Many in vitro and in vivo studies support that green coffee polyphenols (GCP) and beta-glucans (BG) present important hypolipidaemic and hypoglycaemic effects. However, their weight-management/-reducing properties are less clear. Considering that these compounds act on different metabolic pathways, their combination could increase their beneficial health effects. The aim of the present study was to investigate the effects of regularly consuming supplements containing GCP, BG or the novel GCP/BG combination on body composition in overweight/obese subjects without changing their dietary and physical activity habits, hence addressing the difficulty to adapt to lifestyle changes. A randomised, cross-over, blind trial was carried out in 29 volunteers who consumed GCP (300 mg), BG (2.5 g) or GCP/BG (300 mg + 2.5 g) twice a day for 8 weeks. At the beginning and end of each of the interventions, body weight, body mass index, body fat%, intracellular and extracellular water, skinfolds  and body circumferences  were measured. Along the study, volunteers filled out 72 h dietary records, and physical activity was measured using accelerometers. The results show that dietary intake and physical activity were unchanged throughout the study; however, there were no changes in any of the body composition parameters analysed with any of the food supplements. In conclusion, the regular intake of GCP, BG and GCP/BG, without changes in diet and physical activity, is not an efficient strategy to lose weight or induce other positive changes in body composition, although results should be taken with caution as the study was underpowered. Over the last 30 years, the prevalence of obesity has increased significantly, and nowadays around 39% of the world’s population is affected. Obesity is more than an imbalance between energy intake and energy expenditure; thanks to the continuous scientific advances in the field, it is known that obesity is a multifactorial disease involving genetic, epigenetic, environmental and socio-economic factors . MoreoveRegarding DF, both soluble (SDF) and insoluble (IDF) dietary fibre may help to lose weight, interfering in the digestion and absorption of macronutrients, delaying gastric emptying and increasing satiety. However, only SDF can be metabolised by the intestinal microbiota and induce prebiotic effects by promoting the growth of lactic acid bacteria and bifidobacteria ,6. The g2 following the intake of 4.4 g/day of BG-rich barley for 12 weeks  to better estimate the effects produced by the treatment, evaluating how much the variable increased or decreased with respect to the baseline value. Data are shown in the tables as means ± standard error of the mean.Volunteers kept their dietary habits unchanged during the 32 week study, according to the 72 h dietary records . There w2 and the accumulation of CO2 in the air camera. In our study, the measurement of O2 and CO2 was 0.28 ± 0.01 mL/min/m2 and 0.23 ± 0.01 mL/min/m2, respectively. Considering these results, the average resting energy expenditure was estimated to be 1546 ± 110 and 1964 ± 138 kcal/day, for women and men respectively.Indirect calorimetry is based on the principle of gas exchange, and breathing in a calorimeter produces the depletion of OUnquestionably, limiting the energy intake from total fats and sugars, increasing the consumption of fruit and vegetables, as well as legumes, whole grains and nuts, and engaging in regular physical activity are the best strategies to reduce the incidence of overweight and obesity . HoweverThe monitoring of volunteers’ dietary habits showed that the intake of kilocalories, macro- and micronutrients did not change during the study . TherefoRegarding physical activity, there were no differences between the interventions, so the volunteers also followed the study instructions in this respect . PhysicaSaccharomyces cerevisiae, a statistically significant reduction in BMI, body weight (−1.8 kg) and WC was described [t-test was carried out, only the change rate of total body fat% was significantly lower with 5 g/d of the 70% BG [Sound scientific evidence supports that BG may promote health by improving glucose and lipid metabolism as evidenced by the positive opinions issued by EFSA ,17,18. Hescribed , but it escribed . In contescribed . In accoescribed , two outescribed  or with escribed . The resescribed ) or to tescribed , two dose 70% BG , and thuSimilar disagreement exists regarding the antiobesity effects of hydroxycinnamates. In the recent systematic review carried out by Coman and Vodnar  on the eThe idea of combining BG and GC resulted from the attempt to understand if these compounds could exert complementary or synergistic actions, considering that the mechanisms of action of PP and SDF are different, as they affect the absorption and metabolism of nutrients (mainly fats and sugars) differently, with thermogenic effects, promoting satiety, acting as laxatives, etc. (revised in ). In a rKnowing that the GCP/BG food supplement used here had induced changes in total body fat percentage in the dose–response trial previously described , in the Clostridium orbiscindens and Clostridium spp.), Roseburia hominis, and Ruminococcus spp. and a decrease in Firmicutes and Fusobacteria after the administration of whole-grain barley pasta containing 3 g/day of barley BG for 2 months [Bifidobacterium spp. [Another factor that could also affect the bioactivity of BG, GC and BG/GC is their effects on the gut microbiota. As indicated in the introduction, gut microbiota is linked with weight gain via an increase in the ability of the altered microbiota to extract energy from the food, such as in people with obesity. Moreover, an increase in the Firmicutes:Bacteroidetes ratio in overweight/obese subjects has been described in comparison to healthy individuals . BG can 2 months . However2 months  the chan2 months . On the 2 months ). Among 2 months , and tha2 months . However2 months  in healtium spp. . ConsideLimitations: This study was carried out on 29 subjects and thus it was underpowered. It is possible that our study was not long enough, since in a previous 12-week-long intervention significant differences have been observed ,32, althThe regular intake of the studied food supplements containing GCP, BG and GCP/BG, without changes in dietary and physical activity habits, is not an efficient strategy to lose weight or produce other positive changes in body composition in overweight or obese people. However, we do not dismiss the possibility that, if the study had been carried out in a larger group of volunteers, and/or if the interventions had been longer, positive effects would have been observed, considering the health properties of BG and GCP.


Cardiovascular (CV) morbidity and mortality increase along with the progression of chronic kidney disease (CKD). The potential novel biomarkers of cardiotoxicity have been tested with the aim of the early detection of patients at high CV risk, and among them are markers of inflammation, oxidative stress, acute renal injury, and microRNAs. The study analyzed biomarkers in non-dialysis-dependent  and dialysis-dependent (DD) CKD patients. The prospective cohort study included 87 patients who were followed for 18 months, during which period newly occurred CV events were recorded. Cox regression analysis confirmed serum albumin, urea, interventricular septum thickness diameter (IVST), the use of calcium antagonist, and erythropoiesis-stimulating agent to be significant predictors of CV outcome. No significant difference was observed in biomarkers of inflammation, oxidative stress, acute kidney injury , and miR-133a, in regards to the presence/absence of CV event, CV death, and left ventricular hypertrophy. Serum albumin, urea, IVST, and the use of calcium antagonist and erythropoiesis-stimulating agents were confirmed to be factors associated with CV events in CKD patients. Apart from traditional risk factors, new research is needed to define novel and reliable biomarkers of cardiotoxicity in CKD patients. Chronic kidney disease (CKD) has a great impact on global health, as a direct cause of global morbidity and mortality, especially for the occurrence of cardiovascular (CV) diseases, and CKD is recognized as an independent and strong risk factor for their occurrence . CV morbApart from the traditional risk factors that contribute to increased CV risk, the literature data confirmed that non-traditional risk factors play an important role in the very complex pathophysiology of CV events in the CKD population ,7. ThereInflammation and oxidative stress are well-known contributors for atherosclerosis, which is among the underlying mechanisms not only for progressive kidney damage, but also for CV comorbidity ,9,10,11.Catalase, glutathione peroxidase, glutathione reductase, and superoxide dismutase (SOD) are among the main enzymatic antioxidants in the human body . SeveralIschemia-modified albumin (IMA) is also one of the novel biomarkers, and its values were altered in conditions of ischemia, inflammation, and high oxidative stress, and, therefore, it has been tested as a potential biomarker for ischemic heart disease ,19. AccoNeutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule-1 (KIM-1) are most commonly examined from the group of markers of acute kidney injury, and could potentially play a role in the pathogenesis of CKD and the prediction of CV events. It has been described that elevated plasma NGAL in CKD patients may be an independent predictor of future CV events . Also, iGiven that previous research has led to conflicting results, the search for new biomarkers is justified. One of the newest are microRNAs (miRNAs). Previous studies showed the association of various miRNAs with CVD pathogenesis, and altered levels of these biomolecules have been observed in patients with LVH, CAD, HF, PAD, and ischemic cerebrovascular insult (ICVI) ,28,29,30Since the aforementioned biomarkers have not been tested together at different stages of CKD, our study aimed to test different biomarkers as potential markers of cardiotoxicity in CKD (stage 3a–4) and ESRD patients. The influence of applied therapy, comorbidity, echocardiographic parameters, and standard laboratory biomarkers were also analyzed.Our study included 87 patients who developed CKD mainly due to hypertensive nephroangiosclerosis (50%) and diabetic nephropathy (34.5%). Also, the majority of patients had a diagnosis of arterial hypertension (98%) and diabetes mellitus (41.2%), either as underlying renal disease or comorbidity.After the 18-month follow-up period, 52 (57.4%) CV events were registered, including 8 (9.2%) CV deaths. The most common event was angina pectoris de novo 14 (26.9%), peripheral arterial disease for 12 (23.1%), angina pectoris non-stabilis for 8 (15.4%), heart failure for 6 (11.5%), myocardial infarction for 3 (5.8%), and cerebrovascular insult for 1 (1.9%).The clinical characteristics, laboratory results, and applied therapy were compared between patients who developed CV events and those who did not develop CV events . The DD p = 0.001; OR: 0.60, 95% CI 0.446–0.817). For each unit of urea increase of 1 mmol/L, the probability of the occurrence of CV events increased by 1.33 times . With each unit of increase in IVST diameter of 1 mm, the probability of the occurrence of CV events increased by 3.82 times .Cox regression analysis showed that serum albumin, urea, IVST diameter, the use of a calcium antagonist and erythropoiesis-stimulating agent proved to be significant predictors of CV outcome . For eacp = 0.016; OR: 23.33, CI 95% 1792–303,989). The patients with erythropoiesis-stimulating agents as chronic therapy were 99% less likely to develop a CV event .The patients with calcium antagonists as chronic therapy were 23.33 times more likely to develop a CV event compared to patients who did not use it , when the changes are irreversible. Indeed, the authors confirmed that the risk factor for CV morbidity starts very early during CKD progression, being more prominent with stage 3b and lower. Therefore, in recent years, studies have focused on the earlier stages of CKD, at which time preventive measures may yield some promising results.After the follow-up period, a significantly higher number of CV events was registered in the HD group of patients. In agreement with previous knowledge, significant CV events may be observed from stage 3b, increasing to stage 5HD ,3. In thWe have shown that patients with CV events had lower