Fat-free mass and all forms of PA remained unchanged from baseline to 12 months. Hence, there were indications of improvement among completers on psychological variables, lifestyle behaviors, and physical health indicators. The low rate of completion was a concern, and the unchanged levels of PA reflect an important area of focus for future interventions in the context of HLCs.Healthy Life Centers (HLCs) have been established throughout Norway to support lifestyle changes and promote physical and mental health. We conducted a 12-month observational study among participants in an HLC that aimed to improve physical activity (PA) and dietary behaviors, and this study examined predictors of completion, and changes in psychological variables, lifestyle behaviors, and physical health indicators. The participants  reported symptoms of psychological distress (77%) and were obese (77%). No baseline characteristics were found to be consistent predictors of completion (42%). Completers had significant improvements in autonomous motivation for PA ( Moreo2 ,4 and un2 ,7, exerc2  and anxi2 . The WHO2 . Regardi2 , which i2 . Adoptin2 ,14,15, b2 ,17,18 an2 . This stespecially among at-risk populations [and diet, and large effect sizes from interventions that target PA and diet with weight loss as the outcome [Research in the context of HLCs is sparse, and it has yielded mixed findings amid low rates of completion  is a macro-theory of human motivation that examines the factors that are conducive to the initiation ver time . Accordiver time ,39,40,41ver time ,42. Recever time . One stuver time , suggestThe services offered by HLCs have the potential to provide support for participants’ basic psychological needs by providing choices about various activities (support for autonomy) from a menu of customized options (support for competence) that are delivered to individuals and/or groups by employees who are experienced in lifestyle counselling (support for relatedness) . SupportAccordingly, the research questions that guided the current study were three-fold. First, what percentage of participants will complete the HLC services at 6 months and 12 months, and will baseline characteristics such as gender, age, ethnicity, education, work status, diagnosis, BMI, psychological distress, autonomous motivation, and perceived competence predict this completion? Second, will autonomous motivation, perceived competence, and psychological distress increase from baseline to 6 months and 12 months? Third, will lifestyle behaviors  and physical health indicators  improve from baseline to 6 months and 12 months?We conducted a 12-month observational study among participants in an HLC intervention at a site that is in one of the largest cities in Norway. The intervention occurred in a real-world setting and was part of the HLC’s standard program; a control group was not established. Measures were obtained at baseline, 6 months, and 12 months. Our goal was to determine if participants will report changes in psychological variables, lifestyle behaviors, and physical health indicators over time, which may lay important groundwork for more rigorous experimental research in the future.The HLC services were designed to be individually customized, and participants could choose activities according to what they perceived as necessary to support their process of lifestyle change. All participants received individual guidance, which represented the “lowest level” of intervention, and reflections on activity choice, goal setting, and goal striving were important themes in these consultations. The activities in which participants could engage during the 12-month study period included lifestyle courses (13 weekly sessions), food classes (5 weekly sessions), yoga , and discussion groups for exchanging experiences (8 weekly sessions); walking and training groups were offered continuously. All activities and guidance were provided by educated, experienced, and competent HLC employees. By design, the HLC was intended to be supportive of participants’ basic psychological needs, as each participant was given the opportunity to develop a personally tailored process of lifestyle change (supportive of autonomy and competence) in a caring social context (supportive of relatedness).This study was approved by the Norwegian Data Protection Authority and the Data Protection Officer in the municipality where the HLC is located. All participants provided written, informed consent after receiving information about the purpose and procedures of the study, along with information about how their data would be handled in a secure way.All individuals who consulted the HLC during a 20-month period, who met the inclusion criteria, and who agreed to participate were included in the study. Inclusion criteria were being ≥18 years of age  with the ability to speak Norwegian and with the intention to become more active and eat healthier . Participants who reported severe and disabling mental illness  were excluded. Declining to participate in the study did not affect the services that were offered by the HLC.Participants who did not attend their scheduled appointments at the HLC were contacted by phone (and then by letter) with an invitation to make a new appointment, after which non-responsive participants were registered as dropouts. A total of 66 (55%) participants provided data at 6 months, and 50 (42%) participants provided data at 12 months.All assessments at baseline, 6 months, and 12 months were undertaken at the HLC, and participants completed the self-report measures via an online questionnaire that was aided by SurveyXact . All measures had satisfactory reliability (α ≥ .73). Accelerometers were worn by participants during their daily activities outside the HLC.The Health Care Climate Questionnaire [not true at all) to 7 (completely true).ionnaire  was usedThe Treatment Self-Regulation Questionnaire [not true at all) to 7 (completely true).ionnaire  was usedThe Perceived Competence Scale [not true at all) to 7 (completely true).ce Scale  was usedThe Hopkins Symptom Checklist-10 [not at all) to 2 (a little) to 3 (quite a bit) to 4 (extremely). In the Norwegian version of this measure, a mean value > 1.85 indicates the presence of psychological distress [klist-10  was useddistress ,52.Inbody 720  was used to calculate BMI and to measure body composition. Inbody 720 uses direct segmental multifrequency bioelectrical impedance (BIA) for measurements of body components. Assessments were conducted at the HLC by trained personnel, and manufacturer instructions were followed before and during the assessments. Height was measured by a stadiometer and plotted in the Inbody 720 analyzer prior to the assessments. Inbody 720 has been shown to be valid for measuring body composition in general populations and among obese participants [2, healthy weight = 18.5–24.9 kg/m2, overweight = 25.0–29.9 kg/m2, and obese = ≥30.0 kg/m2. The obese category is further classified as follows: obese class 1 = 30.0–34.9 kg/m2, obese class 2 = 35.0–39.9 kg/m2, and obese class 3 = ≥40.0 kg/m2 [An icipants ,54. BMI,.0 kg/m2 ,56, baseThe ActiGraph GT3X  [FL, USA) ,58 accelFL, USA) , we defiThe 30 s chair-stand test was used to measure lower body strength. This test has stable reliability and moderately high correlation with maximum weight-adjusted leg-press performance [formance . AlthougFruit and vegetable intakes were assessed by asking participants how many fruits and vegetables they typically ate every day. Previous research has demonstrated the utility of this approach for monitoring dietary preventive efforts [ efforts .d > 0.45 for independent t-tests, d > 0.26 for dependent t-tests, and a difference in percentages of about 25% with groups of equal size). With a sample size of 60, a dependent t-test would identify change yielding a d = 0.40 (α = .05 and 1 − β = .80) [We conducted power calculations prior to the start of the study, in which a dropout rate of 50% was anticipated based on a combination of clinical experience and previous research ,62. A saβ = .80) ,64.t-tests to examine baseline differences in the continuous variables  and cross tabulation with chi-square tests for independence to examine baseline differences in the categorical variables  between participants who left the HLC before 6 months or 12 months (dropouts) and those who completed activities in the HLC at 6 months and 12 months (completers). Education was dichotomized as low education (primary school and high school) and high education . Work status was dichotomized as able to work  and unable to work due to health (sick leave and disabled). For our second and third research questions, we used paired-samples t-tests to examine changes in psychological variables , lifestyle behaviors , and physical health indicators  from baseline to 6 months and 12 months. Changes in BMI and body composition were analyzed among all participants, among participants with BMI ≥ 25.0, and among participants with BMI ≥ 30.0. Unless otherwise stated, the results are presented as mean (SD). Effect sizes (d) were defined as follows: small = 0.2, medium = 0.5, large = 0.8, and very large = 1.3 [phi) were defined as follows: small = 0.1, medium = 0.3, and large = 0.5 [All analyses were conducted using IBM SPSS Statistics 24 . After ensuring that the data did not violate statistical assumptions, we used means (with standard deviations) and frequencies (with percentages) to describe the continuous and categorical variables, respectively. For our first research question, we used independent-samples ge = 1.3 . Effect ge = 0.5 .A total of 120 individuals  were included and provided data at baseline. As shown in Of the 120 participants who were recruited at baseline, 66 participants (55%) completed activities in the HLC at 6 months and 50 participants (42%) completed activities in the HLC at 12 months. t-tests revealed no significant baseline differences in age, BMI, psychological distress, autonomous motivation, or perceived competence between completers and dropouts. However, the following variables yielded differences that had an effect size ≥ small: ethnicity, work status, diagnosis, and autonomous motivation for diet.Chi-square tests for independence revealed no significant differences in gender, education, ethnicity, work status, or diagnosis between completers and dropouts. Independent-samples Completers reported high average levels of autonomy support from counsellors at the HLC regarding PA at 6 months (6.0 (0.9)) and at 12 months (5.9 (1.0)), and regarding diet at 6 months (5.9 (1.0)) and at 12 months (5.8 (1.0)) .Regarding psychological variables, there were significant increases in autonomous motivation for PA, perceived competence for PA, and perceived competence for diet. A significant decrease in psychological distress was also found. The effect size for these changes ranged from medium to very large. There was no change in autonomous motivation for diet.Regarding lifestyle behaviors, there was a significant decrease in sedentary time at 6 months, which was not found at 12 months. Significant increases in fruit and vegetable intakes were also found at 12 months. The effect size for these changes ranged from small to medium. There