A high prevalence of overweight/obesity among the adolescents was observed according to the CGF (26%), followed by that of the WHO (24%) and then the IOTF (23%) cut-off, with high concordance values between each pair. After adjustment, a significant association was found between physical activity (PAQ score) and lean mass (p = 0.027). No lifestyle determinant was found to be a significant predictor of fat percentage. A high sedentary level increased the likelihood of obesity , while antioxidant-rich supplements were found to protect against obesity . The lifestyle predictors identified in this study may be considered when designing interventions that integrate lifestyle modifications targeting adolescents.The prevalence of childhood obesity in Malaysia has doubled in less than a decade. Moreover, being overweight and obese have been associated with immediate and later comorbidities, thus emphasizing the need to prevent obesity from an early age. This cross-sectional study involved 923 multi-ethnic Malaysian adolescents aged between 15 and 17 years old. Body composition was estimated using bioelectrical impedance analysis. Body mass index (BMI) classification was based on the World Health Organization (WHO) growth reference and the International Obesity Task Force (IOTF) cut-off. Meanwhile, the Child Growth Foundation (CGF) body fat reference was used to classify adolescents’ adiposity. Lifestyle indices including physical activity, sedentary behavior, supplement intake, smoking and alcohol status were assessed  Obesity refers to excessive fat accumulation that might threaten a person's health . The ratFor many countries, the steep upsurge in obesity has been attributed to rapid nutritional transition . The funObesity trends in children and adolescents are of particular concern for the predictions of how the burden of obesity might soon affect the population . OverweiSeveral local studies have been conducted to examine the lifestyle predictors of obesity risk among school-going adolescents living in certain states of Malaysia. Lifestyle determinants including low physical activity and smoking status have been associated with greater odds of obesity , l8. In This population-based, cross-sectional study was conducted among 15–17 years old Malaysian adolescents attending their fourth year (Form 4) of government secondary school education. A stratified random sampling technique was used to select six secondary schools  to represent the national school system, locations, and heterogeneity of the population using computer-generated random number lists. The adolescents were recruited from three regions: the Federal Territory of Kuala Lumpur (metropolitan area), Selangor , and Pahang (eastern region), based on discussions with the Ministry of Education (MOE) of Malaysia , 24. AllThe study was conducted in accordance with the Declaration of Helsinki and approved by the Institutional Ethics Committee of the University Kebangsaan Malaysia Medical Center (UKMMC) (UKM 1.5.3.5/244/FF-2014). Formal approvals were also obtained from the Ministry of Education (MOE), the Department of State Education (JPN), the Department of Orang Asli Development (JAKOA), and the school authorities.2 at age 18 by the IOTF . Body weight was measured in light clothing to the nearest 0.1 kg using a segmental body composition analyzer BC-420 MA . Age- and gender-specific BMI-z scores were calculated based on the least mean square method (LMS) , 26. Bodthe IOTF .r = 0.6 , known as PAQ-C (M) . The quer = 0.6 . The subr = 0.6 . For theRegarding sedentary behavior, the adolescents were asked to report their daily sedentary activities (sitting or lying down) over the previous week, which included the number of hours usually spent per day during school days and on the weekend watching television, playing video games, surfing the internet/using the computer, doing homework/revision, attending extra classes (not within regular school hours), reading, and sitting while playing. Then, the average duration of total sedentary time was calculated for every subject. Due to the insufficient evidence available to specify time limits on sitting in children and adolescents, the recommended sitting time of not more than eight hours per day for adults was applied to classify the duration of sedentary behavior .k = 0.62)  with a substantial kappa correlation coefficient ( = 0.62) . In brieo Biloba , 35. Foro Biloba .t-test was applied for age. Differences in proportions (percentages) were carried out using the χ2 test.At the initial stage, all the variables were tested for non-normality based on histograms and skewness values larger than two or an absolute kurtosis larger than seven . ContinuThe strength of agreement/degree of concordance between each pair of cut-offs was calculated using the kappa (k) coefficient. Cohen's k coefficient values of less than 0.20 are described as poor agreement, values of 0.21–0.40 as fair agreement, values of 0.41–0.60 as moderate agreement, values of 0.61–0.80 as substantial agreement, and values of 0.81–1.00 as almost perfect agreement .via the univariate analysis. Only significant variables in the univariate analysis were entered into the multivariate model. The variance inflation factor (VIF) was calculated for each regression model and detected no severe multicollinearity issues (>10) that warranted corrective measures.A multivariate linear regression model was generated to determine the best combination of lifestyle predictors for body fat percentage and lean mass, while a multivariate logistic regression model was created to determine the risk of obesity after controlling for confounding factors. Potential confounders were predetermined p-value of <0.05 is considered significant.Statistical analyses were performed using the SPSS for Windows version 24.0 . A n = 923). A total of 49.5% (457) participants were female adolescents, while 50.5% (466) were males. The overall composition was 65.4% Malays, 20.6% Chinese, 9.5% Indians, and 4.4% Orang Asli of the Semai tribe . More than half of the sample population reported low levels of physical activity (60%), with 64% being identified as sedentary. In regard to gender, male and female adolescents did not differ by ethnicity, school type, sedentary level, or any type of dietary supplement intake. Female adolescents were less active than males (p < 0.05). Male adolescents reported higher proportions of smoking and alcohol consumption compared to female adolescents (p < 0.05). Male adolescents had higher mean lean mass and PAQ scores (p < 0.005), while female adolescents had a higher mean fat percentage than male adolescents (p < 0.05). There were no significant differences between the genders regarding the mean age, BMI-z scores, and sedentary time.p < 0.05).k = 0.95), while the agreement between WHO-CGF (k = 0.76) and IOTF-CGF (k = 0.77) was good. A good to very good agreement between the three references allowed any subsequent analysis to employ only one reference.R2 = 0.506, p < 0.001).p = 0.027). This model accounted for 53% of the variation in the adolescent lean mass .p = 0.003), while adolescents who took antioxidant-type supplements had obesity odds 0.4 times lower than their non-intake counterparts (p = 0.010).The present study estimated a high prevalence of overweight and obesity in adolescents aged 15–17. The prevalence of obesity in a national study among children and adolescents under 18 (15%) was slightly higher than that reported in this study (11%), probably due to the wider age range .Notably, a higher prevalence of overweight/obesity was discovered with the CGF cut-off (26%), followed by those of the WHO (24%) and IOTF (23%). A previous comparison study also found important differences between the cut-off points for BMI and body fat. The prevalence of overweight/obesity in children and adolescents aged 7–19 from Southern Europe  was 32.3% according to IOTF, 37.3% according to the WHO, and 39.8% according to the CGF references  and exercise (either resistance or endurance) . Althougvia the increased indirect utilization of antioxidants, while the free radicals generated may play a major role in the etiology and development of obesity-related comorbidities. However, the proposed mechanism should consider the limitations of the existing studies , while no lifestyle determinant was found to be a significant predictor of fat percentage. The lifestyle predictors identified in this study could be considered when designing interventions that integrate lifestyle modifications targeting adolescents.


To evaluate the radiological differences between diffuse idiopathic skeletal hyperostosis (DISH) and ankylosing spondylitis (AS) using whole spine computed tomography (CT), including the spine and sacroiliac joint (SIJ). The ossification and bridging of spinal ligament and fusion of the facet joint and SIJ were evaluated in 111 patients who were diagnosed with DISH and 27 patients with AS on the whole spine CT. The number of anterior bridging and shape of bridging (candle-wax-type/ smooth-type) were also evaluated. We further evaluated patients with DISH and AS by matching their age and sex. Complete SIJ fusion was more common in AS, whereas anterior and posterior bony bridging around SIJ was more common in DISH. However, 63% of patients with DISH had a partial or complete fusion. In spinal anterior bony bridging, the majority of patients with AS had the smooth-type, whereas those with DISH had the candle-wax-type. However, some of the patients with DISH (11%) had smooth-type. Intervertebral facet joint fusion is more common in AS. The number of anterior spinal bony bridging was greater in AS than in DISH, especially in the lumbar spine. These results are useful in differentiating DISH from AS and should therefore be considered when making a diagnosis. The prevalence varies between 2.9% in the Asian population aged > 50 years to 42.0% in European men aged > 65 years4. The reported risk factors for DISH are old age, male gender and metabolic factors such as obesity, hypertension and type 2 diabetes mellitus6.Diffuse idiopathic skeletal hyperostosis (DISH) is characterised by calcification and ossification of the anterior vertebral body and peripheral entheses, leading to the bony bridging of multiple vertebral bodies7, which diagnoses DISH in the spine based on radiographic features based on (1) ‘flowing’ ossification of at least four contiguous vertebral bodies, 2) relative preservation of the intervertebral disc space and 3) absence of apophyseal joint ankylosis and sacroiliac joint (SIJ) erosion, sclerosis, or intraarticular osseous fusion8. DISH is also characterised by swelling ossification of the anterior longitudinal ligament1. As the number of bone bridging of the vertebrae increases, spinal ankylosis becomes severe, which sometimes causes unstable three-column spinal fracture by minor trauma9.To diagnose DISH, various criteria have been used in the clinical setting. Of these, Resnick criteria are the most commonly used11. The prevalence varies 0.03–1.8%13. Symptoms usually appear in the 20–30’s and rarely occur after the age of 40 years1. The modified New York criteria show that AS diagnosis is based on radiographic features, including sacroiliitis grade ≥ 2 bilaterally or grade 3–4 unilaterally14. AS is also characterised by annulus fibrosus ossification and adjacent vertebral body bridging anteriorly and laterally known as a ‘bamboo spine’1.Ankylosing spondylitis (AS) is also known to cause severe spinal ankylosis and is characterised by a chronic inflammatory disease and the presence of the HLA-B27 antigen16. However, no previous reports have compared radiological features of DISH and AS using spinopelvic