hemoglobin and albumin values compared to patients without CV events, and this finding is consistent with early claims that anemia and malnutrition are very important nontraditional risk factors for CV diseases in patients with CKD ,32. The In this paper, we confirmed that, with each increase in albumin value by one gram, the probability for the occurrence of CV events decreased by 40%. Therefore, the prevention and early treatment of malnutrition are of vital importance in the preservation of the CV system in CKD patients . Our finUnlike albumin, the role of IMA in the pathogenesis of CV outcome is less analyzed. Albumin is the most abundant protein in human plasma, and acts as a scavenger for divalent metal ions via the N-terminal part of the molecule. In terms of altered albumin, IMA is a form of human albumin in which the N-terminal amino acids are unable to bind to transition metals. IMA formation may occur not only under acute, but also chronic, conditions, such as chronic oxidative stress and inflammation, as observed in dialysis patients. Increased IMA can, in turn, aggravate the severity of oxidative stress and inflammation . In ESRDThe present study confirmed that the number of echosonography parameters related to left ventricular enlargement, such as LVM, LVMI, LVPWT, and IVST, was increased in patients with CV events, but only IVST proved to be a potential predictor of CV events in CKD patients. It was already confirmed that LVH is very frequent in CKD patients, even in the pre-dialysis stage. It may predict the worse CV outcome, particularly in ESRD patients ,43. We nOur study also suggests that the use of calcium antagonists was associated with increased CV morbidity and mortality, and CKD patients who received calcium antagonists were 23 times more likely to develop CV events. Early studies have led to the conclusion that some patients were more likely to develop myocardial infarction if they used fast-acting calcium antagonists to treat arterial hypertension, instead of beta-blockers, and the risk itself increased with increasing the drug dose . The aboThe interpretation of elevated myocardial markers of necrosis, such as troponin, in patients with renal failure, is still controversial, since it could be elevated in some patients with CKD even in the absence of signs and symptoms of myocardial ischemia . In our The role of more recent biomarkers  in the pathogenesis of CV events were promising, at least on the basis of some reports. The present study found no differences in the values of IL-18, SOD, and IMA between patients with and without CV events, finding that is not in agreement with well-known facts about inflammation and oxidative stress as risk factors for CV disease ,50. AlsoThe literature data on the novel biomarker miR-133a have been followed with enthusiasm. As mentioned, various miRNAs were associated with CVD pathogenesis ,28,29,30Early studies have shown that miR-133a is important for the regulation of cardiac hypertrophy and the onset of HF . There wDespite numerous achievements in the field, the mortality of patients with CKD is unsatisfactorily high. Numerous attempts to find newer, more reliable markers of uremic cardiotoxicity that directly affect CV morbidity and mortality have required substantial effort and resources. Still, it seems that ‘classic’ biomarkers  are the most reliable predictor of CV outcome in CKD patients. Therefore, there is still room for new research that would allow us to timely and efficiently prevent CV disease in CKD patients, thus improving their survival.Our study has several shortcomings, such as a small number of participants and a relatively short follow-up period, and both may be a reason for the less conclusive significance of the evaluated biomarkers. Even so, some well-known markers of CV disease have been confirmed in this paper as well. Unfortunately, the predictive role of the newly investigated biomarker has not been confirmed by this study. Still, this study may offer direction for future research for some new biomarkers, potential cardiotoxins that, either alone or in association with traditional ones, may help in understanding the complex mechanism of CV morbidity and mortality.In conclusion, serum albumin, urea, IVST, and the use of a calcium antagonist and erythropoiesis-stimulating gens were confirmed to be factors associated with CV events in NDD and DD CKD patients.2 and signed informed consent of the participants. Exclusion criteria were as follows: previous CV events , the use of corticosteroids or other immunosuppressive therapy, present active inflammation and malignant disease.We conducted a prospective cohort study with a follow-up period of 18 months during which newly occurred CV events were recorded. The study included 87 patients treated at the Clinical Department of Nephrology with Hemodialysis, Zvezdara University Medical Center, Belgrade, from March 2017 to September 2018. Inclusion criteria were as follows: age over 18 years, CKD with eGFR less than 60 mL/min/1.73 mThe sample size was calculated based on the reported prevalence of CAD among CKD patients, and the prevalence of CAD in Serbia for the determination of differences between the two proportions for the statistical significance of 0.01 and the power of 80%. Another 15% was added to the sample since we expected the dropout ,3,4,5,53Arterial hypertension is defined by values of systolic blood pressure ≥140 mmHg and/or diastolic blood pressure ≥90 mmHg according to the European Society of Hypertension guideline in 2021 .2.Hyperlipidemia is defined by elevated values of total cholesterol ≥5.2 mmol/L and/or triglycerides ≥1.7 mmol/L according to the reference values of the local laboratory. Obesity is defined by BMI (body mass index) values greater than 30 kg/m2, no. = 63) and dialysis-dependent CKD patients (DD CKD) . The eGFR was calculated using the MDRD formula  [2. Since our study included patients with eGFR less than 60 mL/min/1.73 m2, we found the MDRD formula to be appropriate for the staging of CKD in our group of patients [According to the level of eGFR, the participants were divided into the following two groups: non-dialysis CKD patients (NDD CKD) stage 3a–4 (eGFR = 59–15 mL/min/1.73 mdisease) . MDRD stpatients ,56. All As CV events are more frequent only starting from stage 3b CKD, we performed the analysis of the biomarker in 51 patients with stage 3b-5HD ,3.During 18-month follow-up period, each newly occurred CV event was registered including either CV disease or CV death. The following events were recorded: myocardial infarction, new onset or worsening of existing stabile angina pectoris, PAD, HF, and cardiac death. Patients with at least one CV event were classified into the group ’patients with CV events’.Symptoms and signs of heart failure, coronary artery disease, peripheral arterial disease, and cerebrovascular insult,Diagnostic procedures: electrocardiography, color Doppler scan, echocardiography, computerized tomography of endocranium,Biochemical findings and biomarkers.Diagnosis of CV event was based on the clinical examination of patient and data from medical records, as follows:2) was obtained by dividing the obtained value of LVM (g) by body surface area (m2). LVMI over 95 g/m2 for women and over 115 g/m2 for males were criteriums for LVH diagnosis. All patients with echosonographically proven LVH were classified as ’LVH patients’.One experienced cardiologist did a standard, transthoracic echocardiographic examination of the heart, and intra-observer variability was 4%. The measurement was conducted on the Toshiba Artida, the SSH-880 CV model of the echo machine following the guidelines of the American Society for Echocardiography . An echoBlood for laboratory analysis for NDD CKD patients was sampled in the morning after 12 h from the previous meal. In dialysis patients, blood was sampled just before dialysis, and after the longest pause from the previous dialysis, and an average of three consecutive measurements is shown. The following parameters were determined using standard laboratory techniques: hemoglobin, calcium, phosphorus, parathyroid hormone, total proteins, albumins, cholesterol, triglycerides, uric acid, ferritin, C-reactive protein, Tnl-Ultra troponin.The tested biomarkers  were determined from the serum and urine (NGAL) by ELISA testing . Blood was sampled at the beginning of the study in tubes without anticoagulants, centrifuged at 4 °C for 10 min at 3000 rpm, serum was separated into cryotubes and frozen at −80 °C until testing. C-reactive protein and ferritin were also tested as markers of inflammation using standard laboratory techniques and assays.For analysis of miR-133a, serum samples were collected into cryotubes and frozen at −80 °C until use. Total RNA was extracted from all serum samples using PureLink viral RNA/DNA mini kit  according to the manufacturer’s protocol. The RNA concentration and purity were determined by UV absorption at 260/280 using spectrophotometer. Reverse transcription was performed using TaqMan microRNA reverse transcription kit  according to the manufacturer’s protocol.Level of miR-133a was measured by quantitative real-time PCR (qRTPCR) using TaqMan miRNA probe . All analyses were performed in triplicate. For all experiments miR-16 was used for normalization as an internal control .Mir-16 is the most frequently used circulating miRNA endogenous control. The inclusion criteria for mir-16 as reference miRNA used in our study were previously reported, and are as follows: (a) detection in all analyzed samples; (b) quantification cycle less than 35, and (c) no significant difference in quantification cycle (Ct) between groups ,59.The qRTPCR was performed on 7500 real-time PCR system  using the following program: 50 °C for 2 min; 95 °C for 3 min; 40 cycles of 95 °C for 15 s and 60 °C for 1 min.miR-133a − CtmiR-16. There is an inverse correlation between value of ΔCt and serum level of miR-133a .The value of ΔCt of miR-133a represents the difference in the level of targeted miR-133a and the endogenous control of miR-16. Further, ΔCt was calculated according to the following formula: ΔCt = Ctp-value of less than 0.05 was considered statistically significant.Statistical analysis was performed using Statistical Package for Social Sciences 22.0 . Data were expressed as means ± standard deviation (SD) for continuous variables and percent values for categorical variables. Differences between groups were analyzed using the following: independent sample t-test, analysis of variance test (ANOVA), independent samples Mann–Whitney U test, Kruskal–Wallis test, and Pearson χ2 test. For correlation of variables Spearman correlation test was used. To test the normality of parameters the Shapiro–Wilk test was used. All variables shown as significant were entered in the Cox regression model with CV event as an outcome variable. A