was no change in LPA or MVPA.Regarding physical health indicators, there were significant decreases in BMI and body fat percentage among all participants, among participants with BMI ≥ 25.0, and among participants with BMI ≥ 30.0. There was also a significant increase in lower body strength. The effect size for these changes ranged from small to large. Fat-free mass remained unchanged.This observational study followed participants in an HLC as they strived toward lifestyle change over 12 months. Results revealed that a concerningly high percentage of participants dropped out of the HLC, as only 42% of participants completed HLC services at 12 months. However, completers showed indications of improvements in psychological variables, lifestyle behaviors, and physical health indicators. Levels of PA did not change. We reflect on these findings below.Across the 12-month study period, 55% of the participants completed activities in the HLC at 6 months, whereas 42% of the participants completed activities in the HLC at 12 months. This may suggest that roughly half of the participants did not succeed at their lifestyle changes, and most participants who dropped out did so without giving notice or reason, and they did not respond to contact attempts from the HLC. There were no predictors of completion, although we encourage caution in the interpretation of these findings due to low statistical power. There were small differences in the variables ethnicity, work status, and diagnosis, and these may have been significant predictors of completion in a larger sample. It is useful to note that low rates of completion have been reported previously with HLCs, although our inability to predict completion stands in some contrast to previous research that has shown that older age and no mental disease are common predictors of completion ,21,23. MThere can be many reasons for dropping out of HLC services, including a lack of time and/or energy to pursue lifestyle changes and frustration from a lack of progress towards one’s goals. Another factor affecting completion may also be the level of positive experiences from attending the HLC. Additionally, the level of psychological distress at baseline was quite high , which cThe current study investigated perceptions of autonomy support and changes in the psychological variables of autonomous motivation, perceived competence, and psychological distress. The HLCs are designed to provide support for participants’ basic psychological needs for autonomy, competence, and relatedness . CompletFinally, the current study examined changes in lifestyle behaviors  and physical health indicators . Although autonomous motivation and perceived competence for PA increased over time, PA levels did not change during the 12-month study period. This may have been due to the high levels of MVPA at baseline. The WHO recommends at least 150 min of such PA per week  , and our findings are contrary to the large effect sizes that were found among interventions that target weight loss. The small decreases in BMI and body fat percentage might be due to complexities around eating regulation and pathological eating [There was a small, although significant, decrease in BMI and body fat percentage, which, coupled with the increase in fruit and vegetable intake, may indicate an overall improvement in the consumption of a healthy diet. Fat-free mass did not change, which suggests that the decreases in BMI were a function of reductions in fat mass. The effect sizes for changes in BMI and body fat percentage were larger among overweight and obese participants. Although participants were not recruited into a weight-reduction intervention, a majority of participants were obese and likely had intentions to reduce and/or stabilize their weight. From this perspective, a small effect size for reductions in BMI and body fat percentage over one year is sobering, although there may be some clinical relevance to these findings given that average BMI among obese participants decreased from 36.1 (obese class 2) to 34.5 (obese class 1). Indeed, relative to obese class 2, both men and women in obese class 1 have an increased life expectancy of more than two years  systematl eating . Qualital eating . Though The intent of an HLC is to empower individuals to move towards sustainably healthier lives, and it seems that the HLC that was examined in the current study was partly successful in doing so. The health services were perceived as need supportive, and completers reported improvements in the psychological variables of autonomous motivation, perceived competence, and psychological distress. The findings with regard to lifestyle behaviors were less clear as fruit and vegetable intakes increased over time but PA did not, which is inconsistent with previous research from SDT ,38 but cThis study has several limitations. First, without an experimental design and a control group, we cannot conclude that a causal association exists between participation in the HLC and the changes that were observed. It is important for future research not only to test for causality, but also to examine directionality between changes in psychological variables, lifestyle behaviors, and physical health indicators over time in the context of an HLC. Nor was it possible to “tease apart” various components of the intervention. Indeed, we simply observed changes over time, which may be attributed to factors that are also unrelated to the HLC. Second, participants were recruited from one HLC only, which limits the generalizability of our findings. Nonetheless, sample characteristics were similar to those of other recent HLC studies ,21, and The findings suggest that the activities provided by HLCs are potentially beneficial in some ways for those who complete services at 12 months since autonomous motivation and perceived competence increased, and psychological distress decreased. These improvements in psychological constructs can indicate an improved foundation for continuing the strive towards a healthy lifestyle. Findings for lifestyle behaviors were less consistent. Completers reported meaningful improvements in diet, but their levels of PA remained unchanged. Although rather high levels of MVPA were maintained, greater effort may have been made to reduce sedentary time and increase levels of LPA. Moreover, physical health indicators, namely, BMI, body composition, and lower body strength, tended to improve. An issue of concern was the high rate of dropout, which could have biased the results in a positive direction. More knowledge is needed about how the service could be adapted or strengthened to encourage a higher level of retention by investigating reasons for dropout Indeed, attrition might be attenuated by a focus on mental health issues in the context of the intervention. As support for the basic psychological needs is likely to be important in this process, it is critical to assess perceptions of need support more systematically and earlier in the intervention.


Obesity is a growing public health problem worldwide. It is a pathological state that degrades the proper functioning of skeletal muscle. Diazoxide treatment and exercise have been shown to generally improve muscle function. However, the effect that each of the different exercise intensities has when combined with diazoxide on the contraction capacity, resistance to fatigue and oxidative stress levels in rat skeletal muscle is unknown. Therefore, this work focused on analyzing which exercise intensity was more efficient in combination with diazoxide in improving muscle tissue and its metabolic capacities. The best results were obtained with low- and moderate-intensity exercise when combined with the drug. These results expected to open a window of time that allows the implementation of a constant and prolonged exercise protocol that completely reverses the harmful effects of obesity on muscle tissue and obesity itself.Obesity is a chronic disease that impairs skeletal muscle function, affects the ability to contract, and promotes the development of fatigue. For this reason, the study of treatments that seek to reduce the harmful effects of obesity on muscle tissue has been deepened. Diazoxide treatment and various exercise protocols have been proposed to protect skeletal muscle against oxidative stress and its effects. However, the intensity and duration of exercise combined with diazoxide that would obtain the best results for improving skeletal muscle function in obese rats is unknown. To this end, this study evaluated the effects of three different exercise intensities combined with diazoxide on contraction capacity, resistance to fatigue, markers of oxidative stress, lipid peroxidation, ROS, and glutathione redox status of skeletal muscle. The results showed that treatments with diazoxide and exercise at different intensities improved muscle contraction capacity by reducing oxidative stress during obesity, with the best results being obtained with low-intensity exercise in combination with diazoxide. Therefore, these results suggest that diazoxide and low-intensity exercise improve muscle function during obesity by decreasing oxidative stress with the same efficiency as a moderate-intensity exercise protocol. Obesity is a chronic disease triggered by multiple factors, including disordered eating, lack of physical activity and sedentary lifestyle, or metabolic, hormonal, or genetic alterations ,2. It ocThe increase in body fat during obesity is directly associated with the appearance of numerous metabolic and physiological processes, such as the modification in fiber type from slow to fast, a decrease in the number of fibers and their diameter; an accumulation of fat between the fibers, trigger insulin resistance, and establish a state of chronic inflammation ,6,7, eveThe increase in oxidative stress manifests itself in a very significant way in skeletal muscle during obesity, with the excessive increase in ROS levels, lipid disorders, and deregulation of antioxidant defense, which is directly reflected in the deterioration of muscle function ,11.ATP), inhibits insulin production and regulates the ability of skeletal muscle to take up glucose; it is a drug used in the treatment of dyslipidemia and obesity [In order to combat the harmful effects that obesity has on muscle tissue, pharmacological and non-pharmacological treatments have been studied in depth. For this purpose, treatment with diazoxide, a drug derived from benzothiadiazines, has been proposed . Diazoxi obesity  since it obesity , improve obesity . Studies obesity , as well obesity ,15. HoweIn the case of non-pharmacological treatments for metabolic alterations associated with obesity, various exercise protocols have been suggested, with effective results obtained to prevent and counteract said alterations . It has Studies have identified that regular exercise is associated with numerous adaptations that improve the functioning of muscle tissue in obesity; it has been observed how fat stored between muscle fibers decreases , attenuaHowever, the response obtained from the exercise will depend on the intensity, duration and the type of exercise applied ; the exeTherefore, it is essential to evaluate the effects that different exercise intensities have on the biochemical and physiological profiles of skeletal muscle during obesity, and to analyze their effectiveness when combined with other treatments that counteract the harmful effects of obesity at the systemic and tissue level, i.e., diazoxide.To this end, the objective was to evaluate the effect of exercise at different intensities in combination with diazoxide on muscle contraction, resistance to fatigue, and oxidative stress of skeletal muscle in obese rats.Male rats of the Wistar strain, weighing 300–350 g, were used at the beginning of the experiments. They were kept in acrylic cages under vivarium conditions at room temperature with periods of 12 h of light/12 h of darkness, with free access to food and water. The animals were randomly assigned to sixteen experimental groups, with n = 6 per group see .