computed tomography (CT), including the whole spine and SIJ. Moreover, no studies have specifically focused on the characteristics and degrees of SI fusion and spinal ankylosis. Hence, this study aimed to identify the difference in the reconstructed whole spine CT images between DISH and AS to radiologically differentiate the disease from each another.Although the pathological characteristics of AS are different from that of DISH, patients with DISH may radiologically present with SIJ fusion and/or smooth bony bridging of the vertebrae in clinical practiceThe Ethics Committee of Tokyo Medical and Dental University Hospital approved this study (M2020-235) and informed consent was waived since it was a retrospective, anonymized study. All methods were carried out in accordance with relevant guidelines and regulations.18. DISH was diagnosed with anterior longitudinal ligament ossification in at least four contiguous vertebral bodies as previously reported18. Previous reports20 demonstrated that pelvic findings were excluded from the diagnostic criteria. Patients with AS were retrospectively enrolled in three institutions. Rheumatologists diagnosed AS using the modified New York criteria20. The inclusion criteria were as follows: age > 20 years; and CT images available to determine the location of ossification, bony bridging, and union in the spine and SIJ. The CT images were obtained with 0.5–1.0 mm thick sections using a 64 or 80-row multi-detector unit. The acquisition parameters were 120–150 kV, using auto exposure control.This study was conducted by the Japanese Multicenter Research Organisation for Ossification of the Spinal Ligament with the assistance of the Japanese Ministry of Health, Labour and Welfare. This study was performed using ossification of the posterior longitudinal ligament whole spine CT database, in which 50% of the patients have co-existing DISHThis study included 138 patients: 111 with DISH  and 27 with AS . In patients with AS, the duration of symptoms was an average of 18.7 ± 10.0 years. In clinical data, 82% of patients with AS were HLA-B27 positive and 36% were C-reactive protein positive (≥ 0.3 mg/dL). Regarding the medical treatment, 75% of patients with AS used biologics . We further evaluated patients with DISH and AS by matching their age and sex .19 , indicating substantial agreement. The whole spine CT data of ligaments, including anterior bony bridging of the vertebral body and spontaneous facet fusion, were also collected. Anterior bony bridging of the vertebral body was defined as candle-wax-type if it bulged > 3 mm from the anterior vertebral body wall and as smooth-type if it was < 3 mm  was used for all statistical analyses, and a P-value of < 0.05 was considered to indicate statistical significance.Differences between DISH and AS were analysed using the Mann–Whitney P < 0.001). Regarding SIJ, 63% of patients with DISH had a partial or complete fusion, whereas 19% of patients with AS did not demonstrate any evidence of SIJ fusion (Table P < 0.001) and posterior bridging  were higher in patients with DISH than those with AS (Table P < 0.001), and anterior bony bridging (P < 0.001) was significantly higher in patients with DISH than those with AS (Table Table P = 0.009). The percentage of candle-wax-type bone bridging was significantly higher in patients with DISH than those with AS  (Table Table P = 0.004), either in T7–T12 (P < 0.001), in the lumbar spine (P < 0.001) and in the whole spine (P < 0.001) (Table P = 0.002), T7–T12 (P < 0.001), lumbar (P < 0.001) and whole spine (P < 0.001) . Conversely, some patients with AS had candle-wax-type bridging (30–70% candle-wax-type: 20.8%). To the best of our knowledge, this is the first study to reveal that anterior bony bridging in patients with DISH occasionally demonstrated ‘AS-like’ bony bridging. Our findings refute that of the previously reported8, i.e. the typical appearance of bony bridging in DISH. Physicians should consider that candle-wax-type bridging is not always present in DISH, and this appearance alone cannot prove its diagnosis.In analysing the shape of spinal bridging, as expected, the rate of candle-wax-type was much higher in DISH than in AS. This result is consistent with that of a previous report8. A previous study showed that facet joint ankylosis in AS is more common than that in DISH, and a small number of cervical ones with DISH were observed in whole-body magnetic resonance imaging20. They showed no thoracic and lumbar facet ankylosis in DISH. Another study reported that thoracic and lumbar facets in patients with AS had more inflammatory lesions24. We also found that both-side facet fusions of the thoracic, lumbar and whole spine were more common in AS than in DISH, and results were similar after age and sex matching (Table In evaluating the facet joint fusion, the DISH diagnosis requires the absence of joint fusion10. Fourth, we could not obtain functional from AS patients included in this study. Large-scale, prospective study is therefore needed to clarify relationships between clinical data and radiological findings.This study has some limitations. First, the sample size of patients with AS was relatively small because AS is uncommon in our country. However, most of the important comparisons reached statistically significant difference. Second, the number of bony bridging and fusion depends on the patient’s age. However, results after age matching showed a similar tendency. Third, although rare, there may be concurrent patients of AS and DISH, as reported in the pastIn conclusion, both sides of complete SIJ fusion are common in patients with AS, and anterior/posterior bridging around the SIJ is common in patients with DISH. However, a considerable number of patients with DISH have SIJ fusion. In the anterior spinal bridging, patients with AS are characterised by smooth bridging, which commonly occurs in the lumbar spine of patients with AS when compared to those with DISH. Conversely, patients with DISH are characterised by candle-wax-type bridging, which commonly occurs in the thoracic spine. However, interestingly, a certain percentage of patients with DISH had smooth-type bridging and some patients with AS had candle-wax-type bridging. Furthermore, a considerable number of patients with DISH showed spinal facet fusion. These facts should be considered when making a diagnosis for AS or DISH.


Acinetobacter baumannii superinfection. The 64 A. baumannii isolates were recovered from COVID-19 patients admitted to ICU at General Hospital “Dr Laza K. Lazarević” Šabac, Serbia, during the period from December 2020 to February 2021. All patients required mechanical ventilation and mortality rate was 100%. The goal of this study was to evaluate antibiotic resistance profiles and virulence potential of A. baumannii isolates recovered from patients with severe form of COVID-19 who had a need for mechanical ventilation. All tested A. baumannii isolates (n = 64) were sensitive to colistin, while resistant to meropenem, imipenem, gentamicin, tobramycin, and levofloxacin according to the broth microdilution method and MDR phenotype was confirmed. In all tested isolates, representatives of international clone 2 (IC2) classified by multiplex PCR for clonal lineage identification, blaAmpC, blaOXA-51, and blaOXA-23 genes were present, as well as ISAba1 insertion sequence upstream of blaOXA-23. Clonal distribution of one dominant strain was found, but individual strains showed phenotypic differences in the level of antibiotic resistance, biofilm formation, and binding to mucin and motility. According to PFGE, four isolates were sequenced and antibiotic resistance genes as well as virulence factors genes were analyzed in these genomes. The results of this study represent the first report on virulence potential of MDR A. baumannii from hospital in Serbia.Since the WHO declared the COVID-19 pandemic in March 2020, the disease has spread rapidly leading to overload of the health system and many of the patients infected with SARS-CoV-2 needed to be admitted to the intensive care unit (ICU). Around 10% of patients with the severe manifestation of COVID-19 need noninvasive or invasive mechanical ventilation, which represent a risk factor for  From the beginning of the COVID-19 pandemic, intensive care units (ICUs) have been overflowed with patients suffering severe clinical presentation of COVID-19 and acute respiratory failure, of which over 10% needed noninvasive and invasive mechanical ventilation . AlthougA. baumannii has prolonged survival and transfer in the hospital environment, such as attaching to various biotic and abiotic surfaces, including surfaces at ICUs so bacterial superinfections caused by A. baumannii may lead to hardly treatable conditions in COVID-19 patients  admitted to ICU at the General Hospital “Dr Laza K. Lazarević” Šabac, Serbia. To the best of our knowledge, this is the first study on the virulence potential of A. baumannii isolates from COVID-19 patients admitted to Serbian hospitals.The aim of our study was to evaluate antibiotic resistance profiles and virulence potential of 2.2.1.n = 64, male:female ratio 1:1) required mechanical ventilation (MV). Besides, Acinetobacter spp. infection was confirmed based on bacteriological analyses of different samples recovered from COVID-19 patients on MV  are presented in Acinetobacter spp. infection (n = 48), were not included in the study. Surveillance program through rectal swabs, was not carried out within ICU, so there is no available data if patients excluded from this study were also colonized by A. baumannii but did not develop the infection. The study protocol was approved by the Ethical Committee of the General hospital “Dr Laza K. Lazarević” Šabac .Sixty-four patients admitted to the ICU of secondary referral hospital “Dr Laza K. Lazarević” Šabac, Serbia, due to severe coronavirus disease 2019 (COVID-19) were included in this study. Patients were hospitalized during the study period from December 2020 to February 2021, and COVID-19 was confirmed by real-time PCR. All patients (ts on MV . The cri2.2.Acinetobacter spp. (n = 64) were collected from blood (n = 28), tip of the central venous catheter , tracheal aspirate , tip of the aspirator (n = 3), and sputum  for searches against GenBank database. The strains labels were assigned randomly to preserve the anonymity of the patients , belonging to m n = 1; . Identifm n = 1; . 