The sex steroid hormones (SSHs) play several roles in regulation of various processes in the cardiovascular, immune, muscular and neural systems. SSHs affect prenatal and postnatal development of various brain structures, including regions associated with important physiological, behavioral, cognitive, and emotional functions. This action can be mediated by either intracellular or transmembrane receptors. While the classical mechanisms of SSHs action are relatively well examined, the physiological importance of non-classical mechanism of SSHs action through membrane-associated and transmembrane receptors in the brain remains unclear. The most recent summary describing the role of SSHs in different body systems is lacking. Therefore, the aim of this review is to discuss classical and non-classical signaling pathways of testosterone and estradiol action via their receptors at functional, cellular, tissue level and to describe the effects on various body systems and behavior. Particular emphasis will be on brain regions including the hippocampus, hypothalamus, frontal cortex and cerebellum. In addition, SSHs play a pivotal role in brain structure formation and cognitive function . FurtherSSHs shape the brain during the critical prenatal and perinatal periods of development  when hormones interact with an immature neural substrate. In this period of life, exposure to SSHs can cause permanent sex differences in brain structures and their functions, which are responsible for the sexual differentiation of the brain and behavior . Prenata2)) via classical and non-classical receptors and their role in different body systems is lacking. In this review, we aimed to sum up what is known and update the latest knowledge regarding the role of T and E2 in various tissues and body systems with specific focus on the brain via classical and non-classical signaling pathways. First, we discuss the specific effects of T and E (E2) via different receptors  on various body systems. Subsequently, we describe the role of T and E2 in hippocampus (HIP), hypothalamus (HYP), frontal cortex (FC), and cerebellum (CER) – selected brain regions associated with important physiological, behavioral, cognitive, and emotional functions.However, a current summary of results of experimental or clinical studies describing the role of testosterone (T) and estrogen , the major androgen, is aromatized to estradiol by the enzyme aromatase and also reduced to the non-aromatizable androgen dihydrotestosterone (DHT) by 5α-reductase .As mentioned previously in the introduction, these hormones play a crucial role throughout the development and exert diverse physiological functions in the body . Moreove2.1.The actions of androgens and estrogens were historically thought to be slow, nuclear processes mediated through hormone receptors located in the cytoplasm complexed to chaperons or in the nucleus, i.e. intracellular AR and ERs . These pMoreover, in the past couple of decades, rapid signaling through 7-transmembrane GPCR receptors, including the androgen receptor ZIP9 and GPER1 has been discovered . The rolThere are numerous studies describing the molecular signaling pathways of these receptors and the effects of SSHs via these receptors on the brain, body and behavior . The res2.1.1.The binding of T and DHT to nuclear AR was first described in 1968 , 1968b. The classical and non-classical signaling pathways via this receptor are activated through androgen hormone ligands, predominantly T and DHT  in the cytoplasm . In the Fast, non-classical actions mediated by AR associated with the membrane in a complex with caveolin 1 have been already discovered . These a2.1.2.Intracellular ERs were first identified in 1958 . Their eThe concentrations of ERs are the highest during the critical or sensitive periods of life , which supports the important role of E in the organization of hippocampal structure during its development . The cel2) has equal affinity to both, whereas estrone preferentially binds to ERα and estriol to ERβ , G-proteins, striatin or Src tyrosine kinases . There al to ERβ .ERα is crucial for the physiological development and functions of many organs and systems, such as reproductive, central nervous, cardiovascular and skeletal systems . The strAs well as AR, ERα can activate the non-classical, rapid signaling cascade through its association with the cell plasma membrane. This signaling, together with the caveolin-binding protein striatin, activates MAPK, phosphatidylinositol 3-kinase and Akt kinase in the vascular endothelial cells leading to higher activity of endothelial NO synthase . These rERβ is important for migration of neurons and glial cells, or neural differentiation of embryonic stem cells, especially for differentiation of midbrain neurons . Further2 as well as T, binds to GPCR receptors.Concerning the non-classical effects of ERβ, in cardiovascular system, especially in cardiomyocytes, ERβ activation stimulates PI3 kinase which increase the modulatory calcineurin-interacting protein 1 gene and protein expression, which subsequently inhibits calcineurin activity increased by angiotensin II and prevent the hypertrophy . The cla2.1.3.2  transcription in breast cancer cell lines  . It has ll lines . In the ll lines . GPER1 sll lines  and can ll lines . Howeverll lines . Regardill lines . Moreovell lines .2 binds GPER1 with high affinity and this activation leads to ERK phosphorylation, PI3K stimulation, intracellular Ca2+ increase, and cAMP production in the MCF-7 breast cancer cell line, which occurs via trans-activation of the epidermal growth factor receptor and results in proliferation  will be discussed.Effects of GPER1 signaling in the brain and other organs are summarized in 3.2 are the enzymes converting T to E2 (aromatase) or DHT (5α-reductase). The dissimilarities in concentrations of androgens, estrogens, aromatase, and 5α-reductase, as well as in expression of SSH receptors  in specific areas of the brain contribute to behavioral heterogeneity between males and females  and neurons throughout the entire nervous system already have receptors for these sex hormones . Prenata females . The rol3.1.2, increases cell density by stimulating neuronal cell proliferation in the HIP  and posterior (neurohypophysis) parts of the pituitary gland ; therefore, it supports body homeostasis by regulation of endocrine system and autonomic behavior . Neurons2, while in adulthood greater conversion of T to DHT occurs in male rats  manage early development of HYP neurons. This management involves the sex specific neuritogenesis and regulation of the process of neuritogenic factor neurogenin 3 expression stimulated by ERα signaling in the HYP . It was 2 promotes many actions in the HYP. For example, it increases the expression of glial cell neurotrophic factor in hypothalamic neurons, but not in astrocytes, through non-classical E action   . E2 alsoabundant . Moreove nucleus . In addiium rise .3.3.Androgens seem to be critical modulators of executive functions in the mesocorticolimbic area, including the prefrontal cortex (PFC) . Early eUnlike higher concentration of T, DHEA is associated with a prepubertal increase in neuron density in various cortical regions, which can positively facilitate cortical functions such as attention and working memory . Male an2 modulates the development of catecholamine activity during neonatal period in male FC   . Moreoveemotions . In adultions  in comparison to adulthood  of male rats, and highest in the female rat HIP in comparison to other brain regions of female rats. During the first PND, the concentrations of E2 decreased in the majority of brain regions and the only sex difference remained in its hypothalamic concentrations , which begin at about 1 year of age  via their classical or non-classical pathways at functional, cellular, and tissue levels, with the main focus on brain regions involved in cognition, including the HIP, HYP, FC and CER. The role of SSHs in modulation of behavior in both humans and laboratory animals is described. SSHs are involved in regulation of many body systems such as reproductive, immune, muscular, cardiovascular, skeletal and neural. SSHs affect these systems differently via different receptors. Although the actions of intracellular AR are central for example for bone healing, glucose and fat metabolism and β-amyloid plaque reduction, the role of membrane-associated AR is pivotal in process of neuronal apoptosis, cell survival and cell proliferation. ERα plays an important role in energy metabolism, insulin resistance, fat accumulation or atherosclerosis. On the other hand, the fast, non-classical actions of ERα result in endothelial NO activation, lipolysis, bone growth and beiging of adipocytes. Intracellular ERβ is involved in neural differentiation and in oncogenesis and metastasis suppression, whereas non-classical ERβ signaling can reverse pre-existing heart failure or inhibit hypertrophy of cardiomyocytes. Concerning transmembrane GPCR receptors, ZIP9 is crucial for male fertility, spermatogenesis, or apoptosis, whereas GPER1 is responsible for proper functioning of gonads in both males and females.SSHs are crucial for the proper development and function of the body in both, males and females. The research regarding the effect of SSHs on different organs and body systems, especially the brain, is moving forward very quickly, and it is important to stay abreast of the latest developments. The present review summarizes the latest information on the effects of SSHs , increases male sexual or aggressive behavior (HYP), regulates executive functions and control of emotions (FC), and decreases neuroticism and regulation of male sexual behavior (CER). E2 increases glutaminergic synapse formation and neurogenesis in HIP, modulates synapse formation, increases axodendritic synapses and axogenesis in HYP, and regulates neurotransmission in the FC and cell growth in the CER. E2 also improves object recognition and spatial memory consolidation , controls aggressive behavior and anorectic actions in females (HYP), and regulates modulates motor memory formation (CER).Regarding the effects of T or E2 in the brain confirm the importance of these SSHs in regulation of structural changes in the brain with an impact on behavior and cognitive function. Plenty of studies have been published describing the molecular signaling pathways of SSHs receptors and their effects on the brain, body systems and behavior. The results of these studies have brought new insights into the neurobehavioral effects of T and E2. However, additional questions have arisen, such as the sex-, age- and tissue-specific role of rapid, non-classical mechanisms involving the GPCR ZIP9 and GPER1 receptors, mainly the brain. The answers to these questions will provide a more complete picture of how SSHs regulate the functional of neural and non-neural systems.Both classical and non-classical actions of T and E


Bacillus subtilis, B. licheniformis, Saccharomyces cerevisiae cell walls and silymarin against the deleterious effects of Aflatoxin B1 (AFB1) and Ochratoxin A (OTA) in broiler chicks. A total of 160 one-day-old Ross 308® broiler chicks were randomly allocated in four treatment groups, with four replicates, according to the following experimental design for 42 days. Group A received a basal diet; Group B received a basal diet contaminated with AFB1 and OTA at 0.1 mg/kg and 1 mg/kg, respectively; Group C received a basal diet contaminated with AFB1 and OTA and MMDA at 1 g/kg feed, and Group D received a basal diet contaminated with AFB1 and OTA and MMDA at 3 g/kg feed. Results showed that ingested mycotoxins led to significant (p ≤ 0.05) reduction in body weight and feed conversion from 25 days of age, induced histopathological changes, increased the pH of the intestinal content, and altered the biochemical profile of birds with significantly (p ≤ 0.05) increased aspartate aminotransferase (AST) values (p ≤ 0.05). On the other hand, the supplementation of MMDA significantly (p ≤ 0.05) improved the feed conversion ratio (FCR) during the second part of the study, diminished biochemical alterations, reduced pH in jejunal and ileal content, and E. coli counts in the caeca of birds (p ≤ 0.05). It may be concluded that the dietary supplementation of the MMDA partially ameliorated the adverse effects of AFB1 and OTA in broilers and could be an efficient tool in a mycotoxin control program.The present study was designed to determine the efficacy of a novel multicomponent mycotoxin detoxifying agent (MMDA) containing modified zeolite (Clinoptilolite),  Mycotoxicosis was first described in 1960 when a shipment of Brazilian peanut meal was used in the diets of poultry and other domestic animals in the UK. This caused an outbreak of an unknown disease that killed more than 100,000 turkeys and was reported as the Turkey “X” Disease. Later, mycotoxins, which are a group of fungal secondary metabolites with diverse biological activity, were identified as the etiological factor . These lAspergillus spp., particularly A. flavus, A. parasiticus and A. nomius, which primarily grow in regions with tropical or subtropical climate [1 (AFB1) consists of the most detected mycotoxins in cereals and has the greatest toxicity. In both, humans and animals, AFB1 is reported to cause carcinogenic, mutagenic or teratogenic effects, and is therefore categorized as human carcinogen group I [1 may induce clinical signs, like mortality, decreased egg production, reduced body weight, and feed efficacy [1 is usually detected at low concentrations in poultry feeds, which may lead to subclinical effects, including slightly reduced production, immunosuppression, and vaccination failure [Among almost 500 identified mycotoxins, Aflatoxins (AFs) are considered the most hazardous for human and animal health. AFs are mainly produced by fungi belonging to the genus  group I . In a vaefficacy . However failure .Aspergillus spp. and Penicillium spp. The group consisted of three metabolite forms (A–C), with Ochratoxin A (OTA) being the most important. OTA is classified by the International Agency