® rodents had a content of 336 cal/100 g with a proportion of 28.507% protein, 13.496% fat, and 57.996% carbohydrate. The high-fat diet ; the visceral and perigonadal fat was obtained and quantified at the end of the treatment and after sacrifice.During the treatment application, weight and glucose were monitored each week. A blood sample was taken by tail puncture  muscles of both hind limbs were dissected and harvested. A pair of muscles were held in Krebs-Ringer solution, and the isometric tension protocol was applied. Simultaneously, the other pair of muscles was stored at −80 °C to later homogenize for future biochemical tests [4, 24.8 mM NaHCO3, 1.18 mM KH2PO4, 10 mM glucose and CaCl2 2.54 mM) and carbogen gas (95% O2 and 5% CO2). The muscle was cleaned and mounted as indicated in the methodology of Gómez-Barroso et al. 2020, in order to apply the fatigue induction protocol, which consisted of pulses of 100 V, 300 ms duration, and frequency of 45 Hz for soleus muscle and 50 Hz for EDL muscle. The stimulation was stopped once the fatigue and tension records were obtained, analyzed and quantified.Soleus and EDL muscles were kept submerged and perfused with Krebs-Ringer solution  and incubated with the H2DCFDA probe at a concentration of 12.5 µM for 15 min at 4 °C [ROS production was determined by reacting these molecules with the fluorescent probe 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA). A total concentration of 0.5 mg/ml of protein was used, which was determined by the biuret method . The pro at 4 °C . The bas2PO4, pH 7.6), and 2 ml of acid solution , the mixture was subjected to three cycles of agitation at low temperatures and then incubated for 15 min in boiling water. Finally, the samples were placed on ice for 5 minutes; after this time, the samples were centrifuged at 7500 rpm for 5 min. The absorbance of each sample was measured at a wavelength of 532 nm on a Shimadzu UV-2550 spectrophotometer using a molar extinction coefficient of 156 mM−1 cm−1 [To determine lipid peroxidation, the levels of thiobarbituric acid reactive substances (TBARS) were analyzed . 0.5 mg/M−1 cm−1 .Muscle glutathione content was evaluated using 0.5 mg/ml sample protein. Samples were resuspended in 0.1 M phosphate buffer, 5.0 mM EDTA-Na2, 0.10% Triton-100X, and 0.6% 5-sulfosalicylic acid pH 7.5 and mixed vigorously.They were then sonicated three times for 20 s on ice, followed by two freeze/thaw cycles, and centrifuged for 10 min at 8000 rpm. For total glutathione, 100 µL of the resulting supernatant was mixed and incubated for 30 seconds with 0.1 M phosphate buffer containing 5 mM EDTA-Na2, 0.1 mM 5,5-dithiobis-2-nitrobenzoic acid, 100 µM glutathione reductase enzyme, and 50 µM NADPH (reduced form). Changes in absorbance were recorded on a Shimadzu spectrometer (model UV-2550 UV VIS) at 412 nm at room temperature. Oxidized glutathione (GSSG) was assayed in 100 µL of supernatant previously incubated with 0.2% 4-vinylpyridine for 60 min. GSH was calculated by subtracting GSSG from total glutathione .p ≤ 0.05 was established. The analysis was performed with GraphPad Prism version 6.0 software .Results were expressed as the mean ± standard error of independent experiments using samples from different animals. Statistical differences between groups were determined by one-way analysis of variance (ANOVA) and Tukey’s post hoc test. A p = 0.024, 0.018) reduction in body weight, between 61–67.5%  in plasma glucose levels and 40%  in visceral and perigonadal fat weight vs. the obese group without treatment.The effect of different exercise intensities, diazoxide, and their respective combinations on body weight, fasting serum glucose levels at the end of each treatment and after sacrifice, and the weight of visceral and perigonadal fat was quantified. After applying the various treatments for 8 weeks, phenotypic and biochemical were observed. p = 0.036, 0.017, 0.0058, 0.041, 0.011, 0.0078). The treatment with the different exercise protocols, combined with diazoxide, increased the force of contraction and the time of resistance to fatigue in both types of muscle during obesity (p < 0.05), observing the best results with moderate-intensity exercise in combination with diazoxide and presenting significant increases of than 60%  vs. the obese group without treatment.To explore the effect of treatment with diazoxide, the different exercise intensities, the combination of both the maximum and total tension, and the time of resistance to fatigue, recording of the isometric tension in soleus muscle and EDL of the different groups was performed. p = 0.029, 0.016) in lipid peroxidation levels and 300%  in ROS levels during obesity. Notwithstanding the treatments applied, the three different exercise protocols and their respective combinations with diazoxide showed beneficial effects on these parameters, with slight variations between them. They present a decrease in both TBARS and ROS levels in both muscles; the low-intensity and moderate-intensity exercise protocols in combination with diazoxide showed the most significant results, presenting reductions greater than 100% (p < 0.05) vs. the obese group without treatment.To evaluate the oxidative stress in skeletal muscle of obese rats, we determined the levels of TBARS as an indicator of lipid peroxidation and the levels of ROS. p = 0.021) was observed, and a decrease in the GSH/GSSG ratio of 71.3% % (p = 0.012) in soleus muscle and 66.88%(p = 0.018) and 54.9% (p = 0.036) in EDL muscle. The various treatments applied in the groups of obese rats improved both total glutathione and the GSH/GSSG ratio. However, the low-intensity exercise treatment in combination with diazoxide presented the best results in both muscles, with significant increases of more than 200% in total glutathione and the GSH/GSSG (p < 0.05). These results show how diazoxide potentiates the effects of low-intensity exercise, increasing antioxidant defense and, therefore, decreasing oxidative stress.To determine if the lower levels of lipid peroxidation and ROS caused by the different exercise and diazoxide protocols in the muscles of obese rats were directly proportional to the changes in the redox state, the glutathione antioxidant defense system  was analThe anatomical and functional alterations of skeletal muscle during obesity are complex: this pathology causes muscle atrophy , metabolIn order to counteract the harmful effects of obesity on skeletal muscle tissue, this study compares the effects of three different exercise protocols, which differed from each other in intensity and duration , togetheThis experimental series was applied to analyze the muscle response to these treatments in the context of diet-induced obesity. The study sought to determine which exercise protocol was most efficient in combination with diazoxide to improve the functioning of muscle tissue and, therefore, its metabolic capacities, thereby opening a window of time that allows the implementation of a constant and prolonged exercise protocol that fully reverses the effects of obesity on skeletal muscle, and eventually obesity itself.Previous studies have shown that obesity induced by a high-fat diet alters many biochemical and physiological parameters, increases body weight and plasma glucose, triggers insulin resistance, and alters the lipid profile in Wistar rats ,11,14,30In healthy models, the literature has reported the best effects for moderate-intensity exercise ,5,32, whAs we have already mentioned, the alteration of the biochemical and physiological parameters produced by obesity triggers dysfunction of the skeletal muscle tissue . In thisHowever, prior studies have shown that diazoxide has positive effects by preventing and even reversing metabolic disorders that are related to obesity, such as improving insulin sensitivity, which has been associated with a rectification in glucose transport and lipid metabolism ,14,15, fSimilarly, exercise has a significant effect on promoting adaptations that improve the metabolic capacities of muscle and its functioning ,17,38, aIn the pathology case, we observed how all the protocols improved the contraction capacity and the fatigue resistance in both types of muscle, which indicates the plasticity capacity of the muscular tissue to adapt to the exercise type ,38. BettObesity increases oxidative stress, eventually damaging muscle tissue function ,39. For In this work, different oxidative stress markers were evaluated to analyze their presence during obesity and determine the effects of the different treatments on these markers. For this, the levels of ROS , the levROS is one of the primary markers of oxidative stress, the excessive accumulation of these molecules is associated with structural and functional damage to cells. During obesity, there is excessive production of ROS ,23. TherOne of the main effects of the increase in ROS is the damage to muscle cell biomolecules, such as proteins, lipids, and nucleic acids . TherefoOn the other hand, obesity increases the levels of lipid peroxidation in muscle tissue; this is associated with the increase in ROS, the increase in adipose tissue and its lipolysis, triggering of lipid peroxidation and causing structural and functional damage of the muscle cell, which interferes with its ability to contract and resist fatigue . HoweverAntioxidant systems in the cells protect them against oxidative stress. Glutathione is included in this system, whose synthesis can be altered by exercise, diet, and age ,43. In tHowever, the different treatments applied during obesity improve the levels and ratio of glutathione in soleus muscle and EDL, with low-intensity exercise combined with diazoxide presenting the best results . By incrIn healthy models, it can be seen how high-intensity exercise presented the most significant increase in antioxidant defense , provingDiazoxide potentiates the effects of exercise, regardless of its intensity and duration. The best results during obesity were obtained by combining the drug with low-intensity exercise, resulting in the treatment with the best effects on muscle contraction and fatigue resistance by reducing ROS levels, lipid peroxidation, and enhancing the redox state of glutathione. In healthy models, we rectified that the best effects occur when a moderate intensity protocol is applied and how diazoxide also potentiates these effects.