16S rRNpatients .2.3.Acinetobacter spp. was determined by PFGE of ApaI digested (3 h at 30°C) genomic DNA as previously described  and Group 2 (IC1), respectively, . Minimum inhibitory concentrations (MICs) were determined for meropenem (1–8 μg/ml), imipenem (1–8 μg/ml), colistin (1–8 μg/ml), gentamicin (1–8 μg/ml), tobramycin (1–8 μg/ml), and levofloxacin (0.25–2 μg/ml). Experiments were done in triplicate. After 24 h incubation at 37°C, cell density was monitored by OD570 measurements using Multiscan FC Microplate Photometer  and MIC values were determinate as the lowest concentration of antibiotic that inhibited bacterial growth. Acinetobacter baumannii strain ATCC19606 was used as a control strain.Antimicrobial susceptibility testing was performed using the broth microdilution method according to the European Committee on Antimicrobial Susceptibility Testing breakpoints , cephalosporinase (blaAmpC), and metallo-β-lactamase (blaNDM-1) in genomes of analyzed isolates (n = 64) was monitored by PCR amplification method as previously described . The Invitrogen Collibri Library Prep kits for Illumina HiSeq was used for the library preparation. The quality of each sequenced genome was checked using FastQC  and obtained annotated GFF files were further clustered by Roary (version 3.13.0) into core genes (selection threshold for hard core genes was presence in >99% of the isolates and for soft core genes threshold was presence in 95–99% of isolates) and accessory genes . Phylogenetic tree was created by Roary and visualized by Phandango.Pan-genome analysis approach was used in order to compare genomes of four sequenced 2.6.A. baumannii isolates (n = 64) were tested for the ability to form biofilm following the protocol described previously  was established as three SDs above the mean OD of the negative control: ODc = average OD of negative control + (3 × SD of negative control) (Collected eviously  with slicontrol) . ODc is 2.7.A. baumannii isolates to mucin was tested as described previously (Mucus consists primarily of water (~95%), and the major non-aqueous component is mucin, while proteoglycans, lipids, proteins, and DNA are also present in smaller quantities . The bineviously , with th2.8.A. baumannii isolates were tested for two types of motility: swarming and twitching. Freshly grown cultures were stabbed with sterile toothpick on the surface of modified Luria Bertani (LB) agar  with 0.4% agar for swarming motility and 0.8% agar for twitching motility, as described previously  as it was reported that motility can be decreased in the presence of light  Mann–Whitney test was used. Analyses were performed in Prism 9 version 9.3.1 and results were visualized as scatter and whicker plots. Dendrogram for analyses of the PFGE patterns was created in SPSS 28.0 for Windows.For comparing a difference in biofilm formation and mucin-adhesion ability among Virulence potential data of tested isolates were summarized and visualized by a heat map created using Rx64 3.5.1 software. The results were approximated on the relative scale ranging from blue as the lowest values, progressing to white, then to red as the highest value.3.3.1.A. baumannii according to 16S rRNA gene sequencing. According to Blast results of selected isolates the percentage of similarity of 16S rRNA genes were in the range 97.82–99.02%. The incidence of A. baumannii infection in COVID-19 patients was 57.14% during the study period.Duration of patient hospitalization, time points of specimen collection, and origin of bacterial isolates are presented in Ethyl alcohol (70%) was used as disinfectant for surfaces and items which may be in contact with the patient, while chlorine-based agents were used for disinfection of floors. Healthcare workers had disposable, single use protective gloves, masks, and gowns to protect patients at ICU from possible further infection. The use of closed aspiration systems was implemented in order to prevent nosocomial infections.The average duration of hospitalization for all patients was 16.86 days, and male COVID-19 patients were hospitalized 16.22 days on average, while female patients spent 17.5 days in the hospital. Mortality rate was 100%.3.2.A. baumannii isolates (n = 64) belong to international clone 2, IC2 (sequence type Group 1).The multiplex PCR performed for identification of international clone belonging revealed that all tested ApaI fingerprint, a dendrogram was constructed for all isolates and shown in ApaI digestion are presented in According to the obtained PFGE ApaI profile of DNA fragments indicating the dominant presence of one pulsotype among the isolates, while only four (6.25%) isolates were different. Although these four isolates  had a very similar ApaI profile to the dominant strain, they differed in at least one ApaI fragment; isolate 1 lacks an ApaI fragment of 242 kb, while a unique fragment of 228 kb is present; isolate 39 has an extra ApaI fragment of 250 kb that could be a plasmid, while isolates 54 and 55 have the same profile, differing from others in that they lack a fragment of 300 kb, and have a new unique fragment of 250 kb (ApaI fragment), it can be assumed that they also originated from the dominant strain during propagation in different patients.Of the 64 isolates, 60 (93.75%) possessed an identical f 250 kb . The obtA. baumannii 1), JAPCYK01.1 (A. baumannii 2), JAPCYL01.1 (A. baumannii 39), and JAPCYM01.1 (A. baumannii 54).According to PFGE four isolates were sequenced: isolate 1, isolate 2 , isolate 39, and, isolate 54 (as it has the same PFGE pattern as isolate 55). Sequences were deposited to NCBI databank: JAPCYJ01.1  according to Oxford nomenclature, and ST2 according to Pasteur nomenclature.3.3.A. baumannii isolates (n = 64) were sensitive to colistin, while resistant to meropenem, imipenem, gentamicin, tobramycin, and, levofloxacin according to the broth microdilution method. According to the available data, resistance to different classes of antibiotics  confirmed multidrug resistance (MDR) phenotype in tested isolates  using ResFinder is presented in A. baumannii one had slightly higher virulence potential comparing to other sequenced isolates , while the blaOXA-58 gene was found in four isolates . Further, the gene encoding for another porin included in carbapenem resistance in A. baumannii, Omp33-36, of all isolates was almost identical to the omp33-36 gene of A. baumannii MS14413 with only one nucleotide substitution at position 113 resulting in amino acid substitution Lys38Thr.Typically for s 6.25%; . ISAba1 3.5.Under the tested conditions only one isolate  was classified as weak (W) biofilm producer. Fifteen isolates (23.44%) were moderate (M) biofilm producers; of those 53.33% (8/15) were recovered from male and 46.67% (7/15) from female patients . Most ofA. baumannii isolates is shown in p < 0.05) according to the Mann–Whitney test.Mucin-adhesion ability expressed relatively to control (non-coated wells in the microtiter plate) of 64 3.6.A. baumannii isolates were tested for twitching and swarming type of motility under the same temperature and light. According to twitching phenotype isolates were divided into three groups (based on the zone diameter around the inoculation point). Only 9.38% (6/64) isolates were characterized as non-motile, while the highest number of tested isolates  were intermediary motile. The 7.81% (5/64) isolates were highly motile, with a zone diameter up to 45 mm. Relative to the type of the sample, the number of motile strains were higher for isolates from blood (hemoculture and tip of the CVC) compared to motile isolates originated from the respiratory tract  and the data are presented in All Examination of swarming-like motility revealed that only 17.19% (11/64) of tested isolates were classified as a motile category. It is interesting that all isolates with the proven swarming ability, had also the ability of twitching motility, and all were originated from blood.None of the isolates showed gelatinase activity.3.7.A. baumannii isolates from COVID-19 patients on MV, heat map was constructed  recovered from clinical samples of COVID-19 patients admitted to ICU who required MV were analyzed in this study.Epidemiology, virulence traits, and antibiotic resistance profiles of 64 Considering that the mortality rate of all patients was 100%, this study revealed that the duration of hospitalization of male patients (average 16.22 days) was shorter than females (average 17.5 days) what is in accordance with data that males are at higher risk of severe form of the COVID-19 and ICU admission .A. baumannii at ICU designated to COVID-19 patients. Clonal spread of A. baumannii highlights the importance of adopting good practices for equipment disinfection, surfaces and management of COVID-19 patients  indicating that they may be derivatives of the dominant strain. According to PFGE pattern four isolates were sequenced and deposited to NCBI database. These isolates  were analyzed for the presence of antibiotic resistance genes and genes encoding virulence factors. Genome analyses of four isolates using Virulence factors database (VFDB) revealed that isolate A. baumannii one has slightly higher virulence potential comparing to other sequenced isolates. MLST analyses revealed that all isolates obtained from Šabac hospital were identified as ST2 according to Pasteur nomenclature. The previous epidemiology studies in Serbia revealed that ST2 circulates in Serbian hospitals, as well as other ST types which were not detected in Šabac hospital  and represent the important virulence factor of A. baumannii  compared to the swarming-like motility identified in 17.10% of all isolates. We found that in the category of blood originated isolates (from hemoculture and CVC) some bacterial strains showed more proficient motility with the greater zone range, up to 45 mm, which contributes to their pathogenic profile. These highly-motile isolates were not detected in any respiratory originated samples . Blood related isolates, compared to respiratory ones, stood out also by showing swarming-like motility. Motility is enabled by the presence of type IV pili, so we could hypothesize that correlation rises due to overexpression of type IV pili-related genes in isolates from blood samples compared to isolates from respiratory samples (Two different types of motilities (twitching and swarming) were tested for all 64  samples .Comparison of virulence potential revealed that seven isolates  stood out from the rest in terms of analyzed phenotypes , mutations or physiological adaptations. The answer to this question can be obtained by a complete analysis of the genomes, transcriptomes, and metabolomes of strains of the same pulsotype that showed different phenotypes. Considering that the mortality rate of all patients was 100%, potentially higher virulence of isolates recovered from male patients may be the reason for the shorter duration of hospitalization of male patients (average 16.22 days) comparing to females (average 17.5 days).In conclusion, we reported the first study from a Serbian hospital about MDR profile and virulence potential of The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found at: NCBI—JAPCYJ000000000, JAPCYK000000000, JAPCYL000000000, and JAPCYM000000000.The studies involving human participants were reviewed and approved by General Hospital “Dr Laza K. Lazarević” Šabac, Serbia. The patients/participants provided their written informed consent to participate in this study.KN and SKN were involved in acquisition of laboratory data, analyses of data, and final approval of the manuscript. MP, GN, and BG were involved in acquisition of bacterial isolates and medical data and final approval of the manuscript. MK and BJ were involved in data analysis, critical revision of the article, and final approval of the manuscript. BF designed the study and was involved in analysis of data, drafting of article, and final approval of the manuscript. All authors contributed to the article and approved the submitted version.This work was funded by the Ministry of Education, Science and Technological Development of the Republic of Serbia by the Contract of implementation and financing of scientific research work Contract No: 451–03-68/2022–14/200161, 451–03-68/2022–14/200042, and 451-03-68/2022-14/200178.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Biological synthesis of natural products from plants has made us an inspiring methodology in the field of science and biotechnology.Catharanthus roseus (L.) G. Don plant leaves (CrPLE) were extracted and characterized by utilizing the phytochemicals estimation, Thin-layer chromatography (TLC), and High-Performance Liquid Chromatography (HPLC) analysis; and further evaluation for an understanding of the biomedical uses of CrPLE was done.The methanolic extracts of The evaluation of the seven phytochemicals designates the presence of secondary metabolites in the CrPLE. The CrPLE (test samples) exhibited the Catechin and Caffeic acid contents of 0.0055 and 0.0149 mg/g respectively. The CrPLE revealed the highest antimicrobial activity and showed a mortal effect against the tested microorganisms. Cytotoxicity of the breast cancer cell lines was exposed that CrPLE as a respectable anticancer specialist and metabolically vigorous cells.C. roseus plants would be appreciated and have incredible contributions to the field of medicinal applications.Consequently, the biological synthesized methanolic leaf extracts of the  Catharanthus roseus plants containsecondary metabolites.