for Research on Cancer as a possible human carcinogen (Group 2B) and is associated with immunotoxic, neurotoxic, hepatotoxic and embryotoxic effects in both humans and animals. OTA is a very stable substance in high thermal conditions and thus is very difficult to eliminate by physical methods, once occurred in the feed [Ochratoxins are secondary metabolites, produced by fungi mainly belonging to genera the feed . OTA is the feed .1 is the most potent hepatotoxic associated with an enlarged, pale and friable liver, with histological findings of fatty infiltration and bile duct proliferation [Long-term epidemiological surveys indicate that a large fraction of global feedstuffs is exposed to more than one mycotoxin ,12. The feration . On the feration . Both myferation ,9,15,16.1 and OTA can lead to less apparent hepatic and more severe kidney lesions, which may be a burden in achieving diagnosis [1 and OTA show weak cytotoxic effect in liver cell lines, when are in combination than alone, suggesting an antagonistic interaction [1 and OTA in concentrations as low as naturally found in foodstuffs.However, when AFs and OTA co-existed in the poultry feeds, their interaction leads to less additive and more antagonistic effects in biochemical parameters of birds, including total proteins, albumins, cholesterol, creatinine, and uric acid ,18,19. Miagnosis . This caeraction . In conteraction ,18. A wi1 in complete feed, with 12% moisture content, for poultry (with the exception of young ones) is 0.02 mg/kg [Recognition that mycotoxins affect health and productivity in poultry farming has set maximum levels or guidance values for mycotoxins. According to directive 2002/32/EC on undesirable substances in animal feed, the limit imposed for AFB02 mg/kg . Further02 mg/kg . The Foo02 mg/kg ,19.Despite many years of research and interventions in pre-harvest, harvest and post-harvest levels, mycotoxins continue to be a considerable threat to the poultry industry . As harv1 [1, they are not equally efficient for other mycotoxins, such as trichothecenes. Therefore, alternative approaches for the amelioration of mycotoxicosis adverse effects in broiler chicks are required [One of the approaches to this global concern is “binding” of mycotoxins in the gastrointestinal tract of animals and reducing their bioavailability, with the use of inorganic adsorbents, such as clay minerals, bentonite and zeolite in animal feed . Clinopt1 ,26. Howerequired . Novel arequired .Bacillus licheniformis CFR1 shows an AFB1 degradation of more than 90% when tested in vitro, whereas yeast cell wall containing beta-glucans and mannan oligosaccharides can efficiently bind with AFB1 up to 90% [Moreover, the use of specific microorganisms, including bacteria, fungi and yeast, or enzyme systems that effectively bio-transform non-binding mycotoxins into some less-toxic metabolites, is a promising alternative. Rao et al.  reportedp to 90% .1 and OTA when co-occurring in broilers feed at realistic or occasional levels. If the dietary supplementation of a novel multicomponent mycotoxin detoxifying agent could ameliorate the negative effects induced by Aflatoxin-B1 and Ochratoxin A on the intestinal ecosystem, then the performance of broiler chicks could be improved. Thus, this study aims to investigate the efficacy of a novel multicomponent mycotoxin detoxifying agent (MMDA) containing modified zeolite (clinoptilolite), B. subtilis, B. licheniformis, S. cerevisiae cell wall and silymarin, on the physicochemical properties of the intestinal content, gut microbiota, histology, serum biochemistry and performance in broiler chickens ingesting AFB1-and OTA-contaminated feed.However, only limited research studies exist, using a combination of the previously mentioned approaches that investigate their ameliorating effects against AFBDaily clinical examination of birds revealed sporadic clinical signs, indicative of mycotoxicosis, including ruffled feathers, diarrhea and growth retardation in groups that received contaminated feed. Clinical signs started on day 17 and were continued until the end of experiment (42nd day). During the experimental period, no mortality was recorded among the tested groups. The effect of mycotoxins and multicomponent mycotoxin detoxifying agent (MMDA) on the performance of birds per treatment group is shown in p < 0.001 for all comparisons). On the other hand, average daily weight gain (ADWG) mean values did not show significant differences among groups at any point of the study. Feed conversion ratio (FCR) alterations between groups and time periods showed a quite different pattern. At the mid-point of the study (25 days of age), the FCR of the three groups receiving AFB1 and OTA were significantly reduced when compared to group A . On the contrary, at the end time point of the study (42 days of age) the FCR improved in the groups receiving the test product (C and D group) in comparison with the B group. The comparisons among groups for the total trial period suggested a numerical improvement of FCR and Adjusted FCR in C and D groups in comparison with the B group. Nevertheless, the FCR comparisons showed a trend for statistically significant improvement in C and D groups when compared with the B group .Feed contamination with Aflatoxin B1 (AFB1) and Ochratoxin A (OTA) did not significantly affect the body weight (BW) of birds for the first two weeks when compared to the controls. However, from the 25th day of the trial and onwards, the BW of animals receiving mycotoxins either with or without the test product was significantly lower than the control group . Such observation was not evident for the lower dosage level group C (1g test product/kg feed). On the other hand, ALT values were significantly improved in group D, irrespective of the factor time, when compared with group B (p = 0.007). Both of the abovementioned evidence sources support a liver-protective effect of the test product when added at the greatest concentration level. However, aspartate aminotransferase (AST) values were significantly increased in the groups that received mycotoxins  in comparison with group A at both sampling time points (p < 0.001) for all comparisons except comparison between groups D and A at first sampling: p = 0.007).Total protein, albumin, alkaline phosphatase (ALP), alanine aminotransferase (ALT) and gamma-glutamyl transferase (γ-GT) values in all experimental groups were not significantly different neither at 25, nor at 42 days of age. A significant increase of glucose levels was observed in group D that received the greater dosage level of the test product in feed (3 g test product/kg feed) when compared with group B (p = 0.042) in the group that received MMDA at 3 g/kg feed when compared to other experimental groups.The results of the gross lesion score are presented in 1 and OTA led to a significant increase in the pH value of the duodenal and jejunal content in comparison with the control group. However, the addition of MMDA at 3 g/kg feed significantly ameliorated the increase of the pH value in the jejunum (p = 0.001). The pH of the ileal content was significantly lower in groups that received MMDA either at high or low dosage level, when compared to the control group . Moreover, the addition of MMDA in 3 g/kg led to a significant increase in the caecal pH. The viscosity of jejunal and ileal content was insignificantly different among groups. However, the highest jejunal and ileal viscosity values were noted in groups that ingested mycotoxins without the test product, whereas the lowest numerical values were observed in group C.The results of the pH and viscosity values of the intestinal content are shown in Samples from the liver, kidney, and intestine, based on specific gross pathology criteria, were collected and pooled from animals of each group for further histopathological analysis. All liver samples from all groups showed multifocal inflammation and hepatocellular vacuolation on day 25 , whereasOn day 25, kidney samples of all groups showed glomerulonephritis alongside focal necrosis . MoreoveThe crypt hyperplasia finding was present in small intestine samples of groups that received contaminated feed, but the overall picture of samples from groups that received the test product  was milder, especially on the 42nd day of the study. Mild lymphocytic-plasmacytic inflammation was present also in groups A and B. Focal lymphocytic-plasmacytic inflammation with a mild occurrence of macrophages and surface epithelial necrosis as well as focal atrophy and necrosis of villi, was observed in group C. Focal atrophy of the villi without findings of necrosis was also present in group D samples. Increased goblet cells were present in group B samples and myelocytes in group D samples.E. coli were not significantly affected by the ingestion of AFB1 and OTA in group B when compared with controls. However, the addition of MMDA at the concentration of 1 or 3 g/kg of feed, significantly reduced the E. coli counts in the caecal content of birds when compared with group B . Counts of Clostridium spp., Lactobacillus spp. and Bifidobacterium spp. were not significantly affected by the ingestion of mycotoxins or the test product.Microbiological analysis of caecal content at the end of the study period is presented in B. subtilis, B. licheniformis, S. cerevisiae cell wall and silymarin fed to broiler chickens ingesting contaminated feed with Aflatoxin B1 (AFB1) and Ochratoxin A (OTA). Results suggested a partial improvement of performance and biochemical parameters, as well as reduced E. coli counts in groups treated with the test product when compared with the group that received only AFB1 and OTA. The group that received the test product at greater dosage (group D) showed an overall more noticeable improvement of particular parameters in comparison with the low-dose group (group C).Mycotoxins are considered the most dangerous contaminants of animal’s feeds as even low levels could lead to deleterious effects. As a mycotoxin occurs in feed it is very difficult to eliminate . Among d1 and OTA on broiler performance [1 in their diets. Thus, it is not surprising that, in the present study, the dietary supplementation with 0.1 mg/kg AFB1 and 1 mg/kg OTA in broiler diets significantly compromised the BW of birds from 25 days of exposure. Moreover, the ingestion of AFB1 and OTA, led to a significant increase in the feed conversion ratio (FCR). The mechanism behind the adverse effects of ingested mycotoxins in BW of birds has not been fully elucidated; however, the aforementioned mycotoxins have been associated with anorexia, listlessness, and metabolic changes, as a result of the liver damage and the decrease of the activity of digestive enzymes in combination with the decrease of nutrient digestibility due to the changes on the intestinal environment [1 inhibits the digestibility of fats by reducing both enzyme activity and bile acid production necessary for their digestion and absorption [Several studies have been conducted to determine the adverse synergistic effect of AFBformance . There iformance  reportedformance  recordedironment ,34. In asorption .1 and OTA on the FCR. The ability of clinoptilolites to diminish the adverse effects of mycotoxins in broiler performance has been previously described. Orgus et al. [1 on feed consumption and body weight gain. Moreover, Nedeljković-Trailović et al. [B. subtilis and B. licheniformis, could degrade the mycotoxins and reduce their absorption, whereas the Saccharomyces cerevisiae cell wall had shown sorption capability against OTA [1 intoxication on the performance of broiler chicks [1 or OTA; thus the effects on performance of broiler chicks were more pronounced, and the respective improvements provided by adsorbents were more noticeable.The addition of MMDA product, ameliorated the adverse effect of AFBs et al.  reportedć et al.  saw thatinst OTA ,39. Furtr chicks . Finally1 and OTA are associated with significant alterations in the biochemical profile of broilers. Mycotoxicosis can be suspected by determining changes in serum biochemistry parameters before clinical symptoms become apparent. The change of these serum biochemistry parameters is indicative of liver pathology [1 in broiler feed led to decreased total protein and increased alkaline phosphatase (ALP) values in the serum of birds. Moreover, decreased serum albumin, cholesterol levels and increased activity of hepatic enzymes, including ALP, alanine aminotransferase (ALT), gamma-glutamyl transferase (γ-GT), have been noted when AFB1 added in broiler diets in concentrations up to 1 mg/kg [1 and OTA in concentrations of 0.1 and 1 mg/kg, respectively, did not significantly alter the biochemical profile of broilers either on 25 days or at 42 days of life. However, a significant increase in aspartate aminotransferase (AST) values, as compared with the control group was noted in both samplings.Studies conducted during the last 15 years revealed that AFBathology . Denli eathology  reported1 and OTA. The liver is the main target organ involved in the detoxification of mycotoxins alongside the kidneys. Blood serum enzymes, including ALP, ALT, AST and γ-GT, are important blood biomarkers of liver activity [1 in broilers [1 activation [Saccharomyces cerevisiae cell wall glucans, can act as nutritional aids and growth promotors, and bind mycotoxins and could act synergically with the modified zeolite adsorbing ability [Our results are in agreement with Pappas et al.  who did activity . These eactivity . Their eactivity . In addiactivity . The addbroilers . In additivation . Moreove ability .Salmonella spp., Campylobacter spp. and Clostridium perfringens [1 and OTA induced a significant increase in the pH of the duodenal and jejunal content. However, the addition of MMDA at both dosage levels significantly reduced the pH value of jejunal and ileal content. Our results are in agreement with Ismail et al. [B. subtilis and B. Licheniformis) are able to metabolize soluble nondigestible carbohydrates into short-chain fatty acids (SCFAs) and lactate which consequently lowers the pH, while the prebiotic Saccharomyces cerevisiae cell wall promotes the proliferation of lactic acid bacteria in the intestine contributing also to the reduction of pH [The main factors that determine the pH of intestinal digesta are the feed composition, the gastrointestinal secretions and the volatile fatty acids, produced by intestinal microbiota. Thus, any factor altering the intestinal mucosa and microbiota could influence the pH of intestinal digesta. The manipulation of digesta pH in broiler chickens could act as a tool to manage the potential for optimum gut health and maximum nutrient absorption since the reduction of the pH may inhibit the growth of acid-sensitive bacteria, such as fringens . In our l et al. , who repon of pH .Viscosity, a physicochemical property of the intestinal digesta, is associated with the feed materials, mainly the undigested part and the digestive chyme. Increased viscosity in the gastrointestinal tract of poultry has been shown to negatively interfere with digestion and absorption of nutrients and consequently reduce performance . In partLactobacillus spp. and Bifidobacteria, which may lead to a less competitive environment for the growth of pathogenetic bacteria like E. coli and Salmonella. In our study, the ingestion of AFB1 and OTA increased the counts of E. coli in the ceca of birds; however Lactobacillus spp. and Bifidobacteria counts were not significantly affected, and this hypothesis cannot be supported. Our results are in line with Jahanian et al. [E. coli, Salmonella spp., Klebsiella spp. and total negative bacteria were markedly elevated by increasing the aflatoxin level in broilers feed. These high numbers of E. coli could be attributed to the favorable intestinal environment created by the increase of the intestinal pH.The gut microbiota plays a vital role in maintaining health and influences the overall performance of chickens. In addition, gut microbiota limits and controls the colonization of foodborne pathogens by a competitive exclusion process. It acts through diverse mechanisms, such as providing nutrients, preventing pathogen adhesion to host cells, interacting with host immune systems, affecting the gut morphological structure, and producing organic acids and antimicrobial agents ,51. The n et al. , who notE. coli and Salmonella has been highlighted [E. coli and Salmonella spp., as a result of the increased hydrophobicity of the mineral surface [B. subtilis, B. licheniformis, Saccharomyces cerevisiae cell wall and silymarin, ameliorated the increase of E. coli, which agrees with the previously mentioned studies.Also, clinoptilolites can absorb certain microorganisms and their toxins. In many in vitro and in vivo studies, the sorption capacity of clinoptilolites for pathogenetic bacteria, including hlighted ,56. Modi surface . In our 1 and OTA at concentrations of 0.1 mg/kg feed and 1 mg/kg feed, respectively, in broiler chicks. In particular, the added mycotoxins deteriorated the FCR, as a result of poor nutrient digestion and absorption, whereas the use of the test product limited the increase of FCR that mycotoxins caused and showed a mild protective effect against mycotoxicosis, in particular in liver function. In addition, the use of MMDA reduced the E. coli counts in the caeca as well as limited the increase of the pH value of jejunal and ileal content, which could act as a favor environment for gut pathogens, such as E. coli, C. perfringens, Salmonella spp. and Campylobacter spp. It can be concluded that the MMDA product, a novel multicomponent myco-toxin detoxifying agent, could positively affect the intestinal ecosystem and thus could be an important part of a mycotoxin control program.Based on the results of the study, we could suggest that the addition of the test product in feed moderately ameliorated the deleterious effects of AFBThe experimental study was performed in the experimental facilities of the Unit of Avian Medicine, School of Veterinary Medicine, Aristotle University of Thessaloniki (AUTh), Greece. Husbandry, euthanasia, experimental procedures, and biosecurity precautions were conducted in accordance with Council Directive (2010/63/EU)  and the B. subtilis, B. licheniformis, S. cerevisiae cell wall and silymarin.The test product was a novel multicomponent mycotoxin detoxifying agent  containing modified zeolite (Clinoptilolite), 1 and 188 mg/kg Ochratoxin A (OTA) was provided by the sponsor of the study  and used for the contamination of basal feed by partial replacement of non-contaminated corn. Contaminated corn was produced after incubation with Aspergillus ochraceus and Aspergillus parasiticus at 25 °C with 13% moisture content for 15 days. After incubation, the corn was autoclaved, dried, and grounded to powder form. The amount of Aflatoxin-B1 (AFB1) and OTA in spiked corn was quantified using liquid chromatography–tandem mass spectrometry (LC–MS/MS).Corn spiked with 12.3 mg/kg AFB1 and 1 mg/kg feed for OTA, which equals to 5 (AFB1) and 10 (OTA) times greater levels than the EU suggested maximum levels [1, AFB2, AFG1, AFG2), diacetoxyscirpenol (DAS), deoxynivalenol (DON), zearalenone (ZEN), OTA, fumonisins  and trichothecenes  for the trial feeds was performed at an accredited testing laboratory , whereas feed nutritional analysis was performed in the Laboratory of Nutrition . Mycotoxin analysis of feed was performed by LC/MS–MS following the method of Li et al. [1, AFB2, AFG1, AFG2, 1 μg/kg for OTA, T-2, HT-2 and DAS, and 10 μg/kg for ZEN, DON, FB1, FB2. Feed analysis was performed with a diode array near-infrared spectroscopy instrument  and included Weende constituents as well as starch, total sugars, calcium, phosphorus. Feed and mycotoxin analysis results are presented in Contaminated feed was prepared with the inclusion of contaminated raw material in flour form into the two types of feed that were used during the trial. The final target was contamination levels of 0.1 mg/kg feed for AFBm levels ,23. Multi et al.  for fumoi et al.  for the i et al. . Detecti1 to humans, special precautions were taken during the preparation and serving of trial feed to the animals in order to minimize the inhalation of dust particles by persons involved in the trial. Therefore, efforts to minimize dust formation during feed preparations and handling, as well as the use of appropriate biosafety equipment (masks) were used by the relative personnel when in contact with contaminated feed. To avoid contamination with previous productions, the feed was manufactured in an appropriate rank order starting with the control diet and with a neutral meal mixing in between each of the diets. The rank of feed production order was group A feed at first followed by B, C and D group’s feed, respectively. Among each production, a quantity of control corn was used for the removal of leftovers in the mixing mill.The production procedure of trial feeds was performed by gentle mixing of the test particles in the feed and inclusion to the remaining feed. Due to the relative toxicity of AFB® (Aviagen®) broiler chicks were randomly allocated into four treatment groups of ten chicks each, with four replicates per group. Broilers in all groups consumed a specially formulated two-phase feed. A starter feed  or occasional (OTA) contamination  conditions [One hundred and sixty (160) one-day-old Ross 308ter feed  was provter feed  up to th®). Temperature and humidity were monitored in each room at two locations at bird level using a temperature-humidity record system .Birds in each group were placed in a pen with a deep litter of wood shavings, which were previously sterilized in an autoclave at 121 °C for 20 min . Each group was kept in a specially designed experimental room , where the temperature, the relative humidity and the lighting program was controlled, following the recommendations of the breeding company . A double-blinded scheme was implemented, thus personnel occupied in clinical evaluation of trial animals and the respective personnel that performed laboratory analysis were unaware of trial grouping and treatments. Bodyweight (BW) was measured on the 1st, 7th, 15th, 25th, 32nd and 42nd day of age. Average daily feed intake (ADFI), Average daily weight gain (ADWG: (Liveweight (end)—Liveweight /days on trial)) and Feed conversion ratio (FCR: ADFI/ADG) were recorded. ADWG and FCR were reported for the periods of the 1st–25th day, 25th–42nd day and 1st–42nd. An Adjusted FCR (Adj. FCR) was calculated based on a standard reference weight of 2500 g, according to the following equation: Adj.FCR = FCR − Y.Y. = (Average slaughter weight − 2500)/50/100. Thus, an effect on the FCR of about 2 points (±0.2) for each 100 g difference between 2500 g and actual slaughter weight was adjusted. For each dead bird of the trial, the date, the age, the live weight and the possible cause of death were recorded.® flexor, Vital Scientific NV, Spankeren/Dieren, The Netherlands) in the Diagnostic Laboratory of the School of Veterinary Medicine, AUTh.Twenty-four birds per treatment group per sampling day were bled via the jugular vein. The blood collected in 10 mL tubes and centrifuged for 3 min at 3000 rpm in order to obtain the serum. Serum albumin, total protein, cholesterol, glucose, alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyltransferase (γ-GT) were measured with an automated clinical chemistry analyzer . Two readings were taken from each sample and the average was presented.g for 15 min to separate the feed particles from the liquid phase. Supernatants (0.5 mL) from each tube were taken and the viscosity was measured in a Brookfield DV-II+ PRO Digital Viscometer . Two readings were taken from each sample and the average was expressed in centipoise (cP).The viscosity of intestinal digesta was determined as described by Tsiouris et al. . The homSamples from the intestine, liver and kidneys were fixed in 10% neutral-buffered formalin for 48–72 h and embedded in paraffin by a routine procedure. Dewaxed 3–5 μm thick sections were stained with haematoxylin and eosin (H–E).During the evisceration process, one cecum from each bird was aseptically removed and forwarded for further bacteriological analysis within 24 h. The content of each cecum was weighted and co-placed by a tenfold quantity of Maximum Recovery Diluent , in a sterile plastic Stomacher bag. Following homogenization for 3 min in a Stomacher bag, 1 mL of homogenate was further diluted, by the 10fold dilution method. Finally, 1ml of each dilution was suspended in a sterile petri dish, in duplicate and covered by the appropriate medium, cooled at 45 °C.Escherichia coli, the ChromoBio® TBX agar  was used, and all green colonies were countered according to the ISO16649-2: 2001 after incubation for 24 h, at 44 °C, under aerobic conditions. Moreover, Lactobacilli were measured at the genus level, according to the ISO 15214:1998 after aerobic incubation for 72 h at 37 °C on de Man, Rogosa and Sharpe agar . Tryptose Sulfite Cycloserine (TSC) agar was used for the enumeration of Clostridium spp. according to the ISO7937:2004. Finally, Bifidus Selective Medium , was used for the enumeration of Bifidobacteria according to the ISO29981:2010. For Clostridium spp. and Bifidobacteria, plates were incubated anaerobically for 48 and 72 h, respectively. The anaerobic environment was generated using Oxoid™ AnaeroGen™ 2.5 L Sachet and was confirmed using Anaerotest® (Merck 1.15112). Results were expressed as base-10 logarithm colony-forming units per gram of caecal digesta.For the cultivation of ® Statistics Ver. 25  and the statistical language R [The statistical analysis was conducted using the statistical software SPSSnguage R . Univarip-values for the fixed component of the model were calculated from a F-test based on the Kenward–Roger approach [p-value (significance) was less than 0.05 (p ≤ 0.05). All the tests conducted were two-tailed  in the sense that the alternative hypothesis is that the measures tested are not equal.The effect of treatment on variables violating the normality assumption was assessed by the non-parametric Kruskal–Wallis test followed by pair-wise comparisons through the Mann–Whitney test using Bonferroni’s correction. In order to examine the effect of treatment on continuous parameters measured at different timestamps, Linear Mixed Effects (LME) modelling was used . All staapproach  in order