Chelon auratus , a native pelagic fish species of the Black Sea. Juvenile golden grey mullets were exposed for 96 h, under semi-static conditions, to dilutions of the reference toxicant 3,5-Dichlorophenol. Results from three reference toxicity tests  indicated that C. auratus is of moderate sensitivity when compared to literature values from EPAs Ecotox database. The protocol described within is intended to ensure Black Sea native organisms are represented by standard hazard assessment practices.In recent years, hydrocarbon exploration and production operations have intensified in the Black Sea. Alongside growth in exploration and production activities, the influence of chemical usage across multiple industrial sectors within the Black Sea environment has become increasingly interesting. The aim of this research was to define a protocol for determining the acute toxicity of chemicals using the golden grey mullet,  Aquatic toxicology, broadly defined as the study of the effects chemicals or materials from natural or anthropogenic sources have on aquatic organisms , has plaToxicity studies in aquatic species have not only documented the susceptibility of individual species to a wide range of toxicants, but also served to highlight several fundamental principles, such as bioaccumulation within the individual organism and biomagnification along the food chain .The principle upon which toxicity tests are based is the recognition that the response of living organisms to the exposure of external stressors  is dependent upon the dose of the stressor . Using tEarly toxicity tests lacked standardization and varied widely in environmental and chemical exposure conditions. This variation in test conditions led to ineffective interpretation of chemical hazard . EffortsAcute toxicity tests are usually designed to evaluate the concentration–response relationship for survival, whereas chronic studies evaluate sublethal effects such as growth, reproduction, behavior, tissue residues, or biochemical effects and are usually designed to provide an estimate of the concentration that produces no adverse effects . Acute tDanio rerio, Pimephales promelas, Menidia beryllina, Cyprinodon variegatus) , as representative native pelagic fish of the Black Sea, with a size and handling tolerance appropriate for laboratory study.As mentioned above, the selected test species was the golden grey mullet [Acclimation procedures are separated into two distinct stages. The first stage is fish acclimation to standard laboratory conditions (this can be ignored if not using wild-caught organisms), while the second stage is conditioning to experimental conditions.Juvenile golden grey mullets  were captured from the wild, near Constanta, Romania , using mesh nets , subsequently placed in 40-L polypropylene barrels (Sterk Plastic), and transported immediately to the laboratory a. DuringThroughout the acclimation period, the water was UV sterilized at a flow of approximately 100 L/h and proper aeration was provided, consisting of an air flow of approximately 300 L/h, in order to obtain around 80–90% dissolved oxygen (DO) saturation. The fish were fed daily with JBL NovoGranoColor mini pellets, at an approximate ration of 2% of total fish biomass in the tank   b, but no3 decanter, which was analyzed for the presence of contaminants for experiments/testing use. The seawater reaches the aquaculture laboratory gravitationally, through a pipeline system. Once in the laboratory, seawater was analyzed before each running test to confirm the ambient conditions of the receiving waters and no contamination. The following parameters were measured: temperature, pH, salinity, dissolved oxygen, nutrients, pollutants , using a Mettler Toledo Seven Excellence multiparameter probe and internationally agreed seawater analysis methods [The dilution water used was natural Black Sea water pumped from a pollution-free location off NIMRD’s premises and stored in an underground 130 m methods ,43,44,45Subsequently, to ensure that the dissolved oxygen saturation content of the dilution water was between 90% and 100% prior to use for fish tests, DO was measured before initiating the testing procedure. Whenever DO saturation was less than 95%, additional aeration for 10–15 min was provided using the air pumps. When reaching levels between 95% to maximum 100% DO saturation, the aeration process was stopped, and the storage tanks were sealed and stored in the temperature-controlled room at test temperature. The water in the tanks was used the following day for test substance dilution preparation.Fish were randomly distributed in the experimental jars, as it is the most reliable method for creating homogeneous treatment groups, reducing potential biases or judgments . The equFor the randomization procedure, the fish were individually extracted from the aquarium in the temperature-controlled room using the fish net, and, following the design of the randomization scheme, placed one by one in the plastic buckets. When reaching the desired number  of indivAcartia tonsa and Skeletonema costatum. In an effort to standardize reference toxicity testing for the laboratory, one test substance  was applied across all testing platforms. Concentrations identified for C. auratus reference testing were defined based on ISO standards described above and reported LC50 literature values ranging from ~1–3.5 mg/L [The reference toxicant used for tailoring the protocol was 3,5-Dichlorophenol (CAS# 591-35-5), a substance commonly used as a reference substance to act as a positive control . The con3.5 mg/L ,53,54,553.5 mg/L .The golden grey mullet 96-h acute toxicity testing was performed in 5000 mL glass jars containing a total volume of 5000 mL of solution. Each jar was covered by a glass lid, in order to avoid contamination, evaporation and escape of the test organisms. Each test jar was labeled with the species initials, treatment group and replicate. Aeration was provided in the jars in order to maintain a DO level higher than 60% [The stock solution for 3,5-Dichlorophenol was prepared on the day of testing by adding 1 g of 3,5-Dichlorophenol to 1 L of natural seawater in a 1 L glass volumetric flask and stirred on a magnetic stirrer for 15 min at 700 rpm. Natural seawater was used as a control medium and as dilution water.A series of 5 dilutions  were prepared at using the 1 g/L stock solution by diluting the stock solution with natural seawater, in 25,000 mL glass carboys labeled accordingly, from 1 to 5 . Each soEvery 24 h, 80% of the tested solution was renewed using freshly prepared stock and dilution solutions. Renewal occurred by siphoning and replacing 80% exposure solutions from each replicate vessel .All resulting wastewater was stored in closed barrels labeled accordingly (“hazardous waste”) and disposed of by a specialized company.Temperature, pH, salinity, dissolved oxygen, and inorganic nitrogen were measured at the beginning of the tests, before the renewal (new medium), and at the end of each 24-h exposure period (old medium) for each replicate and concentration , using aObservations on fish mortality and immobilization were performed and recorded at 24 ± 1 h intervals after the start of the test. Immobilization was considered as the lack of swimming ability . Dead fiAfter the 96-h test period, the surviving fish were euthanized humanely, in compliance with the American Veterinary Medical Association Guidelines for the Euthanasia of Animals . The fis50 at 96 h was calculated using a sigmoid function with the LC50 calculation application from AAT Bioquest [For each concentration, data from each replicate were collected and the mortality rate was calculated after 24 h, 48 h, 72 h, and 96 h relative to the total number of fish used. The LCBioquest   [The first test was treated as a range-finding study, using a relatively broad dose range  to improve confidence in final dose selection for subsequent tests. The range finding test did not meet minimum LChan 50%) . Additiohan 50%) . No morthan 50%) ; consequ50 results obtained from all test runs  were considered relevant, being in the expected range of 1–4 mg/L (50 1.25 mg/L) were excluded from species sensitivity distribution (SSD) calculations due to potential confounding stress from inadequate dissolved oxygen during testing. During all tests performed, temperature, salinity, and pH in the dilution water varied within the range of the Black Sea , NO Romania . At the 50 values were used to generate a species sensitivity distribution (SSD)  and the stressor intensity for C. auratus = 1.6 (1.53–1.7), if not slightly more sensitive than standard laboratory test species to the test substance, 3,5-Dichlorophenol. This indication of sensitivity requires further testing to confirm, as there is a paucity of toxicity data associated with 3,5-Dichlorophenol. Additional assays directly comparing the sensitivity of C. auratus to Danio rerio or Cyprinodon variegatus are recommended be conducted prior to any novel fish species being adopted as standard laboratory test organisms for toxicity assessment.In an effort to relate the results from the reference toxicity assays, data from EPA’s ECOTOX database ,35,36,37on (SSD) . OverallC. auratus as a suitable reference fish species for ecotoxicity tests in the Black Sea proved to be applicable and provides significant support for testing the environmental impact of various xenobiotics  introduced in the environment.The protocol for using the golden grey mullet The primary aim of our research was to set up a protocol for assessing the acute toxicity on pelagic fish species, applicable at regional level, using the golden grey mullet as a native species representative of the Black Sea ecosystem. The selected species showed good adaptability to laboratory conditions, as well as an appropriate size for this type of testing. Juvenile golden grey mullets were exposed for 96 h, under semi-static conditions, with a daily renewal of 80% of the exposure medium, to a series of dilutions of the tested toxicant, prepared by adding the appropriate volume of stock solution in 1000 mL calibrated flasks labeled C0 to C5, filled with natural seawater and well mixed. Five replicates were prepared for each concentration and four organisms were distributed per replicate following a random design.50 of 1.25 mg/L, 1.739 mg/L, and 1.409 mg/L) were calculated using a sigmoid function on mortality data collected throughout the study, and were in line with values reported in other fish species, ranging from 1 to 3.5 mg/L. The tests performed were considered acceptable from the fish survivability perspective, as control mortality did not exceed 20%. The only minor issues that appeared, related to dissolved oxygen concentrations outside the range indicated by the guidelines, were settled by providing proper aeration to the dilution water, as well as in the test jars. The final results (LCC. auratus is likely of moderate sensitivity (HC5 = 0.78 (0.61–1.0) and the stressor intensity for C. auratus = 1.6 (1.53–1.7)), if not slightly more sensitive than standard laboratory test species to the test substance, 3,5-Dichlorophenol. This indication of sensitivity requires further testing to confirm, as there is a scarcity of toxicity data associated with 3,5-Dichlorophenol. We recommend additional assays directly comparing the sensitivity of C. auratus to D. rerio or C. variegatus be conducted prior to any novel fish species being adopted as standard laboratory test organisms for toxicity assessment. Future work needs to be conducted to continue to benchmark the golden grey mullet against standard laboratory test species. Though novel organisms are of high value to specific regions, having the ability to relate novel organism to standard species facilitates extrapolation to other regions as well as standardizes testing across platforms. Moreover, a species sensitivity distribution (SSD) analysis was performed, which indicates that