• Themethanolic leaf extracts of the • CrPLEextracts showed antimicrobial and anticancer activities.Catharanthus roseus plants as a “natural herbal resource” in thepharmaceutical and pharmacological industry has been validated and furtherexpanded.• Theuse of  India has a wide and interesting variety of different biodiversity of beneficial plant sources that are still yet to be investigated totally. Because of the cost and higher optional impacts connected with the misleadingly manufactured prescriptions, the necessity for novel medication stuffs out by the plant achieved an uncommon notice in the current investigation .Catharanthus roseus (L.) G. Don , regularly recognized as Vinca rosea, Rosy or Pink Periwinkle, and Madagascar Periwinkle, a persistent species that are started in Madagascar and are local to Madagascar and Southern Asia [C. roseus, this plant was thoroughly examined with drug associations [C. roseus are applied as normal medications and antimicrobial activities [ern Asia . This plern Asia , 44. Theciations . The seetivities . Since ttivities , the contivities .Catharanthus roseus displays as an anticancer or anti-carcinogenic agent [C. roseus plant is perceived to have different pharmacological exercises of medical importance, for example, mitigating, cancer prevention agent, and anticarcinogenic activities [Earias vittella, larvicidal on Culex quinquefasciatus (house mosquito) and Aedes aegypti (yellow fever mosquito) [ic agent , 58, 60 ic agent , 20. Tootivities , 59; anttivities ; antitumtivities , 65; anttivities , 67; antosquito) , 47. Varosquito) , 23, to osquito) , 61; theosquito) ; prophylosquito) ; therapyosquito) .Catharanthus roseus has numerous pharmacological impacts. Hence, the plant  extracts the plant parts extricates are being utilized against a few infections like loose bowels, Alzheimer’s illness, asthma, hacks, throat infections, sore throat, the anticipation of dementia, water maintenance (edema), diarrhea, ailment, fart, tuberculosis, dyspepsia, tonsillitis, chest torment, digestive torment, toothache, bug’s sting, for outside practice to treat skin issues such as dermatitis, skin inflammation as well as skin break out, expanding (mitigating), mind stimulatory activities, cardiotonic, CNS depressant, hostile to angiogenesis impacts, hostile to feeding, against sterility, against malarial, likewise an intense enemy of microbial, hostile to oxidant action, have against malignant growth, hostile to diabetic, cytotoxic, hypertension, hypolipidemic action and so on [nd so on . This plC. roseus are utilized as prescriptions and in this manner, plants are filled in as an elective source nowadays [C. roseus plant for its capacity to biosynthesize the methanolic leaf extractions. A major aim of this study is to assess the cytotoxic impact of biosynthesized methanolic leaf extracts against human breast cancer cell lines. Our gathering has endeavored to investigate the biogenic synthesis of methanolic leaf extracts from C. roseus plants for biomedical practices.Several engineered mixtures of nowadays . In any C. roseus plant leaves. The leaves of C. roseus show supportive and automatic activities because of the occurrence of pharmacological and phytochemical activities [C. roseus plant leaves extractions (CrPLE) against the bacterial strains - Escherichia coli - ATCC 8739, and Staphylococcus aureus - ATCC 6538; Aspergillus niger - ATCC 6275 and Candida albicans - ATCC 10231. Further, the cytotoxic impacts of a methanolic method of the C. roseus plant leaf extractions (CrPLE) were tried against MDA-MB-231 cell lines. The current examination opens an innovative opening to utilize this strategy for an eco-approachable for the planning and biomedical applications of CrPLE.With such an objective, an undertaking has been thru to join the methanolic extracts exploiting usually existing ordinary things by aapplying the tivities , 43. ConCatharanthus roseus is a flowering plant species, local and endemic to Madagascar, yet developed somewhere else as a fancy and restorative plant. The plant materials were cultivated and followed by using the standard guidelines of plant nurseries of Karnataka State Forest Department, Karnataka State. One such nurseries is well-known as “Dhanavantri Vana”, and generally recognized as ‘Medicinal Plants of Paradise’. Fresh and healthy cultivated leaves of C. roseus plants  were experimented with as described by Kumar et al. [C. roseus were determined utilizing the formula:Methanolic technique for the r et al. , Koel etr et al. , and Syer et al. . WeighedC. roseus plants was experimented with as endorsed by van Der Heijden et al. [The phytochemical screening of achieved methanolic leaf extracts of the n et al. , Nayak an et al. , Jaleel n et al. , and Govn et al. . The scrn et al. , 49.C. roseus plant leaves extracts (CrPLE) [254 with the dissolvable system. Chloroform: methanol (9.5: 0.5) was employed as a solvent system for TLC analysis. The strip or plate is then positioned with this end dunking into the dissolvable blend, taking into consideration that the example spot/zone isn’t drenched in the dissolvable. As the dissolvable moves towards the opposite finish of the strip, the test combination isolates it into different parts. This is called as improvement of TLC plates. The division relies upon a few elements. The plate is taken out after an ideal advancement time and dried, and the spots/zones are identified utilizing a UV chamber and the Rf value is determined utilizing the accompanying formulae:Thin-layer chromatography (TLC) was accomplished the survey to the known purity of  (CrPLE) , 63. 10 C. roseus leaves was marked online, with the assistance of a fine capillary. The distance moved by the solvent is set apart with a pencil and the spots are seen by a UV lamp and set apart as the shape and color they make. The distance traveled by the spots and the distance traveled by the solvent are noted and the retention factor (Rf) value of each spot is determined [In a silica sheet, the mark is made with a pencil about 1 cm range from the lower edge of the end. The fluid concentrate sample of termined . The soltermined . The Rf C. roseus leaves were liquified to the concentration of 1.5 mg/mL, then sieved through a 0.45 μm nylon Phenex syringe strain remover and investigated utilizing a Shimadzu HPLC system  fixed with a reversed-phase column C18 column (Synergi 4u Polar-RP80A Column 250 x 4.6 mm 5um)  utilizing UV detection (UV-VIS detector SPD-10A) set at 210 nm and 280 nm. The portable phase consisted of 3% (v/v) acetonitrile in 0.2% (v/v) orthophosphoric acid (Solvent A) and absolute acetonitrile (Solvent B). An auto-injector (SIL-10A HT) was utilized to inject 20 μL sample volumes onto the HPLC at a flow rate of 1 mL/min. with a incline elution plan as follows: 0–10 min., 0% B; 10–30 min., 50% B; 30–45 min., 50% B; 45–55 min., 60% B; 55–60 min., 0% B [C. roseus were resolved trailed by utilizing a technique of Bramati et al. [Raw extract (10 mg/ mL), methanol chloroform, and hexane (based on solubility) from a stock of n., 0% B . Chemicai et al. , Wagay ei et al. , Lin et i et al. , Liu et i et al.  and Degui et al. .Reference guidelines including caffeic acid and catechin were manufactured in methanol at the concentration of 200 μg/mL for recognition and measurement of the specific substances confined in the extracts. Peaks were recognized in light of the retention time of caffeic acid and catechin standards. Complete coloring matters as a percentage of content was determined from the total peak area by adding from the adjustment diagram . UnidentAlong these lines, a purified solution was examined by a high-performance liquid chromatography system.2 at 37 °C pending blended. The cell lines were separated with the cell separating solution . The feasibility of the cells was monitored and centrifuged. Additionally, the 50,000 cells/ well were cultivated in a 96 well plate and nurtured for 24 h at 37 °C, a 5% CO2 incubator.Human breast cancer MDA-MB-231 strain -CRM-HTB-26 cell line was acquired from the American Type Culture Collection (ATCC) stock cells, then this was cultivated in Dulbecco’s modified eagle’s medium (DMEM) enhanced with 10% deactivated Fetal Bovine Serum (FBS), penicillin (100 IU/ ml), streptomycin (100 μg/ ml) in a moistened atmosphere of 5% COin vitro assay for cytotoxicity, and anticancer action by apoptosis tests utilizing prepared methanolic leaf extracts of C. roseus plants were experimented by succeeding the beneath referenced events.Assessment for anti-microbial action, cell cycle studies by flow cytometry, C. roseus plants (CrPLE) utilizing the agar well diffusion technique [Minimum inhibitory concentration (MICs) is the minimum dosage of a compound prevent microorganism development and was calculated based on cultures comprising a changeable dosage of the methanolic leaf extracts of echnique , 49, 59.The anti-microbial present in the plant extract is permitted to diffuse out into the medium and communicate in a plate newly cultivated with the test organisms. The resulting zones of inhibition would be consistently roundabout and the distance across of zone of inhibition can be measured in millimeters. The agar utilized was Meuller-Hinton agar for bacteria, Potato Dextrose agar for fungi, and Yeast Peptone Glucose (YEDP) Agar for yeast that is thoroughly tried for configuration and pH. This strategy is fine reported as the standard sectors of inhibition have been calculated powerless in addition to safe qualities.Staphylococcus aureus - ATCC 6538 and Escherichia coli - ATCC 8739 cell suspension were ready and grown on tryptone soup, and cultures were nurtured for 24 h at 37 °C. The cell suspensions of the cultures were acclimated to 1–2 x 106 cells/ ml. The methanolic leaf extracts of C. roseus plants are utilized as the test compounds with samples (20 μl and 10 μl), standard as Ciprofloxacin (20 μl) for S. aureus and E. coli were added to the 5 mm well on agar plates [Aspergillus niger ATCC 6275 and Candida albicans ATCC 10231 spore suspension were ready and developed on potato dextrose broth, and cultures were nurtured for 5 to 7 days at room temperature (27 °C). Once more, the methanolic leaf extracts of C. roseus plants are utilized as the test compounds with samples (20 μl and 10 μl), standard as Itraconazole (20 μl) for A. niger and C. albicans were added to the 5 mm well on agar plates.Petri plates (with a diameter of 90 mm) comprising 20 mL Meuller-Hinton agar were cultivated utilizing cotton swabs with 24 h (old) culture of the microbial strains. Wells were cut (10 mm diameter) and 50 μL of various concentrations of a test sample. The plates were then nurtured at 37 °C for 24 h. The anti-microbial action was examined by estimating the diameter of the inhibition zone shaped around the well against the test microorganisms , 53. Morr plates . BesidesAfter incubation, the experimented plates were detected for a zone of inhibition around the wells. The antimicrobial action was assessed by estimating the diameter of the inhibition zone of the tested microorganisms , 51, 63.Levels of cells in different phases of the cell