Analysis of a national database suggests that incidence of hospitalized babesiosis patients stable and deaths are low. Babesia spp. are tickborne parasites that cause the clinical infection babesiosis, which has an increasing incidence in the United States. We performed an analysis of hospitalizations in the United States during 2010–2016 in which babesiosis was listed as a diagnosis. We used the National Inpatient Sample database to characterize the epidemiology of Babesia–associated admissions, reflecting severe Babesia-related disease. Over a 7-year period, a total of 7,818 hospitalizations listed babesiosis as a primary or secondary admitting diagnosis. Hospitalizations were seasonal (71.2% occurred during June–August) and situated overwhelmingly in the Northeast and Midwest. The patients were predominantly male and of advanced age, which is consistent with the expected epidemiology. Despite a higher severity of illness in more than (58.5%), the mortality rate was low (1.6%). Comparison with state reporting data suggests that the number of hospitalized persons with babesiosis increased modestly during the observation period. Babesia spp. are tickborne intraerythrocytic apicomplexan parasites responsible for the clinical infection babesiosis. Babesia microti, the leading cause of human babesiosis, is endemic in the northeastern and north-midwestern United States  from the NIS, the largest publicly available inpatient healthcare database in the United States. The NIS was developed as a federal–state–industry partnership by the Agency for Healthcare Research and Quality for the Healthcare Cost and Utilization Project (HCUP). Data before 2012 used a 20% stratified probability sample of hospitals rather than discharges , academic designation (teaching vs. nonteaching), and bed size. Hospitals were categorized as small, medium, or large according to the criteria defined by HCUP, which were based on region, urban–rural designation, and teaching status , using survey analysis commands applying the sampling weights as determined by HCUP.We used diagnosis and procedure codes from the International Classification of Diseases, Ninth Revision, Clinical Modification (ICD-9-CM), from 2010 through the third quarter of 2015. After the third quarter of 2015, billing codes switched from ICD-9-CM to the International Classification of Diseases, Tenth Revision, Clinical Modification (ICD-10-CM). Babesiosis cases were identified by ICD-9 code 088.82 or ICD-10 code B60.0. Transfusion-transmitted babesiosis infections could not be identified independently because there are no specific ICD-9 or ICD-10 codes for that diagnosis. We described demographic and clinical characteristics as counts, percentages, mean (SD) and median (interquartile range) as appropriate. We stratified results into ICD-9 (2010–2015q3) and ICD-10 (2015q4–2016) data. We analyzed the geographic distribution, demographics. and seasonality of We also performed a graphical comparison of the number and incidence of babesiosis cases reported to CDC during 2011–2016 to compare the overall trends in reporting. The CDC data that were included were reported by individual state health departments; cases were reported by the state of residence, which might not have been the state of exposure.To test specificity, we performed a sensitivity analysis that restricted the assessment to hospitalizations in which babesiosis was listed in the top 5 diagnoses. The analysis also excluded admissions associated with a primary diagnosis of Lyme disease.Given that the NIS is a de-identified, publicly available dataset, this study was deemed exempt from review from the Johns Hopkins Institutional Review Board. This analysis was conducted in accordance with the HCUP data use agreement guidelines.During a 7-year period, babesiosis was listed as an admitting diagnosis for 7,818 hospitalizations, of which 4,648 (59.5%) listed babesiosis as a primary diagnosis and 3,170 (40.5%) as a secondary diagnosis . Annual Of all hospitalizations, 2,325 (71.2%) occurred in summer (June–August); 6,616 (92.7%) occurred in the Northeast and Midwest. New England  and Mid-Atlantic  were the leading regions. The admitting hospitals were predominantly urban , and admissions were overwhelmingly nonelective .A greater severity of illness was reported in 4,574 (58.5%) hospitalizations; risk for death was assessed as major or extreme for 1,339 (17.2%) hospitalizations . The lea+ 7.3 days , acute respiratory failure (p<0.001), and disseminated intravascular coagulation (p = 0.001) when compared with nonfatal hospitalizations. The mean length of stay was 5.8 7.3 days . The aggAt least 1 erythrocyte transfusion was reported in 1,560 (20%) hospital admissions . TransfuA total of 1,953 (25%) babesiosis-related hospitalizations listed Lyme disease as a concurrent diagnosis; 276 (3.5%) listed Lyme disease as a primary diagnosis. Neither disease severity nor mortality differed for those hospitalizations in which only babesiosis was listed, compared with those hospitalizations in which both babesiosis and Lyme disease were listed (p>0.05). Rates of respiratory failure, heart failure, disseminated intravascular coagulation, and mean length of stay did not differ between those with and without a concurrent diagnosis of Lyme disease.Other notable concurrent diagnoses in babesiosis-associated hospitalizations were anaplasmosis and ehrlichiosis (658 [8.4%]); these 2 entities are combined because it was not possible to distinguish them on the basis of the coding in use. Malaria, a clinical and morphologic diagnostic mimic of babesiosis, was reported in 52 (0.7%) babesiosis-associated hospitalizations. In 560 (7.2%) of babesiosis-associated hospitalizations, the patients were noted to have decreased splenic function or were asplenic.When we restricted the hospitalizations to those in which babesiosis was listed in the top 5 diagnoses and a primary diagnosis of Lyme disease was excluded, the number of admissions changed from 7,818 to 6,903. However, all analyses remained comparable –3.Babesia-related hospitalizations occurred in the New England and Mid-Atlantic states, and two thirds occurred in the summer (June–August). More than half of all patients were >65 years of age, and almost two thirds were male. A concurrent diagnosis of Lyme disease was reported in one quarter of all babesiosis-related hospitalizations. Reported clinical complications, notably acute renal failure, were common. Consistent with a selection for severe cases that warrant hospital admission, a high proportion of patients experienced major or extreme severity of illness and were deemed to be at high risk for death. Nonetheless, the overall mortality rate was low. Despite an upward trend in annual cases of babesiosis that have been reported to CDC, Babesia-related hospitalizations appeared stable or modestly increasing during the study period.Our findings offer a nationally representative estimate of in-hospital babesiosis in the United States. During a 7-year period, most (≈85%) Babesia-related hospitalizations in this report are consistent with what is known about the parasite’s general epidemiology in the United States. Specifically, B. microti is widely endemic in the Northeast and upper Midwest. B. microti is the most common species causing human babesiosis in the United States and worldwide. Other species  and variants  have been reported in the United States but are comparatively rare , yet hospitalization follows a period of incubation, symptom onset, and progression. Delayed diagnosis and misdiagnosis further account for hospitalizations in late summer and fall. An additional explanation for delayed presentation may be transmission of Babesia spp. attributed to bites from adult ticks, which are active into the fall , increased physician awareness and reporting may play a larger role than an increase in the number of cases.Our findings provided new insights about the overall health burden of babesiosis in the United States, including the number of cases, the severity of illness, and the financial costs incurred by the disease. The overall rate of severe babesiosis requiring hospital admission is increasing, albeit slowly. These findings complement those of Menis et al. who analyzed US Medicare-related claims pertaining to babesiosis during 2006–2017, thus describing a significant increase, from 4 claims/100,000 beneficiaries in 2006 to 9 claims/100,000 beneficiaries in 2017  does not correlate well with the general epidemiology of this infection, whereby an increase in cases of babesiosis has been observed over the past 3 decades  was low. The observed rates of clinical complications in our study differ from prior reports, which tended to cite pulmonary sequelae  as the most common severe complication of babesiosis  . Erythrocyte transfusions are administered for severe anemia, so they are an index of severe disease, especially for transfusion-transmitted babesiosis, which carries a high death rate (19%)  (data not shown). Although Lyme disease poses a greater health and economic burden, given a greater number of cases and persistence of complications of illness, deaths from Lyme disease, unlike babesiosis, are rarely encountered , exposure to similar tick habitats where I. scapularis and Amblyomma ticks co-exist, or a misdiagnosis. Another limitation is the estimation of hospitalizations for transfusion-transmitted babesiosis. Unfortunately, rates of transfusion-transmitted babesiosis cannot be quantified because causality cannot be established; although one can deduce whether a transfusion was administered, it is not possible to determine whether infection was ascribed to the index transfusion using the database alone. Given the nature of this analysis, we have been cautious not to overinterpret the findings. For example, we cannot be certain whether admissions are increasing in general, whether the demographics of those who are likely to require hospitalization for babesiosis is increasing , or whether babesiosis is simply being recognized in patients because of greater awareness of symptoms.A limitation of our study is that the analysis is confined to hospitalized case-patients. By design, hospitalizations offer insight into the most severe cases . Although our findings are informative, quantifying the complete burden of disease given this highly selected sampling is difficult. Furthermore, the sampling approach does not include all hospitalized patients, nor does it include all hospitals; however, it is a validated, robust sampling approach, correlating well with other national survey methods (In conclusion, we found that there has been a modest increase in hospitalized patients with babesiosis in the United States, yet the associated death rate appears to be low. Nonetheless, the overall health burden, particularly for selected patient subsets who are at risk for severe or even fatal disease, remains a concern.Additional information on epidemiology of hospitalized patients with babesiosis, United States, 2010–2016.