A total of 36 original articles have been selected from five academic databases, including Web of Science, PubMed, Scopus, EBSCO, and Google Scholar, covering 78,674 participants in 19 nations, and yielding 60 effect sizes  based on the random effects model via Comprehensive Meta-Analysis (CMA) software. The results showed that the prevalence of anxiety, depression, and stress among remote learning students during the COVID-19 pandemic was as high as 58, 50, and 71%, respectively. Besides, the moderator analysis found that (1) the prevalence of anxiety and depression among students in higher education was significantly higher than that of students in elementary education. (2) an increasing number of medical students and students in emergency remote learning context suffered from mental stress than their non-medical and traditional distance learning counterparts. Thus, the COVID-19 pandemic triggers concerns related to physical health and mental disorders, especially for remote online learning students. The current situation should be brought to the forefront by educators to develop psychological interventions for relieving students’ anxiety, depression, and stress during the pandemic period.The coronavirus disease 2019 (COVID-19) pandemic has influenced all aspects significantly, and an estimated 1.5 billion students across the globe have been forced to keep up with online courses at home. Many recent empirical studies reported the prevalence of mental health problems among students caused by remote learning during the COVID-19 pandemic, but a few studies aggregated these results. Therefore, to strengthen statistical power, the article aimed to examine the prevalence of anxiety, depression, and stress among remote learning students during the COVID-19 pandemic  The disease was named coronavirus disease 2019 (COVID-19), which first spread across China and then across 216 nations, areas, or territories worldwide . Facing via an alternate delivery mode because of crisis circumstances  and all the possible combinations of these keywords were input in the search bar with the following string: (“COVID-19” OR “Coronavirus”) AND (“Online learning” OR “emergency online learning” OR “remote learning” OR “distance learning” OR “e-learning”) AND . The final search date of this study was 1 November 2022.The criteria for screening resulting articles were as follows: (1) studies that were published during the COVID-19 pandemic; (2) studies that were empirical research; (3) studies that reported the detection rate of anxiety, depression, or stress among remote learning students during the COVID-19 pandemic; (4) studies that reported the accurate sample size; and (5) studies that specified whether students had undergone online learning or emergency online learning. After applying these inclusion criteria, a total of 36 studies, yielding 22 anxiety effect sizes, 17 depression effect sizes, and 21 stress effect sizes, were selected in this meta-analysis  are crucial evaluation criteria. If the Q test shows significant or I2 results higher than 75%, it indicates that high heterogeneity exists, and a random effects model should be selected; otherwise, a fixed effects model should be chosen  software. To aggregate the overall prevalence rate, the software first transformed input ratio data into logit data through the formula on−case . There won−case .3.The results of the study are presented in the following subsections. The Results section starts with description of the characteristic of the study sample, followed by an investigation and measurement of heterogeneity and publication bias. The Results section ends in displaying the combined effect, sensitivity, and moderator analysis of the present meta-analysis.3.1.Overall, 60 total effect sizes from 36 different studies were extracted, and 78,674 participants were included in this meta-analysis. Specifically, anxiety included 22 studies and 64,072 participants, generating 22 effect sizes; depression comprised 17 studies and 22,618 participants, yielding 17 effect sizes; stress incorporated 21 studies and 29,364 participants, generating 21 effect sizes. These 36 studies were conducted from 19 nations , covering both developed and developing countries from Asia, Europe, North America, and South America.3.2.2 results exceeded 75% in anxiety , depression , and stress  studies, indicating that the random effects model should be chosen as the statistical model in this study , depression (p = 0.04), and stress (p = 0.04), with publication bias manifesting in any of the three clinical symptoms. Therefore, the trim-and-fill method, one of the most popular means, was opted for correcting publication bias , which indicates that the prevalence of anxiety hardly changed with time lapse; (2) the moderating effect of whether attending emergency online learning (p = 0.09), development level (p = 0.63), and students’ major (p = 0.92) were all not significant; and (3) the moderating effect of educational level (p = 0.03) was significant. Prevalence of the anxiety of university students (63%) was more than two times that of the k-12 students (29%).In terms of moderator analysis, several variables were set as moderator types to assess whether a third variable causes a significant difference. In terms of anxiety see , the resb = −0.13, 95% CI = ), which also indicates that the prevalence of depression hardly changed over time; (2) the moderating effect of development level (p = 0.40) and students’ major (p = 0.92) were both not significant; and (3) the moderating effect of educational level (p = 0.04) was significant. The prevalence of depression in university students (53%) was much higher than that of k-12 students (34%).As regards depression see , the resb = 0.63, 95% CI = ), which also indicates that the prevalence of stress scarcely variates with time; (2) the moderating effect of educational level (p = 0.59) and students’ major (p = 0.25) was both not significant; and (3) the moderating effect of whether attending emergency online learning (p = 0.03) and development level (p = 0.00) was significant. The stress prevalence of students under emergency online learning contexts (89%) was much higher than that of students under traditional online learning contexts (67%). In addition, the stress prevalence of students in a developed country (93%) was more than 1.5 times higher than their counterparts in a developing country (56%).For the third mental health problem, stress see , the res4.Based on the above findings, we have discussed below the possible explanation of these outcomes , combining it with related prior literature. In addition, limitations are discussed for guiding follow-up studies in the future.4.1.The present study is the first meta-analysis on the prevalence of anxiety, depression, and stress among remote learning students during the COVID-19 pandemic. It synthesized the evidence on the empirical studies associated with online learning and mental health problems, such as anxiety, depression, and stress during the COVID-19 pandemic period. After a rigorous literature search and screening, a total of 36 studies with 78,674 participants from 19 nations were included and generated 60 effect sizes . This meta-analysis showed that the prevalence of anxiety, depression, and stress among remote learning students during the COVID-19 pandemic was high, 58, 50, and 71%, respectively.via meta-analysis. In addition, Comparing the current result with previous analogous meta-analysis, one study found that the prevalence of anxiety, depression, and stress among remote learning students during the COVID-19 pandemic is significantly higher than that of the general population during the pandemic, students during the pandemic, and students before the pandemic. More specifically, Based on the mentioned data comparison, student groups, online learning, and the COVID-19 pandemic are three factors that lead to a higher prevalence of anxiety, depression, and stress. First of all, children and young people nowadays are facing unprecedented challenges that are unacquainted with previous generations’ cognition, and it is exactly the time in one’s life that confronts the peak risk of suffering from mental health problems . Accordi4.2.In the moderator analysis, several variables are set as moderators in the current meta-analysis, including publication year, whether attending emergency online learning, educational level, development level, and students’ major. It is the first study to explore the moderating effect of study publication year on the prevalence of anxiety, depression, and stress among remote learning students during the COVID-19 pandemic. This meta-analysis concluded that the moderating effect of publication year was not significant, which also indicates that the prevalence of anxiety, depression, and stress scarcely variates with time after the outbreak of the COVID-19 pandemic. The result shows a different trend from that before the pandemic. Before the start of the COVID-19 pandemic, studies found that the level of anxiety and depression among adolescents increased substantially from 1997 to 2017 in China and from 2011 to 2018 in America . The samWhether attending emergency online learning is also an innovative variable studied in this meta-analysis. The result showed that the moderating effect of whether attending emergency online learning was significant for the prevalence of stress , but we failed to find the moderating effect in the prevalence of anxiety and depression among online learning students during the COVID-19 pandemic. Compared to traditional online education, the emergency pattern is usually a temporary shift to another delivery mode due to crisis circumstances . For thiEducational level is the third moderator variable in this meta-analysis. The result showed that the moderating effect of the educational level was significant for the prevalence of anxiety and depression among e-learning students during the COVID-19 pandemic. The anxiety and depression prevalence of university students was significantly higher than that of the k-12 students. This result partly conforms to the finding in another meta-analysis, which found that people with a higher level of education experienced a higher level of anxiety, depression, and stress . There aAs regards the development level, the result showed that the moderating effect of development level was significant for the prevalence of stress among e-learning students during the COVID-19 pandemic. The stress prevalence of students in a developed country was more than 1.5 times higher than their counterparts in a developing country. The current result is exactly opposite to the finding among the general population, which found that people in underdeveloped and developing countries suffer from more psychological problems . It is pThe last moderator variable in this meta-analysis is students’ major, which shows no significant difference between students in medical and other diverse majors in the prevalence rate of anxiety, depression, and stress during the COVID-19 online study period. This result is in discordance with the previous study. Reviewing preceding research comparing mental health between medical and non-medical students, there are two contrary findings before and after pandemic. Before the outbreak of the COVID-19 pandemic, more medical and non-medical students reported worse mental health status due to perceiving greater academic pressure . However4.3.Despite this study being a pioneering work exploring the prevalence of anxiety, depression, and stress among remote learning students during the COVID-19 pandemic, several limitations exist in the present meta-analysis. First, the number of studies on emergency remote learning is insufficient: only three for anxiety and stress and one for depression, which leads to the moderating effect of whether attending emergency remote learning on the prevalence of depression cannot be examined. Therefore, more searches under emergency remote learning can be collected to be further analyzed in the future. Second, there are only two categories for students’ major variables: medical and diverse. Students in different majors confront their own specific challenges that other disciplines cannot notice. Thus, scholars should also pay attention to students’ mental health in other majors, such as business, literature, art, and science. Third, only published articles were collected in this study. It is relatively difficult to avoid publication bias in meta-analysis since it is a secondary analysis based on abundant original literature. However, including unpublished articles could lower bias. Fourth, the current research is yet to cover all types of mental health problems. Only anxiety, depression, and stress were regarded as representative indicators reflecting remote learning students’ mental status; therefore, many psychological disorders, such as distress and self-injury, can catch attention in the future. Fifth, all original studies in the current meta-analysis were periodic, which could only reflect participants’ mental health over a period of time. However, psychological status changes as time goes on. Therefore, future follow-up studies could be conducted to explore the overall psychological impact of the COVID-19 pandemic on students in multiple disciplines over a more forward-looking period.5.Over the past 3 years, the COVID-19 pandemic has triggered psychological problems afflicting people worldwide. This study conducted a meta-analysis based on 36 original articles and 78,674 participants across 19 nations and found that the prevalence of anxiety, depression, and stress among remote learning students during the COVID-19 pandemic was as high as 58, 50, and 71%, respectively. The prevalence rate of anxiety and depression among students in higher education was significantly higher than that of students in elementary education. Besides, significantly more medical students and students in emergency remote learning contexts suffer from mental stress than their non-medical and traditional distance learning counterparts.Therefore, instructors, schools, and governments should take notice of the current situation and figure out ways to collaboratively help online learning students relieve anxiety, depression, and stress against the backdrop of the COVID-19 pandemic. Instructors, as the group with the closest connections to students, could alleviate students’ negative emotions by designing flexible coursework and test-taking, having organized online courses, and showing care about students’ mental health . From thThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.TX is responsible for putting forward ideas, collecting and analyzing data, and drafting the paper. HW is responsible for supervising the whole process and revising the paper. 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.