cycle with the mixtures of treated and untreated (controlled) populations were estimated through flow cytometry utilizing the FACS Caliber .in vitro assay typically experimented with the monolayer cell culture was trypsinized and the total of cells was acclimated to 5.0 x 105 cells/ ml utilizing separate media encompassing 10% FBS in half-area flat-bottomed microtiter plates. To each well of the 96-well microtiter plate, 100 μl of the watery cell suspension  was supplemented. Later 24 h, when a partial monolayer was shaped, the supernatant was spun off, splashed the monolayer once with medium and 100 μl of various trial dosages of trial samples were supplementary to the fractional monolayer in microtiter plates.An 2 atmosphere. The decrease of tetrazolium salts was broadly acknowledged as a solid method for inspecting cell expansion. The yellow tetrazolium MTT -2, 5-diphenyltetrazolium bromide) was diminished through metabolically dynamic cells, partially employing the activity of dehydrogenase catalysts, to create decreasing counterparts, for instance, NADH and NADPH. The resultant intracellular purple formazan can be solubilized and evaluated thru spectrophotometric analysis. Test estimates the cell multiplication amount and then again, when metabolic occasions lead to programmed cell death or corruption, the decrease in cell viability.The plates were then nurtured at 37 °C for 24 h in a 5% CO2 atmosphere for 24 h.Cells cultivated in T-25 flasks were trypsinized and suctioned into a 5 mL centrifuge tube. The cell pellet was acquired by centrifugation at 300 x g. The total of the cell was changed, utilizing DMEM HG medium, with the end goal that 200 μl of suspension confined around 10,000 cells. To each well of the 96 well microtitre plate, 200 μl of the cell suspension was supplemented and the plate was nurtured at 37 °C and 5% CO2 atmosphere for 24 h. The plate was eliminated from the incubator and the drug-containing media was suctioned. 200 μl of medium encompassing 10% MTT reagent was then supplemented to each well to get a final concentration of 0.5 mg/mL and the plate was nurtured at 37 °C and 5% CO2 atmosphere for 3 h. The culture medium was eliminated deprived of upsetting the crystals designed. Then 100 μl of solubilization solution (DMSO) was supplementary and the plate was caringly shaken in a gyratory shaker to solubilize the formed blue formazan [Later 24 h, the spent medium was aspirated. 200 μl of various test dosages of test drugs were supplemented to the respective wells. The plate was then incubated at 37 °C and 5% CO50) values was produced from the concentration-response curve for the cell line [The optical density was estimated utilizing a microplate reader at a wavelength of 570 nm and 630 nm. The percentage growth inhibition was determined, after deducting the background and the absolute, and the dosage of the test drug expected to prevent cell growth by 50%  cells and dispensed with by pipette. Supplanted with a new culture medium to the original volume. Offered cells actuate apoptosis with 40 and 80 μg/ml of C. roseus leaves, and incubate for 24 h. Afterward, gathered cell culture medium into 15-mL tubes. Utilizing a policeman, the cells were confined from the dish and added 1 mL of medium to each well, and transported the contents to the 15-mL tubes. Centrifuged and disposed of the supernatant. Splashed the cells twice with cold PBS and afterward resuspend cells in 1 mL 1X Binding Buffer at a dosage of ~1 x 106 cells/mL. 500 μL of cell suspension is aliquoted and 10 μL of PI and 5 μL Annexin V is supplemented. The suspension is incubated for 15 minutes at RT in the dark. Post incubation, the cells were examined through a flow cytometer at the earliest opportunity (within 1 hour).The day preceding enlistment of apoptosis, plated 1 X 10C. roseus were prepared and finally yielded 2.89 g from the 20 g of the dried sample powder.The biological synthesis of natural products has made us a thrilling methodology in the arena of the usual science of natural science and biotechnology. The methanolic leaf extracts of the C. roseus plant leaves (CrPLE) perform as redox agents and the results were qualitatively determined as ‘positive’ signs for the presence and ‘negative’ signs for the absence of phytochemicals. Among the 12 tested phytochemicals screening, the existence of seven phytochemicals for instance alkaloids, carbohydrates, flavonoids, glycosides, phenols, steroids, and tannins indicate the clear existence of secondary metabolites in the CrPLE . The methanol and chloroform (solvent systems) are polar and non-polar solvents respectively and are used for the identification of straining in the various amalgamations constructed on the polarity of the substance and their ratio, the number of bands obtained, and their retention factor (Rf) value is given in the following Table Thin Layer Chromatography analysis of methanol extracts of via HPLC analysis. Analysis by linear phase chromatography using a methanol-water gradient system revealed the presence of a solo top, with a retaining time of standard (catechin) and sample (methanolic leaf extracts of the C. roseus plants) is 2.437 and 2.310 min respectively  is 1.897 and 1.807 min respectively  displayed catechin and caffeic acid contents of 0.0055 and 0.0149 mg/g respectively. Both contents are the form of organic compounds present as a secondary metabolite of the C. roseus plants that consists of a phenolic functional group.The absorption maxima in UV-spectrophotometric analysis were recorded at 250 nm which helped in easy analysis C. roseus have shown moderate to highest inhibitory activity against these mentioned microorganisms through the agar well diffusion technique , Ciprofloxacin  compared to the remaining studied species. Thus, the methanolic leaf extract of C. roseus plants exhibited the highest antimicrobial action and established with fatal outcome against the S. aureus at different dosages. Also, our study exposed the CrPLE exhibited substantial antimicrobial activities and therefore, could be useful for biological practices.The methanolic leaf extractions of que Fig. . A zone C. roseus plant leaves exhibited cytotoxicity towards MDA-MB-231 cell lines  treated at 40 μg/ml and 80 μg/ml has induced early and late programmed cell death in MDA-MB-231 cells with 20.59, 20.90% of early apoptotic cells and 13.17, 22.89% late apoptotic cells respectively  can probably modify the programmed cell death protein expression and activate apoptosis in MDA-MB-231 cells.A diagram of the flow cytometry investigation of MDA-MB-231 cells with Annexin-V fluorescein isothiocyanate (FITC) of which, in the lower left corner are the living cells Fig. . By undeely Fig. . StandarThe Indian subcontinent has the greatest richness and highest diversity in the flora of flowering medicinal plants. Most of the world’s population is mainly basically relying on therapeutic plants, the most extraordinary source is lifesaving drugs. About 80% of the world population takes an interest in the utilization of customary medication founded on plant materials, the therapeutic plants assume a powerful part in human medical care. They stay to be a significant useful help for working on the soundness of individuals and in the better treatment of several diseases.C. roseus plants had exposed the existence of seven phytochemicals for example alkaloids, carbohydrates, flavonoids, glycosides, phenols, steroids as well as tannins indicating that the rich presence of auxiliary metabolites. This is in understanding that of prior investigations by different workers [C. roseus with different plant parts like leaves by Govindasamy and Srinivasan [The phytochemicals present in methanolic leaf extracts of the  workers , 41, 63.inivasan  and Wagainivasan , flower inivasan , root byinivasan , and steinivasan .Catharanthus roseus is one of the significant therapeutic plants, very much perceived in Ayurveda and other customary treatment systems. This is recognized for its antitumor, antidiabetic, antimicrobial, antioxidant, and antimutagenic effects [C. roseus displayed the presence of carbohydrates as well as phenols as a typical phytochemical. effects , 41. As  effects , the antCatharanthus roseus is notable for its pharmacological significance as it creates a wide spectrum (≥130) of terpenoid indole alkaloids (TIAs) whereas just C. roseus is known for creating such a wide scope of alkaloids and they are extremely interesting to be delivered by some other plants in such amounts as produced in C. roseus. These alkaloids contain vinblastine and vincristine generally utilized in anticancer chemotherapies [herapies .C. roseus plant source [C. roseus contains the greatest elevated Rf value (0.70) and exhibited greyish color which demonstrates the existence of vindoline [C. roseus comprises the Rf value of 0.77 which compares to the presence of greyish color demonstrating the occurrence of vindoline. The methanolic extract of C. roseus plant produces the typical light blue color under UV light (365 nm) and contrasted colors oscillated from brown to green observed under visible light. This is in agreement with the earlier discoveries by Nisar et al. [C. roseus plants.The retention factor (Rf) value of the plant extracts ranged from 0.77 to 4.47 and colors fluctuated from brown to green. Yellow spots were recognized in the unrefined methanol extracts . The thet source . The metindoline . By lookindoline , we haver et al. , 41. It The absorption maxima in UV-spectrophotometric investigation were recorded at 250 nm aided in simple examination employing HPLC analysis. HPLC examination showed that several noticeable mixtures contained in the unrefined extract were available in the methanolic fraction, in which catechin and caffeic acid were recognized as significant. i.e., the CrPLE of catechin and caffeic acid were present in the low concentration when contrasted with the principles. In contrast to the findings of Goboza et al. , the CrPC. roseus uncovered that methanolic extract was the top dissolvable to remove the phenolic compounds. These discoveries forms concurrence as per past examinations by Rasool et al. [C. roseus. Thus, the utilization of therapeutic plant sources in the treatment of several diseases is credited with the existence of chemical ingredients such as phenolic substances and alkaloids in C. roseus that have various attributes which add to the helpful impacts of plants. This proposes that the biomolecules acquired from the CrPLE may perhaps act as oxidizing/ reducing agents/ specialists utilized for different applications. Hence, the current study was valuable and supportive for the uses of medications, plans, restorative, and possible proposals for the assembling of therapeutic agents and agricultural products.HPLC chromatogram of the methanolic extract disclosed the higher dosages of the selected phenolic compounds. Polyphenolic compounds are available in plant sources as optional metabolites as well as their incorporation in human food has been documented to have amazing medical advantages. Polyphenolic compounds can search for reactive oxygen species (ROS) and reactive nitrogen species (RNS) in natural frameworks. A measurable polyphenolic evaluation of the different concentrates of l et al.  and Kabel et al.  that repEscherichia coli, gram-positive (+) bacteria for example Staphylococcus aureus, and fungi for instance Candida albicans and Aspergillus niger. The methanolic leaf extract of the C. roseus (CrPLE) revealed strong inhibitory activity against the microorganisms such as the gram-negative (−) bacteria – E. coli, the gram-positive (+) bacteria – S. aureus, and fungi – C. albicans and A. niger. Tested CrPLE