Toxoplasma gondii. T. gondii infects neuronal, glial, and muscle cells, and chronic infections are characterized by the presence of cysts, in the brain and muscle cells, formed by bradyzoites. T. gondii is capable of synthesizing L-DOPA, a precursor of dopamine. Dopamine is a neurotransmitter that is key in the etiology of neuropsychological disorders such as schizophrenia. Previous studies have shown high levels of IgG Toxoplasma antibodies in schizophrenia patients. Many published studies show that the prevalence of toxoplasmosis is higher in schizophrenia patients. In this study, we aimed to identify the prevalence of Toxoplasma infection in patients with schizophrenia and the relationships between, sociodemographic factors and the Brief Psychiatric Rating Scale. A total of 27 schizophrenic patients were included and IgG anti-T. gondii was determined in serum samples by ELISA. The Brief Psychiatric Rating Scale, sociodemographic factors were associated with seropositivity. We found that the prevalence of Toxoplasma antibodies was 51.7%. In the Brief Psychiatric Rating Scale, statistical significant association (p = 0.024) was found in Item 13 which is related to motor retardation, however, the association turned non-significant after of correction for multiple tests or after of analyzed with a logistic regression p = 0.059, odds ratio (OR) = 2.316 with a 95% confidence interval [0.970 to 5.532]. Other association was not found between toxoplasmosis and others factors. The prevalence of toxoplasmosis on our population under study was significantly higher than that reported by general population or other group of Mexican schizophrenia patients.Toxoplasmosis is a disease, which was discovered in 1908, caused by the intracellular parasite  Toxoplasma gondii. The infection can be acquired by various mechanisms such as vertical transmission from mother to child and orally through cysts present in raw or undercooked meat  The relative frequencies (percentages) of the responses given by the patients, 15 positive and nine negative to 2) (p = 0.869) or with Fisher’s exact test (p = 0.893). The mean age of the women was 46.3 years with a standard deviation of ±17.5 years, the minimum age was 20 years, and the maximum age was 71 years; in the case of the men, the mean age was 38.3 ± 14.6 years with a minimum age of 16 years and a maximum age of 64 years. The median ages were 47.5 years for women and 33 years for men. When comparing the ages of men and women to the Student’s t-test and the Mann–Whitney non-parametric test, we did not observe any statistically significant differences .There was a total of 12 women and 15 men in the sample studied. The distribution by age group was similar, i.e., approximately one-third of the patients were younger than 30, one-third were in the range from 30 to 49 years old, and the remaining third of adults were aged 50 and over. Non-statistically significant differences were observed in the distribution of age groups between genders with the chi-square test , four patients were employees, and one patient was a merchant. Toxoplasma antibodies and sociodemographic characteristics  were analyzed using contingency tables and no statistically significant effect was detected The associations between anti-Finally, when using the Pearson and Spearman correlation coefficient between the ELISA results (UI/mL) and the Brief Psychiatric Assessment Scale to assess 24 schizophrenia patients, there was no relationship between these two measurements 2 = 32.42, (p < 0.0001); Is was also higher that prevalence reported in mentally ill patients the prevalence was 38.52%, which was significantly higher, X2 = 10.76, (p < 0.0005) [In our study, we found the current prevalence was 51.7%, which was higher than 27.97% in the general population, X 0.0005) ,22.Toxoplasma seropositivity in patients with schizophrenia was mainly associated with chronic infection [We only determined IgG antibodies, since most studies have shown that nfection ,16,19. Tnfection  or a minnfection .Toxoplasma infection of the brain undoubtedly affects neuro-inflammatory processes, including microglial activation, the levels of inflammatory cytokines, and the number of peripheral immune cells occurring during the infection. All these biochemical and cellular processes alter behavior in various ways and might affect the risk of schizophrenia and the course of the disease [In this study, we investigated the associations of toxoplasmosis with each item on the BPRS. No association was significant after the correction for multiple tests.  disease ,24,25.Our study, indeed, found an increased prevalence of toxoplasmosis in schizophrenia patients, however, the number of participants was probably not high enough for the detection of the effect of toxoplasmosis on the course of the disease.Elevated antibody levels have also been linked to schizophrenia as compared with other patient groups ,24. The Statistical Diagnostic Manual, fourth edition [The inclusion criterion was the provision of a blood sample, which was obtained from 29 patients diagnosed with schizophrenia at the Psychiatric Service of the “Fray Antonio Alcalde” Civil Hospital in Guadalajara, Jalisco, Mexico, from March to August 2019. That met the criteria of the  edition .The exclusion criteria excluded patients who had a history of drug use or whose family did not accept their participation.The data obtained from patients were immunodeficiency, evidence of neurological disease, blood transfusion, transplants, behavior, and treatment.The items on the Brief Psychiatric Rating Scale include the following; BPRS01, somatic concern; BPRS02, psychic anxiety; BPRS03, emotional isolation; BPRS04, conceptual disorganization (incoherence); BPRS05, self-contempt and guilt feelings; BPRS06, tension, somatic anxiety; BPRS07, mannerism and strange Postures; BPRS08, greatness; BPRS09, depressive humor; BPRS10, hostility; BPRS11, suspicion; BPRS12, hallucinations; BPRS13, motor retardation; BPRS14, lack of cooperation; BPRS15, unusual thought content; BPRS16, dullness, affective flattening; BPRS17, excitement; BPRS18, disorientation, confusion. Scores and meaning of the responses to the items comprising the Brief Psychiatric Assessment Scale (BPRS) are as follows: (1) not present, (2) very mild, (3) mild, (4) moderate (5) moderate-severe, (6) serious, and (7) very serious.The sociodemographic factors included age, birthplace, residence, marital status, occupation, educational level, and socioeconomic level .T. gondii  antibody titers were determined in all samples by ELISA. Plates were read at 450/620 nm. Optical density values obtained were plotted along a standard curve to determine the levels of antibody titers (IU/mL). Samples with titers less than 6 IU/mL were considered to be negative and those with titers ranging from 9 to 200 IU/mL were considered to be positive for latent infection. Samples with values greater than 200 IU/mL were checked twice. All tests were performed in accordance with the manufacturer’s instructions.Blood samples were processed in the Neurophysiology Laboratory at the University Center for Health Sciences, University of Guadalajara. The serum samples were obtained by centrifugation and kept frozen at –20 °C until they were processed. IgG anti-SPSS Version 20.0 and SPSS (v. 18) packages  were used to perform all statistical analysis. Quantitative variables included age, the BPRS scores, and the ELISA test results. The Shapiro–Wilk test of “normality” was applied as well as measures of central and dispersion tendency for the seropositivity ELISA results. The statistical significance between the two groups (positive vs. negative to ELISA) for the differences observed in these variables was obtained with the Student’s t-test for independent samples and with the Mann–Whitney U test. We calculated the Pearson (r) and Spearman (Rho) correlation coefficient between the ELISA values and the BPRS test scores. Linear regression was also performed between the total BPRS score and the ELISA values of these subjects.The qualitative variables included age groups . There were two BPRS groups, i.e., schizophrenic patients with a BPRS score greater than or equal to 20 points and those patients with a score of less than 20 points.Qualitative variables were analyzed using contingency tables, contrasting positive and negative results for ELISA from patients. The statistical significance with Fisher’s exact test and the odds ratio were also calculated. Multivariate analysis, namely the logistic regression, was also used for the assessment of the others factors.Toxoplasma infection with any monitored factors. These negative outputs are most probably caused by rather low sample size.In conclusion, the prevalence of latent toxoplasmosis observed in schizophrenia patients was significantly higher than that reported earlier for General population in Mexico. We did not found significant effects of toxoplasmosis on the symptoms of schizophrenia measured with BPRS, or a significant association of the


Furthermore, resilience is negatively related to perceived stress . It is critical in the current crisis to recognize those at risk of developing mental illnesses, taking into consideration the various socioeconomic classes, as well as to maintain and improve the general public’s mental health using appropriate psychological interventions.Facing the COVID-19 pandemic, individuals are experiencing severe mental distress. Thus, during the last year, drastic changes occurred in everyday life of every human being. Following social distancing and economic insecurity, significant increases in mental health concerns  have developed. The objective of this study was to explore the anxiety, perceived stress, and resilience in a population presenting at the general practitioner, during the COVID-19 pandemic. Data were collected between February and April 2021 and 440 individuals who presented to the general practitioner were evaluated. Concerning anxiety level, almost half of the respondents  scored above the threshold value on the anxiety scale . Having a low level of resilience, as well as experiencing a high level of stress, are both predictive of the occurrence of high anxiety ( The novel Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) virus spread rapidly to all countries over the world, and the World Health Organization declared an international pandemic on 11 March 2020 ,4. For eRegarding anxiety symptoms, the COVID-19 pandemic could be seen as a stressor that elicited a strong anxiety response among people in the epidemic regions ,18. A meThe present pandemic will affect some more than others, with a highly heterogenous impact on incidence and severity of anxiety disorders. There are quite diverse frameworks when talking about resilience , but accAddressing questions regarding the psychological processes associated with the COVID-19 pandemic, such as the risk of anxiety, depression, or even self-harm and suicide, the impact of media consumption upon mental wellbeing, as well as the protective individual measures for a mentally healthy life, a position paper urged the need for novel population-based studies establishment on mental health and COVID-19, in order to ascertain its potential consequences and the alleged mitigation measures . NeverthShedding light on the role of the general practitioner in the screening of anxiety disorders, given the increasing prevalence of these ailments in the context of the COVID-19 pandemic is of great value. Therefore, by adjusting primary care services to the contemporary socio-cultural background, early detection and management of anxiety disorders will be facilitated. On the other hand, there is still