Diabetic retinopathy (DR) is one of the most common complications of type 2 diabetes mellitus. The current study investigates the composition, structure, and function of gut microbiota in DR patients and explores the correlation between gut microbiota and clinical characteristics of DR.A total of 50 stool samples were collected from 50 participants, including 25 DR patients and 25 healthy controls (HCs). 16S ribosomal RNA gene sequencing was used to analyze the gut microbial composition in these two groups. DNA was extracted from the fecal samples using the MiSeq platform.Bacteroides, Megamonas, Ruminococcus_torques_group, Lachnoclostridium, and Alistipes, and decreased levels of Blautia, Eubacterium_ hallii_group, Collinsella, Dorea, Romboutsia, Anaerostipes, and Fusicatenibacter genera were observed in the DR groups. Additionally, a stochastic forest model was developed to identify a set of biomarkers with seven bacterial genera that can differentiate patients with DR from those HC. The microbial communities exhibited varied functions in these two groups because of the alterations of the above-mentioned bacterial genera.The microbial structure and composition of DR patients were different from that of HCs. The microbial richness of gut microbiota in DR was higher than that of normal individuals. The alterations of microbiome of DR patients were associated with disrupted Firmicutes, Bacteroidetes, Synergistota, and Desulfobacterota phyla. In addition, increased levels of The altered composition and function of gut microbiota in DR patients indicated that gut microbiome could be used as non-invasive biomarkers, improve clinical diagnostic methods, and identify putative therapeutic targets for DR. A large number of bacteria colonize human guts. The total number of bacteria in the adult intestine is 10 times greater than the number of human cells . These mEubacterium ventriosum, might occur in individuals with age-related macular degeneration  .Although previous studies indicated the alterations of gut microbiota on diabetes mellitus in detail , the corIn this study, we collected fecal samples from the DR patients and healthy controls (HCs) and delineated the community structure of the gut microbiome of DR patients based on 16S ribosomal RNA (rRNA) gene sequencing. In addition, the differences in the composition and function of the gut microbiota between patients with DR and HCs were investigated systematically. We aimed to develop a non-invasive method for diagnosing and identifying potential microbial targets to improve our understanding of pathogenesis and therapeutic approaches for DR.1 The protocol in this study conformed to the Declaration of Helsinki. Informed consents were obtained from all the subjects.This clinical study was approved by the ethics review committee of Shanghai Tenth People’s Hospital, Shanghai, China (Ethics Approve No. SHSY-IEC-5.0/22K100/01) and registered in the China Clinical Trials Registration Center (No. ChiCTR2200058763).A total of 25 DR patients and 25 age- and sex-matched HCs were enrolled in this study. The participants included resident men and women . They were examined by ultra-wide-field scanning laser ophthalmoscopy (UWF-SLO) and optical coherence tomography angiography (OCTA). In addition, fundus fluorescein angiography (FFA) and indocyanine green angiography (ICGA) were conducted in all DR patients. The inclusion criteria of the experimental group were as follows: (1) age 25–70 years; (2) fulfilling the 1999 World Health Organization (WHO) diagnostic criteria for T2DM; (3) DR was diagnosed based on the International Clinical Diabetic Retinopathy Disease Severity Scale (2002). Regardless of the level of severity, patients with retinopathy were classified into the DR group; (4) provided informed consent; (5) did not have any other intraocular diseases. The spouses of patients with DR were recruited as the HC group, all the participants have no obvious dietary differences from their spouses, they had no ocular diseases other than refractive errors, no diabetes, and no other major systemic diseases. In addition, the difference of age between the healthy individuals and their spouses was ≤3 years.The exclusion criteria were as follows: (1) usage of antibiotics within 6 weeks before participation in the study; (2) usage of probiotics and prebiotics within 3 months prior to fecal sampling.; (3) history of abdominal surgeries; (4) daily alcohol consumption >30 g; (5) significant immunodeficiency, serious kidney or liver disease; (6) breastfeeding or pregnancy.In addition, we confirmed all subjects had relatively similar diet structures using a standardized 4-week recall questionnaire, which was discussed with a dietician.Body mass index (BMI) was calculated at the beginning of the study. The fecal samples were collected in cold, sterile stool containers, transported to the laboratory within 2 h of collection, and stored at −80°C until microbiota analysis.® Soil DNA Kit  according to the manufacturer’s protocol. The quality and concentration of DNA were determined on 1% agarose gel electrophoresis and a NanoDrop® ND-2000 spectrophotometer , respectively. The hypervariable region V3–V4 of the bacterial 16S rRNA gene was selected as the target for amplification with primer sequences 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′) on an ABI GeneAmp® 9700 PCR thermocycler . The PCR reactions were performed using TransStart Fast Pfu DNA Polymerase  in a 20 μL reaction, consisting of 2 μL of 2.5 mM dNTPs, 0.4 μL Fast Pfu polymerase, 0.8 μL each primer (5 μM), 4 μL of 5× Fast Pfu buffer, and 10 ng of template DNA. The AxyPrep DNA Gel Extraction Kit  was used to purify the amplicon products, and all samples were amplified in triplicate. Then, the purified amplicon products were pooled in equimolar amounts, followed by paired-end sequencing on an Illumina MiSeq platform  according to the manufacturer’s instructions .Total microbial DNA was extracted from frozen fecal samples using E.Z.N.A.2 was used to cluster the optimized sequences into operational taxonomic units (OTUs) with 97% sequence similarity cutoffs. Then, the most abundant sequence was selected as the representative sequence. Due to the individual differences among various samples, the sequences of all samples were randomly selected into a uniform data body according to the lowest sequence number to compare each sample at the same OTU serial number level. Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt2) was used to predict the metagenomic function.Raw FASTQ files were demultiplexed, quality-filtered, and merged using the following steps. First, to obtain clean data, the 300-bp reads were truncated at any site if the average quality score was <20, with a 50-bp sliding window, and reads with quality scores <50 were discarded. Second, only overlaps >10 bp were assembled based on their overlapped sequence, with the maximum mismatch ratio set to 0.2. Third, the samples were distinguished based on the barcode/primers and barcode matching, followed by adjustment of the sequence direction. Uparse v7.1P-value <0.05 indicated statistical significance.Based on the OTU information, a-diversity was evaluated by the species indices , calculated using Mothur v1.30.1, and β-diversity was used to explore the diversity of the microbial structure. Principal coordinate analysis (PCoA) and partial least squares discriminant analysis (PLS-DA) was performed to determine the similarity among the microbial communities in various samples using Vegan v2.5-3 package. The linear discriminant analysis (LDA) effect size (LEfSe) was used to identify the differences in taxa between DR and HCs (phylum to genera). In this method, a non-parametric factorial Wilcoxon rank-sum test was performed to detect the features with significant differential abundance, and the effect size of each feature using LDA was calculated. Statistical analyses were performed with R software (v.3.4.1). Statistical analyses were performed using SPSS v25 . P > 0.05, A total of 50 participants, including 25 DR patients and their healthy spouses, were recruited in this study. Three samples from healthy subjects were excluded due to insufficient size or inadequate PCR amplification. The researchers have tried PCR amplification several times, but these samples did not meet the requirements and hence, 47 stool samples were tested. All patients with DR were diagnosed with T2DM for >10 years; the longest duration was >30 years. No statistically significant differences were detected in age and BMI between the two groups , indicating that the microbial richness of gut microbiota in patients with DR was higher than that of normal individuals , species evenness indices (Shannoneven and Heip), and species diversity indices (Shannon and Invsimpson), were used to test the index values between the two groups. We found significant difference in species richness indices (sobs index) between the two groups  overlapped and were observed in all groups. Of the 20 genera detected only in the HC group, 84.55% was the no-rank_f_Selenomonadac and un-classified_f_Streptococcaceae genera. A total of 45 genera were detected in the DR group, with 84.15% belonging to Sarcina, Holdeman, Pseudomonas, and Catenibacterium  on the fecal microbiota composition from the phylum to the genus level using LDA to estimate the effect size of each taxon that were differentially represented in the two groups. Additionally, 101 bacterial taxa showed significant differences in the relative abundance in DR and HC groups, with 68 distinct microbial taxa in the DR group. These taxa belonged to 25 main families  as the biomarker to distinguish the two groups.Blautia, Bacteroides, Megamonas, Romboutsia, and Anaerostipes as biomarkers was assessed. We found that the combination of these microbial biomarkers could discriminate DR patients from the HC group with an AUC of 0.85 (95% CI: 0.73–0.97)  curve. At the highest AUC value (0.97), 7 species were ranked according to importance . Figure 73–0.97) .To examine the functional changes of the microbial communities in the DR and HC groups, we used PICRUSt2 to infer the metagenomes from the 16S rRNA data and predict the functional profile of the microbial community. PICRUSt2 is a software tool that provides a starting point for understanding the altered functions within a microbiota flora. In the current study, a total of 6507 Kyoto Encyclopedia of Genes and Genomes (KEGG) ortholog (KO) genes and 335 KEGG pathways from the entire data set were predicted , 3. In aHippocrates, the father of modern medicine, postulated that “all diseases begin in the gut” . The humMicrobial community diversity analysis is essential to quantify the bacterial composition and relative abundance of a specific community, including alpha diversity (within communities) and beta diversity (inter-community). In this study, we found that patients in the DR group had distinct gut microbiota compared to the HC group based on the increased microbial richness and altered microbial composition. In addition to the higher sobs indices in DR than in HC, other alpha diversity indices, including Ace, Chao, Shannon, Heip, and Shannon, did not reach statistical difference, although an upregulated trend was observed in Ace and Shannon indexes. Also, the DR group showed significant alterations in beta diversity (PCoA and PLS-DA) compared to the HC group, indicating an obvious distinction between the microbiota composition of these two groups. This finding was consistent with previous studies, wherein the association between Firmicutes and Bacteroidetes had been reported in diabetic patients , and theWe observed the community structure of the gut microbiome in DR patients utilizing high-throughput gene sequencing. DR was characterized by alterations in specific OTUs assigned to the families, Lachnospiraceae, Bacteroidaceae, Selenomonadaceae, and Peptostreptococcaceae. A total of 45 genera in DR compared to only 20 in HC were obtained. Thus, we speculated that the microbiota in DR might exhibit a complex and high pathology diversity, proposing that the onset and progression of diabetes and its retinal complications are related to the alterations in gut microbiota.Furthermore, the alterations in the microbiome of DR patients were related to disturbances in the phyla Firmicutes, Bacteroidetes, Synergistota, and Desulfobacterota. Our results were consistent with those of a previous study on diabetes, which showed a significant reduction in Firmicutes and Clostridia (members of the phylum Firmicutes) and an abundance of Bacteroidetes in the gut of patients with T2DM compared to non-diabetic individuals . SimilarGut dysbiosis is a multifactorial disease. In healthy individuals, the intestinal wall prevents the migration of microorganisms and their metabolites from the intestinal lumen to blood circulation. However, gut dysbiosis may relieve the barrier effect of the intestinal lining and cause the leaky gut syndrome. Bacteroidetes are Gram-negative bacteria, whose cell wall is mainly composed of lipopolysaccharide (LPS) and associated with the pathogenesis of diabetes. Bacteroidetes release bacterial endotoxin and LPSs , and triBlautia, Eubacterium_hallii_group, Collinsella, Dorea, Romboutsia, Anaerostipes, and Fusicatenibacter and increased Bacteroides, Megamonas, Ruminococcus_torques_group, Lachnoclostridium, and Alistipes were observed in the DR group. However, these results were not consistent with those reported in other studies on diabetic patients without retinopathy . Most DR patients in the current study used metformin and other drugs for their treatment for many years. Another possible reason is the existence of gut-retina axis, which might lead to slight but critical change in the intestinal microecology of diabetic patients with or without retinopathy. Lactobacillus genus was enriched in participants with diabetes and was more abundant in the type 2 diabetic patients than in the DR patients. However, no studies have yet explored these potentially causal bacteria in animal models of diabetes or DR; thus, the effects of these bacteria on DR require further study in basic research setting and clinical research with large sample sizes.At the genus level, decreased inopathy . Among tdiabetes . The altEubacterium_hallii_group , is capable of butyrate production  can be found below: NCBI, The studies involving human participants were reviewed and approved by the Medical Ethics Committee of the Shanghai Tenth People’s Hospital of Tongji University. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.JB and ZW participated in the data analysis and writing of the manuscript. TW, YZ, and YX participated in data collection and revising the manuscript. QP designed the study and wrote and revised the manuscript. All authors contributed to the article and approved the submitted version.