disclosed intense antimicrobial action compared to all organisms. The zones of inhibition showed moderate to highest inhibitory activity against the methanolic leaf extracts from the C. roseus plants and exhibited more than 50% inhibitory action towards these mentioned organismal strains. Consequently, the methanolic leaf extract of C. roseus plants unveiled the highest antimicrobial activity and established fatal outcome for S. aureus at various doses. Consequently, our study has shown that the CrPLE displayed the most noteworthy antimicrobial activities such as antibacterial and antifungal actions, and hence, could be helpful in natural applications. Accordingly, alcoholic extract of C. roseus could be utilized as a likely antimicrobial source [C. roseus towards some human pathogenic microorganisms (S. aureus and E. coli) as well as pathogenic fungi  with slight variation. The sturdiest inhibition movement of the leaf extract was detected towards the S. aureus (15 mm zone) at 100 mg/ml of leaf extricate antimicrobial action of this plant.Drug plants assume a critical part in the revelation of new medications utilized in flow medication and tracked down a powerful medication for different diseases. Antimicrobial susceptibility studies were directed against gram-negative (−) bacteria such as l source , 49, 63.l source  from theCatharanthus roseus is an important therapeutic plant source for drugs since the majority of bacterial microbes are creating opposition against large numbers of presently accessible antimicrobial medications. Plants have ended up being critical normal assets for compelling chemotherapeutic specialists and offering a wide range of action with more prominent accentuation on the preventive activity. The anti-cancer and antimicrobial properties of C. roseus have been examined the microorganisms such as Pseudomonas aeruginosa NCIM 2036, Salmonella typhimurium NCIM 2501, and Staphylococcus aureus NCIM 5021. So, extracts from these plant leaves can be utilized as a prophylactic specialist for a large number of pestilence infections [fections , 49, 63.C. roseus plant material either separately, the antimicrobial action found in this current study. The discovery of a strong cure from the plant beginning will be an incredible movement in microbial contamination treatments. This study offers help to the plant’s customary and elective use against different diseases and infections. Additionally, the utilization of regular items has been urged because of less or no aftereffects, cost adequacy, and improvement of protection from traditional engineered antibiotics. Henceforth, this study holds significance in involving therapeutic plants as an elective hotspot for treating different diseases [Because of the attribution of optional metabolites of different synthetic compounds existing in the diseases . SupplemC. roseus plant leaves presented a cytotoxic interaction against MDA-MB-231 cell lines. The samples of CrPLE for 24 h treatment showed an IC50 value of 57.64 μg/ml inhibition in MDA-MB-231 cells. The expansion of cell demise with an expansion in the grouping of test intensifies connotes that the methanolic extracts of the C. roseus plants were seen as successful. Further, the differences in the IC50s of the CrPLE in vitro were reflected in the effectiveness of the extracts in the MDA-MB-231 cell line model. Bioactive components of the genus Catharanthus have a place overwhelmingly with the seven phytochemical classes that are not able to display cytotoxicity [Cytotoxicity assay has turned into a significant examination for showing a compound of pharmacological significance. The methanolic extracts produced from toxicity . Perhapsin vitro on a wider scope of malignant growth cell lines including A2780 (ovarian), H460 (lung), A431 (skin), MIA PaCa-2 (pancreas), Du145 (prostate), HT29 (colon), MCF-7 (breast), BE2-C (neuroblastoma), SJ-G2, U87 and SMA (glioblastoma) at small amounts  [Catharanthus roseus is also likewise has a perceived as “magic plant”, “flower of immortality” by the Germans, “the flower of death” by the Italians, the “violet of the sorcerers” by the French, and “an emblem of friendship”. Henceforth, the plant parts  removed are being utilized compared to a few significant disorders [It exhibited that the n-butanol fraction confined the most elevated degrees of saponins and phenolics, and had the most grounded cancer prevention agent limit among the tried CrPLE . The n-b0 μg/mL) . Like thisorders , 56.Catharanthus roseus comprises a scope of dimeric indole alkaloids with huge antitumor exercises. These alkaloids have been originated to have programmed cell death-actuating action towards tumor cells in vitro and in vivo facilitated by nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and c-Jun N-terminal kinase (JNK) pathways, in which DNA injury and mitochondrial brokenness assume significant parts. In this manner, the acceptance of programmed cell death through cathachunine occurred through a ROS-dependent mitochondria-mediated intrinsic pathway as opposed to an outward pathway and was managed by the Bcl-2 protein family. It applied a powerful antitumor impact on human leukemia cells by the enlistment of programmed cell death and employing an inborn trial [From apoptotic examination by flow cytometry, CrPLE might conceivably change the apoptotic protein articulation and activate apoptosis in MDA-MB-231 cells. Comparable sort results were acquired from examinations directed by Widowati et al. . Cathararn trial , 65.Catharanthus roseus plants comprise different alkaloids, carbohydrates, saponin, flavonoids and phenolics, and phytochemicals such as vincristine, raubasin, vinblastine, vincolinine, vinacardine, leurocristine, catharanthamine, etc.. It had revealed the existence of different pharmacological activities for instance anticancer, antidiabetic, antiulcer, antioxidant, antimicrobial, antihyperglycemic, antihypertensive, antidiabetic, and wound healing, etc. [Catharanthus roseus is a significant evergreen therapeutic spice utilized for the most part for therapy of malignant growth of cancer and diabetics. Researchers have gathered data on the conventional purposes, phytochemical ingredients, and pharmacological possessions of plants. Along these lines, they have perceived the well-being-advancing activities of this versatile plant and it could likewise give hints to the disclosure of a novel principal substance of drug significance [Therapeutic plants are utilized in the conventional treatment of individuals in India for quite a long while because of homegrown items with fewer aftereffects and are financially savvy. ng, etc. . Catharaificance .C. roseus plant’s roots, stems, leaves, and flowers can be utilized in conventional medication and progressed pharmacology, and gives the promising lead to the revelation of intense antimicrobial mixtures in helpful and dietary use all around the world. The outcome of the current examinations comprises strong proof to approve folkloric utilization of these C. roseus plants as a solution for different diseases. Consequently, the current study has been valuable and accommodating for the utilization of drugs, therapeutic and expected applications, and in the assembling of different herbicides, pesticides, and agricultural fields.The inferences of the study affirmed the remedial strength of C. roseus plants manages documentation for various auxiliary metabolites, wherein they assume a noticeable part as industrially significant naturally dynamic particles having different pharmacological possessions. Catharanthus roseus also exhibited ordinary anti-microbial activity, and cytotoxic and anticancer properties. Besides, this plant species showed a critical cytotoxic impact of methanolic leaf extracts on the MDA-MB-231 cell lines. This could be additionally affirmed that phytochemical constituents found in the C. roseus plant may perhaps add to the current cytotoxicity. The present study gave important data on phytochemistry, hostile to microbial action and cytotoxicity of C. roseus, and the necessity for the extra need for additional investigation of DNA discontinuity and caspase measure studies, pharmacology, and toxicology, in a trial creature model.Description of methanolic leaf extracts of


Fagonia indica in letrozole-induced PCOS young adult female rats. HPLC was carried out to find the phenolic and flavonoid content of the ethanolic extract of Fagonia indica. Twenty-five female rats were taken and initially divided into two groups: group I (control group) and group II (PCOS group). PCOS was induced by letrozole given orally by gavage. Body weight was recorded weekly and vaginal cytology was analyzed daily. After induction of disease, the PCOS group is further divided into four groups (n = 5): group II (positive control with PCOS), group III (metformin 20 mg/kg treated group), group IV (ethanolic extract of Fagonia indica 500 mg/kg treated group), and group V (metformin plus Fagonia extract). At the end of experimental period, the blood sample of each rat was collected and serum was separated by centrifugation. Afterwards hormonal analysis, lipid profile and liver functioning tests were performed. Ovaries were removed and preserved for histopathological findings while the liver of each rat was stored for the determination of antioxidant potential assessment. Fagonia indica was found to possess quercetin as one of the major flavonoid phytoconstituents. The plant extract exhibited its beneficial effects by restoring hormonal balance, lipid profile, and liver functioning markers. Treatment with F. indica reduced body weight, resolved ovarian cysts, and showed positive effects on follicular growth. Treatment with plant also increased the levels of antioxidant enzymes. This study validates the potential of Fagonia indica for the amelioration of metabolic, as well as, hormonal disturbances that occurred in PCOS.Polycystic ovarian syndrome is a multidisciplinary endocrinopathy of reproductive-aged women that provokes insulin resistance, hyperandrogenism, cardiovascular problems, obesity, and menstrual complications. The present study was designed to investigate the effectiveness of ethanolic extract of  Hyperandrogenemia is a salient feature of PCOS, and a major contributor to cosmetic anomalies including hirsutism, acne, and male pattern alopecia in affected women. Polycystic ovarian syndrome is a complex endocrine and metabolic disorder  that is Lifestyle modifications for PCOS treatment in obese patients include moderate exercise (≥30 min/day), reduce psychological stressors, weight reduction, calorie deficit diet of 500–1000 kcal/day, decrease caffeine consumption, and low glycemic index (GI) diet . CurrentGlycyrrhiza glabra, Mentha spicata, Panax ginseng, flaxseed, Aloe vera, Chaste berry, White poeny, cinnamon, milk thistle, Matricaria chamomilla, N-acetyle cysteine, D-chiro-inositol, Kacip fatimah, Astragalus polysaccharide, Apium graveolens, and Cinnamon zeylanicum [Nowadays, many physicians and patients are inclined towards herbal treatment due to these abovementioned adverse effects of conventional treatment and cost of the treatment. The pharmacological and therapeutic effectiveness of medicinal plants is due to the presence of different phytoconstituents in these plants and these can be used for various ailments , 15. In ylanicum , 17.Fagonia indica is a member of Zygophyllaceae family. All of the species of this family are herbs and shrubs/shrublets [Fagonia is commonly known as dhamasa booti. Fagonia has a great medicinal potential to treat a lot of diseases. It is known to possess following activities such as antioxidant, thrombolytic, antimicrobial, antidiabetic, antitumor, anti-inflammatory, hepatoprotective, antihaemorrhagic, laxative, and antiulcerogenic potential. Fagonia has been used traditionally for the treatment of menstrual problems for thousands of years. Practitioners of traditional medicine have used this plant for the treatment of PCOS in different areas of Pakistan. Due to its folklore use in PCOS, it is worthwhile to explore its potential in PCOS treatment. The current study investigates the efficacy of ethanolic extract of Fagonia indica for the treatment of letrozole-induced PCOS rats.hrublets . FagoniaFagonia indica was collected from a town called Tarnol Phatak situated in Islamabad, Pakistan. The plant was collected in March 2021. It was identified and authenticated by the botanist, Dr. Mansoor Hameed at University of Agriculture Faisalabad and was preserved under a specific specimen number 071-21-01 in the herbarium of Department of Botany, University of Agriculture Faisalabad, for the further reference.The extraction was carried out by the simple maceration method. The plant was air dried and soaked for 14 days in absolute ethanol  in 1 : 5Fagonia indica.HPLC was carried out to find the phenolic and flavonoid content of the ethanolic extract of Total phenolic content was measured by using Folin–Ciocalteu reagent. 