scarcity in the amount of research in Romania on the incidence of anxiety disorders in the context of the COVID-19 pandemic, noteworthy to mention being the fact that the vast majority of these studies were conducted online.The first case of COVID-19 infection in Romania was detected on 26 February 2020  and caseThis is a cross-sectional study and data were collected between February and April 2021 and 440 individuals who presented to the general practitioner were evaluated. The study sample size was estimated using the Raosoft sample size calculator. A minimum of 340 participants were required at a margin error of 5%, with a 95% confidence interval (CI), for a population size of 2900  at a 50% response distribution.Demographic and medical data were collected, and participants filled in Zung Self-Rating Anxiety Scale (SAS), Connor-Davidson Resilience Scale brief version with 10 items (CD-RISC-10), and Cohen’s Perceived Stress Scale (PSS). The study was developed in accordance with the World Health Organization (WHO), the Declaration of Helsinki, European Union legislation, and the ethical principles of clinical research of the Guidelines for Good Clinical Practice (ICH-GCP). All participants received and signed a written consent form before entering the study. The protocol and the informed consent were approved by the Ethical Committee of College of Physicians of Bacau .Demographics—Participant gender , age , living location , marital status , number of children , level of education , occupational status , and the weekly working hours were assessed by quota sampling. Furthermore, the impact of the COVID-19 pandemic on revenues was evaluated. Comorbidities—Participants were asked if they have any underlying health conditions (somatic and psychiatric). Those who declared having a somatic or a psychiatric disorder in the previous questions were requested to specify the diagnosis.Behavior—Lifestyle physical activity was evaluated through four levels of intensity: inactive, mild, moderate, and intense. Moreover, tobacco and alcohol consumption habits were evaluated.COVID-19 status, reported for oneself and others, as well as vaccination status were assessed .Psychiatric assessment. SAS was used to assess anxiety levels in participants. It contains 20 items that cover the most common symptoms of anxiety, with a ratio between emotional and somatic symptoms of 5/15, this being the reason for which SAS is considered one of the best scales that can identify somatic symptoms . Each syThe CD-RISC-10 consists of 10 statements describing different aspects of resilience . The scaPerceived stress was assessed with Cohen’s Perceived Stress Scale (PSS) that measured the extent to which participants felt life situations to be stressful . Cohen’sp < 0.05.For data analysis IBM SPSS version 22 was used. The association between qualitative variables used Pearson chi-square test and Fisher’s exact test. One-way ANOVA parametric test, Kruskal–Wallis, and median non-parametric tests were considered for multiple comparison of quantitative variables. For all analyses, the level of statistical significance was set at The final sample consisted of 440 participants, 65.7% female (N = 289) and the most representative age group was 35–64 years old ; three qAbout 53.4% of cases (N = 235) suffered from a pre-existing physical illness, mainly cardiovascular diseases (34.1%), rheumatic or musculoskeletal conditions (12.5%), as well as diabetic and metabolic disorders (11.8%). Regarding mental disorder, 7.0% (N = 31) reported to have a pre-existing diagnostic, more frequently being major depressive and anxiety disorders .Most respondents stated that they do regular mild or moderate intensity exercise  . RegardiRegarding SARS-CoV-2 infection, 18.4%  of the participants stated that they were infected themselves, 44.5 % reported knowing people with an infection in their close social environment (family or friends), and 10.5% had close people who died of COVID-19. At the time the study was conducted, 18.9% of respondents were vaccinated and 41.6% wanted to get vaccinated, however 35.2% of the sample did not want to get vaccinated and the main reason for refusing to take the vaccine was the lack of trust in the vaccines (84.1%). The questionnaire contained 5 questions that assessed how the pandemic affected the physical condition, the mental state, the professional activity, interpersonal relationships, and the quality of sleep. Respondents were asked to respond using a 5-point Likert scale, where 1 means not at all, and 5 means completely affected. Average scores and standard deviations are presented in The main concerns associated with the pandemic context were evaluated through a multiple-choice question. COVID-19 stress related factors are fear of illness/death of close people , concerns regarding the capacity of the health system , social isolation , economic impact , fear of illness/death , fear that the basic protective measures  are not enough , concerns regarding the risk of becoming unemployed , as well as social stigma associated with COVID-19 infection . The major source of information on COVID-19 was the traditional media, including television, newspapers, and radio broadcasting (63.20%), and from the medical staff in health care settings (51.80%), followed by Internet including blogs, news websites, and social media (37.70%), and friends, co-workers, and family members (22.70%).p < 0.001) and respondents under 25 years old and those 65 years of age or older are more likely to experience symptoms of anxiety (p < 0.001). Concerning anxiety level, almost half of the respondents (49.3%) scored above the threshold value on the SAS  , with a p = 0.608). Higher resilience was associated with age between 25 and 54 years old (p < 0.001)  , from 3 < 0.001) .p = 0.708. Higher perceived stress levels were reported at respondents under 25 years old and over 55 years old (The PSS-14 score in the study group ranged from 1 to 44 and the mean score (Q25–Q75) was 22.09 (SD = 7.292) . No signears old .p = 0.011) and lower resilience (p = 0.008) than respondents from the countryside. Lower level of education is statistically significant correlated with anxiety symptoms, perceived stress, and low resilience. Furthermore, unemployed and retired respondents associated higher anxiety and lower resilience. Based on expectations, study participants whose incomes have declined in the current pandemic context, associated an increase in anxiety and perceived stress levels, as well as decreased resilience. Regarding weekly working hours, respondents who work less than 20 h per week report the highest levels of anxiety and stress, as well as the lowest levels of resilience, even though we would expect a busy work schedule to be associated with these feelings. (Respondents living in the urban areas reported higher stress levels (eelings. .p < 0.001) and perceived stress (p = 0.001), as well as a low degree of resilience (p = 0.011). Furthermore, a low degree of resilience was associated with the fear of disease and death (p < 0.001). Concern of stigmatization by others in the event of infection was associated with higher levels of anxiety (p = 0.013).Concerning the respondents’ primary worries about the pandemic, fear of sickness or death of individuals in their immediate circle was associated with elevated levels of anxiety (p < 0.001), higher perceived stress , and lower levels of resilience . Use of the traditional media as a primary source of information regarding COVID-19 was significantly associated with higher anxiety levels , stress , and poor resilience , which are all results that run parallel to one another. Mortality owing to COVID-19 has been linked to advanced age and concomitant chronic illnesses, which have been identified as the most significant risk factors ,40. In ap < 0.001) during the pandemic and to have a substantially poorer coping ability than those without a pshychiatric illnesses (p < 0.001). Action control deficits were observed in people with pre-existing psychiatric disorders like anxiety and depression, OCD, or conduct disorder [People who have pre-existing mental disorders, as has been shown in previous research , are mordisorder . This medisorder ,45. Feardisorder . This fedisorder .p = 0.05. Knowing a loved one with a confirmed COVID-19 case was not linked to an increased incidence of anxiety, contrary to previous research [For statistical purposes, the sample was divided in three groups: 56.6% reported that they do not know anyone in their entourage who was infected/died of COVID-19 (group A), 32.9% knowing people with SARS-CoV2 infection in their close social environment (group B), and 10.5% had close people who died (group C). If groups A and B had similarities regarding anxiety levels (44.97 and 44.23), those knowing someone who died of COVID-19 (group C) had a higher anxiety level (47.81%), research ,49,50. HExcept for investigations involving healthcare professionals, this is one of the first studies to investigate the prevalence of anxiety in Romania, as well as the relationship between anxiety and perceived stress and resilience. Nearly 40% of respondents reported a mild level of anxiety, while over 10% reported scores indicating moderate to severe symptoms. Prevalence of anxiety among respondents is higher compared to other studies ,51. For Finally, major predictors of higher anxiety related to the pandemic outbreak included demographic factors, like being female, being younger than 25 and older than 65, being unemployed or retired, having a low level of education, and having decreased revenue. These demographic variables were also associated with positive screening for GAD or depression in other online surveys during this pandemic ,50. AddiThere were some limitations to this study. First and foremost, because the samples were obtained from only one general practitioner in Romania, it is prudent not to assume the extension of the results worldwide. Secondly, the results were only investigated at a specific point in time. This means that the findings of the study are simply a “snap-shot” of the mental health of the individuals who presented to their primary care physician for treatment. The third limitation is that we did not examine all the factors that could have an impact on the participants’ levels of anxiety, stress, and resilience. More in-depth and prospective studies on anxiety, stress, and resilience will be required in the future. As a fourth point, due to the cross-sectional study design, the causal correlations between the variables should be interpreted with care. It is advisable that further research should be conducted to confirm the validity of the current findings.During the last year, the COVID-19 pandemic has triggered a global state of emergency. According to our findings, patients who attended their general practitioner had a high level of anxiety, which was linked to a variety of predictors, including gender and age, the presence of psychiatric or somatic comorbid conditions, along with socioeconomic and occupational status. Thus, in the current crisis, it is critical to identify individuals predisposed to anxiety disorders across diverse groups and layers of populations. An established screening method for anxiety in the primary care units and the development of a guideline dedicated to general practitioners on the recommendations of psychotherapeutic and psychopharmacological interventions, including telemedicine, is mandatory. Leveraging these possibilities is essential for managing the increased mental health treatment demands emerging as a result of the COVID-19 pandemic and minimizing existing disparities in accessing evidence-based mental health care.