An experiment was conducted to determine the standardized ileal digestibility (SID) of amino acids (AA) in fish meal (FM) and blood-derived protein sources including spray-dried porcine plasma (SDPP), porcine red blood cells (PRBC), and blood meal (BM) fed to growing pigs.Ten barrows  surgically fitted with T-cannulas at the distal ileum were allotted to a duplicated 5×4 incomplete Latin square design with 5 experimental diets and 4 periods. Four experimental diets were prepared to contain FM, SDPP, PRBC, or BM as the sole source of nitrogen. A nitrogen-free diet was prepared and included to estimate the basal ileal endogenous losses of AA. For the 7-day experimental period, pigs were fed for 5 days as adaptation, and ileal digesta samples were collected for 9 hours on days 6 and 7.The SID of crude protein in BM (48.0%) was less (p<0.05) than in FM, SDPP, and PRBC . Pigs fed the diet containing BM had less (p<0.05) SID of AA, except isoleucine and proline, than those fed the diet containing FM, SDPP, or PRBC. Among FM, SDPP, and PRBC, there was no difference in the SID of crude protein and all AA, except isoleucine. The SID of isoleucine in PRBC and BM  was less (p<0.05) than in FM and SDPP . The SID of lysine in FM, SDPP, PRBC, and BM was 85.4%, 84.9%, 89.7%, and 51.9%, respectively.The SID of most AA was not different among FM, SDPP, and PRBC, but BM had lower SID of most AA than FM, SDPP, and PRBC. Protein sources derived from animal byproducts have been used in the modern swine production system, especially for weanling pigs because of their high concentrations of digestible crude protein (CP) and amino acids (AA) as well as functional components. In addition, weanling pigs are susceptible to anti-nutritional factors in soybean meal such as glycinin and β-conglycinin, leading to a limited inclusion in diets immediately after weaning . Thus, fDuring the meat processing, blood from slaughtered animals can be collected, processed, and dried to produce blood meal (BM), which can be used as a protein source in swine diets. On the other hand, collected blood can be treated with anticoagulant and centrifuged to separate plasma, which can be further processed to produce spray-dried animal plasma (SDAP) . It has Protocols of the animal experiment were reviewed and approved by the Purdue University Animal Care and Use Committee .2). Pigs were divided into 2 replicates based on BW  and allotted to a duplicated 5×4 incomplete Latin square design with 5 experimental diets and 4 periods. Pigs had free access to water via nipple drinkers.A total of 10 barrows were surgically fitted with T-cannulas at the distal ileum based on the method reported by Dilger et al , followeFive experimental diets were prepared based on cornstarch and sucrose . Four diIndividual BW of pigs were measured at the beginning of each experimental period to calculate the daily amount of feed offered which was 4% of mean BW within replicates. Pigs were fed twice a day at 0800 and 1700 hours. For the 7-day experimental period, pigs were fed for 5 days as adaptation, and ileal digesta samples were collected for 9 hours on days 6 and 7. Ileal digesta samples were collected via T-cannulas by attaching plastic sample bags  containing 10 mL of 10% formic acid. Attached plastic samples bags were changed every 30 minutes and immediately stored at −20°C. At the end of each experimental period, frozen ileal digesta samples were slightly thawed, pooled within pigs, and subsampled. Collected subsamples were stored at −20°C before further analyses.Frozen ileal digesta samples were freeze dried before the chemical analyses. Test ingredients, experimental diets, and freeze-dried ileal digesta samples were finely ground (<0.75 mm) using a centrifugal grinder . The concentrations of dry matter (DM) in ground ingredients, diets, and freeze-dried ileal digesta samples were analyzed by drying at 105°C for 24 h in a forced-air drying oven  , and theThe apparent ileal digestibility (AID) of CP and AA in test ingredients were calculated by the index method suggested in Kong and Adeola :in and Crout represent the concentration of Cr (g/kg DM) in experimental diets and ileal digesta, respectively; AAin and AAout represent the concentration of CP or AA (g/kg DM) in experimental diets and ileal digesta, respectively. Data from pigs fed NFD were used to estimate the BEL [g/kg DM intake (DMI)] of CP and AA using the following equation:where CrTo calculate the SID of CP and AA in test ingredients, the AID of CP and AA were corrected for the estimated BEL of CP and AA using the following equation:Data were tested for normality by univariate procedure of SAS , and data outside of 2.5 times interquartile range were considered outliers. Thereafter, data were analyzed by mixed linear models procedure of SAS. Model included experimental diet as fixed variable and replicate, period within replicate, and pig within replicate as random variables. Pairwise comparison with the Tukey’s adjustment was conducted to separate the estimated least squares means among experimental diets. Experimental unit was the individual pig. Significance of the model and difference was determined by p<0.05.All pigs were healthy throughout except one pig fed NFD during the last experimental period, which was removed from experiment. Also, data from 2 pigs fed NFD and 1 pig fed the diet containing PRBC were detected as outliers and therefore treated as missing observations in the dataset.On an as-fed basis, the concentration of CP in test ingredients ranged from 658 g/kg for FM to 968 g/kg for PRBC . The conThe AID of CP and AA, except isoleucine, in BM were less (p<0.05) than in the other test ingredients . There wThe BEL of CP in pigs fed NFD was 19.9 g/kg DMI . The BELThe analyzed CP, AA, and ash concentrations in FM are consistent with the previously reported values ,14,15. IFish meal and SDPP have been widely used in diets for weanling pigs due to their high quality of protein and favorable AA contents for animal growth ,19. AlthThe SID of CP and AA, except isoleucine, were not different among FM, SDPP, and PRBC, which may indicate that FM, SDPP, and PRBC tested in this experiment have similar AA availability for pigs. However, it should be noted that the functional properties of FM, SDPP, and PRBC are different when fed to weanling pigs despite the similarity of SID of AA. Fish meal has been used in weanling pig diets mainly because it contains highly digestible and balanced AA as well as poly unsaturated fatty acids  and becaThe BEL of CP and AA observed in this study are comparable to the values summarized in previous studies ,21,22. TThe SID of CP and AA in SDPP observed in the current study are in agreement with NRC  and Wu eThe information for the SID of CP and AA in PRBC as well as blood cells is scarce, but the SID of CP and AA in PRBC are less than those in spray-dried blood cells reported by Almeida et al . This diSeveral processes in the production of BM, such as heat treatment, pH control, or filtration, may negatively affect the availability of protein and AA . In addiBased on the results of this experiment, the null hypothesis of this study was rejected, and it was concluded that the SID of CP and most AA in BM were less than in FM, SDPP, and PRBC. The SID of CP and AA, except isoleucine, were not different among FM, SDPP, and PRBC. The SID of isoleucine in PRBC and BM was less than in FM and SDPP. This study revealed that FM, SDPP, and PRBC have similar digestibility of AA, except isoleucine, all of which contain high quality AA balance to support the optimal growth of pigs.