0.5 mL solution of the extract (0.05 g/5 mL) was admixed with 0.5 mL of Folin–Ciocalteu reagent and then added 7.5 mL of deionized water. This mixture was allowed to set at 25°C for 10 min after that 1.5 mL of 20% sodium carbonate (w/v) was added. Then, the mixture was heated in a water bath at 40°C for 20 min and cooled it in ice bath. Finally, the absorbance was recorded at 755 nm wavelength by using a spectrophotometer. Gallic acid was used as a standard, so the total phenolic contents were calculated by using the standard curve of gallic acid (100–1300 ppm). TPC was measured as gallic acid equivalent (GAE), and the results were expressed as mg/gm of dry matter [3 was added. The mixture was again kept for 5 min at room temperature. After 5 min, 2 mL of 1 M NaOH was mixed and distilled water was used to make up the volume. The absorbance was recorded at 510 nm by using a spectrophotometer. Catechin was used as a standard, so the total flavonoid contents were calculated by a calibration curve for catechin (10–130 ppm). The TFC was measured as catechin equivalent (CE) [The method described by Dewanto  was adopent (CE) .Twenty-five healthy female Wistar albino rats (150–200 grams) were acquired and kept in the animal house of the Faculty of Pharmaceutical Sciences, Government College University of Faisalabad (GCUF). Standard conditions (12 hrs light/dark cycles at room temperature 25 ± 5°C with 45–55% humidity) were maintained. Standard laboratory diet and water ad libitum were given to rats. The experiments on animals were performed according to the guidelines of the Ethical Review Committee ERC-21-886, Government College University, Faisalabad. The experimental design was approved by Advanced Studies and Research Board (ASRB) under the study number IRB-21-19886.After one week of acclimatization period, letrozole (1 mg/kg) in suspension form (letrozole suspended in 0.5% carboxymethylcellulose (CMC)) was given for 7 weeks to rats for the induction of PCOS. This dose was chosen according to protocols suggested in a published article by Rezvanfar et al. . During n = 5). Groups were labeled as I, II, III, IV, and V , progesterone (ng/dL), prolactin (ng/mL), and testosterone (ng/dL) levels were measured by RIA utilizing the kit of Beckman Coulter, Inc. USA. The serum luteinizing hormone  concentration was determined by the ELISA method utilizing the kit of Pointe Scientific Inc. USA. Serum estrogen (pg/mL) levels were evaluated by using the kit of ALPCO, USA, and serum insulin (μlU/ml) levels were determined by using the kit of Calbiotech Inc., USA.Serum hormone analysis was performed by radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA) kit methods. The blood sample was taken by cardiac puncture and then, the serum was separated out by cold centrifugation. Serum follicle-stimulating hormone .It is commonly observed that in polycystic ovarian syndrome, the levels of antioxidant enzymes, e.g., catalase (CAT), reduced glutathione (GSH), and superoxide dismutase (SOD) are altered and therefore were evaluated in the current study. The liver tissues were homogenized to prepare liver homogenates by using 0.1 M phosphate buffer saline (pH 7.4). Supernatant was obtained by centrifuging the mixture at 800 rpm at 4°C for 30 min. This supernatant solution was used for the determination of different antioxidant parameters  .F. indica extract stock solution (5 mg/mL in methanol) was also made. Different sample dilutions  were made using the F. indica extract stock solution. Similarly, 200 mg/L stock solution of BHT (butylated hydroxytoluene) was prepared and using this BHT stock solution, different sample dilutions  were prepared. Subsequently, added 4 mL of DPPH solution in both plant samples and BHT samples. The mixture was kept at room temperature for 30 min in dark, and the absorbance was recorded at 515 nm using a spectrophotometer. The absorbance of the blank (DPPH radicle without antioxidant) was also recorded [Stock solution of DPPH (2.4 mg/100 mL methanol) was prepared. recorded .p value was less than 0.05.The data were demonstrated as mean ± SEM, and the results of different groups were compared by analysis of variance (ANOVA) using GraphPad Prism 7.04 followed by the Tukey's multiple comparisons test. The results were considered statistically significant when the F. indica showed the presence of phenolic contents, i.e., synaptic acid (1.603 mg/gm), benzoic acid (2.785 mg/gm), and traces of chlorogenic acid. While, flavonoid contents in ethanolic extract of F. indica included myricetin (0.672 mg/gm) and quercetin (0.904 mg/gm)  F. indica was determined as 109.56 ± 0.124. The TPC is expressed as gallic acid equivalent (GAE).The TPC of the ethanolic extract of F. indica was determined as 35.10 ± 0.08. The TFC is expressed as catechin equivalent (CE).The TFC of the ethanolic extract of Throughout the study period, changes of the estrous cycle  were regular at regular time periods in group I, indicating a normal estrous cycle. Animals with PCOS delayed the estrous cycle and remained in the diestrus phase for a prolonged time. Animals of group III who were treated with metformin remained in the diestrus phase as most of the cells found in vaginal smear were leukocytes, a characteristic feature of the diestrus phase. Examination of groups IV and V showed the estrous phase of cycle that consists majorly of cornified epithelial cells .Fagonia indica-treated groups, respectively   .At the end of the study period, group II rats generally gained more weight than group I rats. Body weight of control rats were increased by 18% of its original weight, while the weight of PCOS rats was increased by 35%, i.e., almost the double of weight gain of the rats in the control group. The weight difference among all groups was mostly statistically nonsignificant .p < 0.05) in LH levels as compared to group I . LH levels were found decreased significantly in group III (p < 0.05) and group V, (p > 0.05) while a nonsignificant decrease was observed in group IV, when compared with group II (Group II (PCOS) showed significant increase (group II .p < 0.0001) reduction was observed in FSH, estradiol, and progesterone concentrations in group II, when compared with group I. The levels were significantly improved (p < 0.0001) in all treated groups as compared with group II  decrease in serum prolactin concentration was observed in group II (PCOS-induced group) as compared to group I (control group). There was a significant increase in the prolactin level in group III (p < 0.01); however, nonsignificant elevation was found in other treated groups (A significant (d groups .p < 0.05) increase was observed in testosterone concentration of group II (PCOS control rats) when compared with group I . The data showed significant reduction in testosterone levels in all treated groups as compared with group II (A significant (group II .p < 0.001) increase was observed in serum insulin concentration of group II, when compared with group I. The data showed significant reduction in group III and IV (P < 0.001), while nonsignificant reduction was found in group V, when compared with group II (A significant (group II .p < 0.0001) as compared to group I. While treatment groups showed significantly lessened levels of total cholesterol as compared with group II.In group II, total cholesterol (TC) levels were significantly increased (p < 0.0349) was noticed in the triglycerides level compared with group I. However, there was a significant reduction (p < 0.0001) in triglycerides (TG) of group III and nonsignificant reduction was noticed in group IV and V in comparison with group II.In group II, significant increase (p < 0.0001) in high-density lipoprotein (HDL) level of group II in comparison with group I. While a nonsignificant increase in the HDL level was observed in group III, IV, and V when compared with group II.There was a significant decrease (p < 0.0001) as compare to group I. A significant reduction (p < 0.0001) was found in the levels of LDL in group III, IV, and V, when compared with group II (Low-density lipoprotein (LDL) level in group II was significantly elevated , alanine transaminase (ALT), and alkaline phosphatase levels were elevated significantly in group II as compared to group I. There was a significant reduction in the levels of AST and ALT in group III, IV, and V when compared with group II. The levels of alkaline phosphatase is found ameliorated in group V while a nonsignificant difference was observed in group III and V as compared to group II .p < 0.001) in group II as compared to group I. Results showed that in all the treated groups, oxidative stress biomarkers were increased significantly (p < 0.05) when compared with group II , catalase (CAT), and glutathione (GSH) were estimated in liver samples. The levels of all these parameters were decreased  and somatic cells . Interaction of these cells decides the development and formation of oocyte-containing follicles, ovulation of egg, and formation of corpus luteum. Follicular development is regulated by hypothalamic-pituitary-ovarian axis (HPO). In this axis, hypothalamic gonadotropin-releasing hormone (GnRH) stimulates the secretion of pituitary hormones, e.g., FSH and LH . Follicuotential , 36. A votential .Fagonia indica contains different phenolic and flavonoid phytoconstituents that have potential to treat signs and symptoms of PCOS.Herbal drugs have shown excellent results in management of PCOS having steady therapeutic effects with least side effects. They increase immunity of body and regularize menstrual cycle . The ethThe PCOS rat model was successfully developed by administration of letrozole. It is a nonsteroidal and highly potent aromatase inhibitor . CurrentF. indica has shown antioxidant, antiobesity, antilipoprotein and antidiabetic activities. It regularized the imbalanced reproductive hormones as well. So, the present study was aimed to explore its therapeutic potential on PCOS rat model. F. indica have antioxidant potential due to the presence of its flavonoid phytoconstituents [Fagonia indica and are known potent antioxidants [F. indica elevated the levels of CAT, GSH, and SOD enzymes.Previously, tituents . Quercetoxidants , 42. Antoxidants . Quercetoxidants . The resF. indica abolished the irregularity of estrous cycle and controlled the cyclicity. These changes might be attributed to decreased FSH leveled and increased testosterone leveled in PCOS group. Treatment with F. indica enhanced the levels of FSH and reduced the levels of testosterone. Furthermore, combination treatment exhibited remarkable outcomes in group V by restoring the nearly normal physiology of ovaries. Previous evidences suggested that quercetin possessed innate ability to rectify hormonal imbalance and subsequent metabolic disorder that occurred in PCOS [Histology of vaginal smear is the key determinant of ovarian physiology. Vaginal smear test is indicative of disease induction in group II as the leukocytes were present abundantly showing the diestrus stage of estrous cycle. PCOS rats remained for a prolonged period in their diestrus stage. While, treatment with  in PCOS .