differences in central macular thickness were more noticeable in the three amblyopic groups than in the control group. These findings suggest that the study of inter-ocular asymmetry in functional and anatomical parameters may be valuable.Correlation analysis revealed several statistically significant, moderate correlations among some functional parameters, as well as among several anatomical parameters, but no correlation was disclosed between functional and anatomical parameters, with the exception of BCVA and central macular thickness in the strabismic group. Thus, for instance, whereas in amblyopia both BCEA values were moderately correlated with BCVA, BCEA and the FAZ area did not display any significant correlation, which may suggest that the FAZ area does not influence fixation stability, although further research is needed to confirm this hypothesis. Indeed, the lack of correlation between fixation stability and the FAZ area has been previously reported for other retinal pathologies ,27,28, bThis study had some limitations, mainly a reduced sample size and slightly underpowered analysis, considering a required estimated sample size of 18 per group. Unfortunately, these patients are relatively young and sometimes found it difficult to cooperate with the measurements and could not be included in the study. In cases of wide-angle strabismus, certain tests could not be performed due to saccadic re-fixational eye movements that prevented correct captures for scans. In addition, the manual nature of some of these measurements may contribute to reducing the reproducibility of some of the findings. Thus, FAZ area measurements were performed manually, following the border of the vascular and the avascular textures shown in the OCT-A image, as previously described . There aIn addition, although the TNO test has been documented to be adequate to measure stereoacuity in children aged 3 to 6 years old , there aIn conclusion, children with amblyopia have more fixation instability compared to normal controls. Although BCEA 95% and 63% show a strong correlation, these values are not interchangeable. Fixation instability is associated with reduced visual acuity, but not with the FAZ area, suggesting a complex interaction between structural and functional changes in amblyopia, which supports further investigation.