This study aimed to examine the effect of taurolidine irrigation on preventing surgical site infection by comparing the spinal infection rate after spinal fusion surgery using vancomycin powder application and taurolidine irrigation. Of 1081 participants, 369 underwent taurolidine irrigation, 221 underwent vancomycin powder application, and 491 were controls. Of the 20 surgical site infections (1.85%), 14 occurred in the control group (2.85%), 5 in the vancomycin group (2.26%), and 1 (0.27%) in the taurolidine group. Among the various variables, age at the time of surgery, smoking, surgical site, and hemovac removal time were significant in the univariate logistic regression. The final result was derived after variable selection using the stepwise method. In the univariate model, the odds ratios were 0.09 and 0.79 in each of the vancomycin and taurolidine groups compared to that of the control group. In the multivariate model, the odds ratios were 0.09 and 0.83 in each of the vancomycin and taurolidine groups compared to that of the control group. The preventive effect of vancomycin powder application was not statistically significant. However, the vancomycin group showed a less effective tendency than the taurolidine group. Taurolidine irrigation may be a good substitute for the vancomycin powder application. Surgical site infection is a common healthcare-associated infection and complicates up to 10–20% of operations with considerable strain on healthcare resources ,4,5,6.Spinal surgery is challenging and tends to be invasive; therefore, it has a higher risk of infection than other orthopedic surgeries. Advances in surgical instruments, surgical techniques, and anesthesia have made spinal surgery increasingly available for difficult and complex spinal diseases. Unfortunately, diagnosing and treating more complex spinal diseases results in a higher infection rate after surgery. The infection rate after a simple discectomy or posterior laminectomy is approximately 1%, while that after spinal fusion is 2–5%. Adding posterior instrumentation to spinal fusion increases the risk of infection by 2.4–8.5%. The most frequently infected spinal fusion is anterior-posterior combined spinal fusion ,8,9.Locally applied antibiotics date back to the mid-1800s when Joseph Lister began using carbolic acid as an antibiotic on surgical wounds. The modern use of local antibiotics in orthopedics began with the use of polymethylmethacrylate (PMMA) cement impregnated with antibiotics to treat infected joint prostheses. The use of locally applied antibiotics allows delivery of high concentrations of antibiotics without significant risk of systemic toxicity and has been shown to be effective against biofilms. In addition, vancomycin powder application or aqueous gentamycin irrigation is used as an antibiotic without a carrier and gel, sponge, or calcium sulfate-containing antibiotics are also used as local antibiotics ,11.Since vancomycin’s first application in 2011, it has been used locally in many medical institutions to prevent surgical site infections. However, the effects of local vancomycin administration were not constant. Moreover, the pharmacokinetics of vancomycin powder application remains unclear ,13,14.Taurolidine is an antimicrobial agent frequently used in experimental and clinical studies to prevent intra-abdominal adhesions and sepsis. Initially, it was used to obtain anti-inflammatory and antibacterial effects in surgery and wound infection treatment and to prevent infection caused by central vein catheters. However, there are few studies on the effect of taurolidine in orthopedic surgery. In particular, research is yet to be conducted to determine whether taurolidine can reduce the risk of infection after spinal fusion ,16,17.Therefore, this study aimed to compare the effect of vancomycin powder application to that of taurolidine irrigation in preventing spinal infection at the surgical site after spinal fusion.Patients who underwent anterior or posterior spinal fusion treatment for degenerative diseases of the cervical, thoracic, and lumbar spine at the Sinchon Severance Hospital and Gangnam Severance Hospital between February 2018 and February 2022 were included in this study. Patients with infectious diseases, such as infectious spondylitis, intervertebral discitis, abscesses in the spine, primary or metastatic cancer, open fractures or wounds, and a history of infectious diseases in other body parts and those taking immunosuppressants or antibiotics before hospitalization were excluded from this study. This retrospective study was approved by the clinical trial review board of the hospital. Of 1081 participants, 369 underwent taurolidine irrigation, 221 underwent vancomycin powder application, and 491 were controls. The control group did not receive either vancomycin powder or taurolidine irrigation.In all patients, 1 g of a prophylactic antibiotic that was administered after surgery was administered intravenously within an hour of surgery. Furthermore, most patients were administered 1 g of cefazolin as a preventive antibiotic. The operating room environment, including temperature, humidity, and ventilation, or the surgeon, assistant, and nurse was the same for all participants, and all participants wore disposable surgical clothes. After anesthesia, one assistant wiped the surgical site with 7.5% betadine solution and then disinfected the surgical site with 10% betadine solution and alcohol again. The exposed area was sealed using Surgidrap. After the main procedure was completed, sufficient saline was irrigated in all cases, and taurolidine or vancomycin powder was applied before the drainage tube was inserted. In the case of taurolidine , one bottle of taurolidine was heated to 37 °C, 750 mL of saline was mixed with 250 mL of taurolidine, and a total of 1 L was irrigated with bones, muscles, fascia, subcutaneous fat, and dermis using an irrigation spoid at the surgical site. In the case of vancomycin , vancomycin powder was finely crushed, and approximately 2/3 was applied to the bones and muscles. The remaining powder was applied to the subcutaneous fat and dermis. The amount of vancomycin applied varied between 0.5–2 g depending on the wound size and surgical type; however, most patients were administered 1 g. After surgery, the same preventive antibiotics used preoperatively were administered intravenously until the first postoperative day.Participants’ characteristics, including sex, age, height, weight, body mass index, history of smoking, high blood pressure, diabetes, kidney disease, respiratory disease, preoperative white blood cell (WBC) and neutrophil counts, history of spinal surgery and spinal infection, postoperative blood sugar level, instrumented fusion level, bone graft type, bleeding during surgery, blood transfusion, time of hemovac removal, and volume of hemovac drainage were collected. Since posterior implantation was performed in all participants, it was determined by following up for 90 days after surgery according to the WHO standards . In addip-value ≤ 0.05.A statistical analysis of the Kruskal–Wallis test was conducted for numerical variables between groups, and the chi-square test was conducted for categorical variables. Univariate logistic regression was performed to determine the factors affecting the infection status. After univariate logistic regression was performed for each independent variable, multivariate regression analysis was performed. Variable selection was performed using the stepwise method for significant variables in the univariate logistic regression, and the final model result was indicated. Statistical analysis was performed using SAS version 9.4 . Statistical significance was set at a 3/μL (range 2.29–13.62 × 103/μL) and 4416.40 N/μL (range 1178.52–8748.29 N/μL), respectively. The median amount of bleeding during surgery was 350 cc (range 10–2500 cc), and 200 patients (18.50%) received blood transfusions during surgery. Regarding bone graft type during surgery, 952 patients (88.07%) received autobone grafts, and 129 (11.93%) received autobone and allobone grafts. The median time of hemovac removal after surgery was 3 postoperative days (range 1–11 days), and the median cumulative amount of hemovac was 380.40 cc (range 10–2697.80 cc). After surgery, the median serum glucose and albumin levels (g/dL) were 128.5 (range 53–304) and 3.5 (range 1.70–5.00), respectively. Comparing patient information among the three groups showed no statistical difference in all variables except surgical site and preoperative ANC.Patients’ characteristics and the descriptive statistics between the patient groups are summarized in Of the 20 infected patients, infection occurred in the lumbar vertebrae (60%) in 12 cases, the lumbosacral vertebrae (20%) in four cases, and the thoracolumbar vertebrae (20%) in four cases.Fourteen cases (70%) of superficial infections and six cases (30%) of deep infections occurred. Regarding superficial infections, 10 cases, three cases, and one case were observed in the control, vancomycin, and taurolidine groups, respectively. They were treated using secondary sutures after debridement and sufficient irrigation in the ward or operating room, depending on the wound size. The mean postoperative infection date was 14 ± 5 days.Regarding deep infections, four cases, two cases, and no cases were observed in the control, vancomycin, and taurolidine groups, respectively. They were treated using secondary sutures after debridement and sufficient irrigation in the operating room. The mean postoperative infection date was 16 ± 6 days.Staphylococcus aureus . The remaining (35%) were caused by gram-negative bacteria in five cases of Escherichia coli isolates , one case of Klebsiella pneumoniae, and one case of Pseudomonas aeruginosa  of infections were caused by ruginosa .p-value of taurolidine was 0.022; therefore, it can be said that the use of taurolidine affects infection. The risk of infection in the taurolidine group was 0.09 times lower than that in the control group. In the multivariate model, the p-value of taurolidine was 0.02; therefore, it can be said that taurolidine affects infection while correcting the correction variables . The risk of infection in the taurolidine group was 0.09 times lower than that in the control group. In the vancomycin group, the p-value was less than 0.05, and there was no significant difference; however, in the univariate model, the risk of infection in the vancomycin group was 0.79 times higher than that in the control group. In the multivariate model, the risk of infection in the vancomycin group was 0.83 times higher than in the control group  methane] was introduced in the 1970s and was originally used as an antibiotic to prevent bacterial infections in the abdominal cavity of patients with peritonitis . In addiStaphylococcus aureus, Staphylococcus epidermidis, Bacteroides fragilis, and group C streptococci [Staphylococcus aureus, Staphylococcus epidermidis, and Streptococcus species. Therefore, we assumed that taurolidine could contribute to reducing the risk of infection after spinal fusion surgery.Taurolidine has a unique spectrum of antibacterial activity against gram-positive and gram-negative bacteria and fungi ,30. Taurptococci . Tauroliptococci . The mosThis study showed the effectiveness of taurolidine irrigation in over 1000 patients who underwent spinal fusion. Of the 369 patients who received taurolidine, only one patient was infected. Compared to the patient group treated with vancomycin powder, infection occurred with a much smaller probability in both patient groups without vancomycin. In addition, comparing the control and taurolidine groups, the odds ratio was 0.09 in the univariate regression analysis and 0.09 in the multivariate regression analysis, showing very effective infection prevention by taurolidine. In this study, no side effects of taurolidine were found.The risk factors for postoperative infection can be largely divided into patient and surgical factors, including revision surgery, infectivity in other areas, diabetes, smoking, obesity, malnutrition, poor immune function, and long–term hospitalization before surgery ,33,34,35Surgical factors included the type of surgery, presence of instrumentation, the extent of dissection, time of surgery, and amount of bleeding. Wimmer et al. reported no infections in 276 anterior approaches; however, 3.8% of infections occurred in posterior approaches . In thisPreventive systemic antibiotics should also be considered. In this study, cefazolin was administered to patients before and after surgery at the same dose and at the same time. Preventive antibiotics are not administered as a concept to sterilize tissue, but as a concept of helping to keep the load of microorganisms infected during surgery below the host defense level and are not preventing infection through postoperative incision wounds . TherefoThis study had some limitations. First, it was impossible to present an appropriate use of preventive taurolidine except for intermittent irrigation at the surgical site with a 0.5% taurolidine injection heated to 37 °C. Second, because this was a retrospective study, the same number of patients could not be matched for each surgical site. Finally, it was impossible to include all the diverse risk factors for spinal infection.Surgical site infections after spinal fusion can lead to serious morbidity and mortality. This study showed that taurolidine irrigation could effectively prevent infection at the surgical site after spinal fusion. It may be an excellent substitute to compensate for the complications and problems associated with vancomycin powder application.