α hydroxylase enzyme levels, which is used in the reaction to synthesize the androgen from progesterone [F. indica nearly normalized the LH levels.Serum LH levels are increased in PCOS because of disrupted hypothalamic-pituitary axis that activates PI3K/Akt pathway causing the overexpression of ovarian CYP17A1 gene along with 17-esterone . Previouesterone . CurrentF. indica reduced serum levels of total cholesterol, TGs, and LDL while, caused an increase in HDL levels. This may be attributed to quercetin content of the ethanolic extract of Fagonia indica [F. indica also have hypoglycemic effect [F. indica reduced insulin resistance.Current study showed that a indica . Proposea indica . Querceta indica . PCOS pac effect . CurrentFagonia indica at the molecular and genetic levels. Future studies may also emphasize on identification of phytochemical constituents responsible for the found effect.Furthermore, this study can be extended to find out the effects of ethanolic extract of Fagonia indica had the potential to ameliorate polycystic ovarian syndrome at the dose of 500 mg/kg which is statistically comparable with metformin. This property might be attributed to antioxidant activity and improvement in liver enzymes and lipid profile, along with amelioration of hormonal enzymes by F. indica.Current study showed that the ethanolic extract of


Biomarkers are needed for frailty, a common phenotype often associated with muscle loss in older people. Plasma gelsolin (pGSN) is a protein largely synthesized and secreted by skeletal muscle.To investigate whether pGSN could be a biomarker of the frailty phenotype and predict mortality.n = 3490) has been followed since the 1960s. In 2010/11, frailty phenotypes by modified Fried criteria were assessed. pGSN was measured in a convenience subset  and re-measured in survivors (n = 127) in 2017. Mortality through December 31, 2018 was retrieved from national registers. Regression models were used for analyses.A homogenous cohort of males  association between pGSN  and frailty. After multivariable adjustment, higher pGSN levels were associated with lower odds of having contemporaneous phenotypic prefrailty  and frailty . By 2018, 179 males (38.2%) had died, and higher baseline pGSN predicted a lower 7-year mortality rate . pGSN concentrations in 2010/11 and 2017 were correlated .Of 469 males, 152 (32.4%) were robust, 284 (60.6%) prefrail, and 33 (7.0%) frail in 2010/11. There was a graded (Higher baseline pGSN concentrations were associated with a persistently robust phenotype and lower mortality rate over 7 years in a cohort of octogenarian males with high socioeconomic status and may be a promising laboratory biomarker for the development of a frailty phenotype. The calcium-dependent role of gelsolin in the regulation of the actin cytoskeleton was first described by Yin and Stossel in 1979 . HoweverBecause skeletal muscle is a major source of gelsolin circulating in serum , we hypoThe HBS cohort has been described in detail previously , 14, andIn brief, 3490 men, mostly business executives born between 1919 and 1934, participated in volunteer health check-ups during the 1960s and early 1970s. The cohort has been followed-up using national registries, regular postal questionnaire surveys with good response rates, and clinical examinations in random subcohorts. The present analysis originated from a convenience sample of 525 home-dwelling men who were investigated with questionnaires  and clinical examinations including laboratory tests in 2010/11, with subsequent 7-year mortality follow-up. Of the 525 men, we had plasma gelsolin available for 505 men, and the frailty phenotype could be assessed in 469 men. The 469 men (89% of 525) form the analytical group for the present mortality and frailty analyses.Multimorbidity was defined as the presence of at least 2 out of 8 chronic diseases : coronarAfter the mailed survey in 2010/11 we clinically studied 525 survivors willing to visit the clinic, completing the investigation with a blood venous sample for laboratory analyses. Because the time between postal questionnaire and clinical examination varied, contemporary lifestyle, mental, and functional data were updated through an interview by the study nurse.The clinical investigations included measured height, weight, waist circumference, blood pressure, pulse rate, tests for physical function , handgrip (a handheld dynamometer), and Mini-Mental State Examination (MMSE). Weight-adjusted waist index, reflecting fat and muscle mass, was calculated as previously described . RoutineIn 2017, the questionnaire, clinical and laboratory examinations including pGSN measurements were repeated for 127 men of the cohort. As in 2010/11, this was a convenience sample of HBS survivors willing to visit the clinic. Accordingly, they represent a group of well or relatively well-functioning survivors.2.Shrinking: Weight loss > 5% between 2007 (closest data before 2010/11) and 2010/11, or current BMI < 22.0 kg/mWeakness: Hand-grip strength (cut point 27 kg) . Exhaustion .Low activity  .Robust, prefrailty and frailty phenotype were defined in 469 men according to the 5 criteria described in the Cardiovascular Health Study (CHS) by Fried et al. , with thResponders with 3–5, 1–2, and zero criteria were classified as frail, prefrail, and robust, respectively.In the 2017 examinations, frailty status was defined as above, except that weight loss was calculated from weight measured in 2010/11.Participants were linked to electronic health records of the Digital and Population Data Services Agency. This agency keeps a registry of all citizens in our country and thus determination of vital status was reliable with no loss to follow-up. We retrieved the total mortality of the study cohort through December 31, 2018.p < 0.05). pGSN was studied both as a continuous variable and as divided into thirds with the bottom tertile as the reference. The results are presented as odds ratios (OR) with their 95% confidence intervals (CI).Statistical analyses were performed with NCSS 2020 software . Continuous variables are shown as means with standard deviation (SD), standard error (SE), or medians with interquartile ranges (IQRs). Spearman rank correlation and analysis of covariance (ANCOVA) were used to compare groups. Logistic regression was used to examine associations of gelsolin and other baseline covariates with frailty and prefrailty. Covariates were clinically meaningful variables which in univariate analyses were significantly related to frailty phenotype  with their 95% CIs. To assess the independent role of gelsolin, effect estimates were adjusted for age and frailty phenotype in 2010/11. Receiver operating characteristic (ROC) curve was constructed for pGSN and frailty. In all analyses, p-values < 0.05 (two-sided) were considered to indicate statistical significance.n = 469) in 2010/2011 , 152 (32%), 284 (61%) and 33 (7%) were assessed to be robust, prefrail and frail, respectively. Baseline characteristics according to frailty groups are shown in Table p < 0.001) associated with mortality risk (Table p = 0.023) between the groups. Of the routine laboratory variables, only plasma homocysteine levels differentiated robust men (lower homocysteine) compared with prefrail and frail men (p = 0.016 between groups). hs CRP concentration and lipid values were not significantly different between frailty groups.Of the analytical sample (p = 0.93), but there was a graded and statistically significant (p = 0.018) association between frailty status and gelsolin level, with robust men having the highest values  57.9 ug/mL (IQR 52.2 to 64.0), respectfully and followed a Gaussian distribution. pGSN levels did not correlate with age . After adjustment for age, higher pGSN, both as a continuous (per SD) and as a categorical variable in tertiles, was associated with a significantly lower risk of mortality , 125 44%), and 23 (70%) men died in the robust, prefrail, and frail groups, respectively . Also in 2017 there was a graded association between mean age-adjusted gelsolin concentrations and frailty status but in this smaller group of survivors the association was not statistically significant (p = 0.33).In the 2017 examination of 127 surviving men , 35 (27%) were robust, 81 (64%) prefrail, and 11 (9%) frail, and the groups are compared in Table In our octogenarian, home-dwelling male cohort, a higher pGSN level was associated with a robust phenotype independently of other clinical characteristics associated with frailty. Higher baseline pGSN level also predicted lower total mortality over 7 years of follow-up. Among a large set of laboratory variables, including hsCRP, pGSN was a strong independent biomarker for the frailty phenotype. This independency suggests that lower pGSN values are mainly due to lower muscle mass and reduced production rather than increased consumption by low-grade inflammation related to aging (“inflammaging” ). StudieThe robust association between low gelsolin and frailty is important because reliable laboratory biomarkers of phenotypic frailty for clinical use are currently lacking. Operational definition of frailty phenotype  based onp = 0.006).Because pGSN largely originates from myocytes , our resThere are no established reference values for pGSN in an older population; in our ELISA analysis, the median concentration of healthy adults has been 56.8 ug/mL, falling in the range of the present older cohort Table . The pGSLimitations of our study include the inclusion of a community-dwelling all-male-all white cohort with high socioeconomic status—limiting generalizability—and the lack of details regarding comorbidities and hospitalizations. Further studies including women and non-white participants are warranted to examine the generalizability of our findings. We did not define frailty phenotype using the exact CHS criteria, but the validity of our definition was supported by its predictive validity for mortality and association with fall tendency. Gelsolin values in our cohort were higher than concentrations in severe diseases probably reflecting the relatively high functional status of the participants . Area under the curve was significantly high considering that gelsolin was used as a single biomarker of a multi-factorial frailty phenotype. Future studies are needed to find a possible frailty biomarker score where gelsolin could be one important component. Finally, our analyses cannot distinguish cause and effect, i.e., exclude the possibility that high pGSN would actually protect from frailty. This directionality would be plausible considering the pleiotropic effects of gelsolin –4. A phyOur results suggest that plasma gelsolin predicts long-term mortality among octogenarian males and is a promising biomarker for phenotypic frailty. These findings should be considered hypothesis-generating rather than definite as our study was based on a relatively small sample size and did not include replication in an independent population.