An oral acute toxicity study was carried out following OECD guidelines. Hepatotoxicity was induced by the administration of ethanol for 4 weeks. Hepatic enzymes and oxidative stress biomarkers were determined using commercial diagnostic kits. ∗∗p < 0.01) reduced SGPT. Similarly, serum SGOT and ALP were significantly decreased by MECW . All used doses of MECW significantly increased antioxidant enzymes GSH and SOD. MECW (600 and 800 mg/kg) significantly promoted CAT levels in liver tissues; whereas, it significantly diminished oxidative biomarker, MDA. Histopathological observations of the liver showed improvement in the architecture of hepatic cells having signs of protection with a reduced number of inflammatory cells, vascular degeneration and congestion, cellular degeneration, necrosis, and significant reduction of fatty cells accumulation. Acute toxicity study resulted in the well-tolerability and safety of used doses of MECW (200–1000 mg/kg) in rats.  Treatment of rats with MECW (800 mg/kg) showed the highest reduction of body weight (4.76 ± 0.372 vs. 16.92 ± 0.846) and liver weight (3.06 ± 0.128 vs 5.55 ± 0.311). Treatment of rats with MECW at 200, 400, 600, 800, and 1000 mg/kg significantly (Clerodendrum wallichii extract against ethanol-induced liver injury in the laboratory rats model. Our study clearly demonstrated the hepatoprotective effect of  Medicinal plants and isolated bioactive compounds are a good hub for the discovery and development of novel therapeutics. Due to the adverse effects and toxicity issues of synthetic drugs, scientists all over the world are emphasizing natural medicines and phytocompounds for the treatment of chronic as well as emerging diseases such as diabetes [cancer [, immunological disorders [, hyperlipidemia [, obesity [, neurological diseases [, inflammation [, diarrhea [, infections [and renal impairment [Clerodendrum wallichii Merr (family: Lamiaceae) is a tall ornamental bushy shrub with pendulous white flowers which is native to Southern Asia and Southern China, found in hilly areas [Clerodendrum wallichii is found in open forests on mountain slopes. The leaves of the plant have been used in the traditional system of medicines for the treatment of fever, skin infections, inflammation, diarrhea, and dysentery [C. wallichii were reported to possess stigmasterol, β-sitosterol, clerosterol, clerodolone, and 24(S) ethyl-cholesta-5,22,25-trien-3β-ol [Clerodendrum wallichii leaves was reported to contain cubic type and square type calcium oxalate crystals [diabetes –7, cance [cancer , 9, immuisorders –17, hypeipidemia , 19, obe obesity , neurolodiseases –23, inflammation , 25, diadiarrhea , 27, inffections , and renpairment . Clerodely areas . In Chinysentery . Some trysentery . Phytochysentery , 33. Theen-3β-ol , 34. Powcrystals .Clerodendrum sp. was reported to contain cardiac glycosides, terpenoids, anthraquinones, proteins, flavonoids, saponins, tannins, lignin, phenol, and alkaloids [Clerodendrum wallichii was reported to have analgesic, antioxidant, and cytotoxic activities [Clerodendrum sp. which resulted in the presence of 68 phytocompounds; among them, 29 were found to be unique [Phytochemical analysis of lkaloids . Methanotivities . The hydtivities . Alcoholtivities . Kar et e unique . The maje unique .Clerodendrum species, pharmacological and biological investigations on Clerodendrum wallichii are scarce. No scientific investigation has been conducted to evaluate its effect on the liver and/or its functional activities. Therefore, we aimed to assess the effect of methanol extract of the leaves of Clerodendrum wallichii (MECW) against ethanol-induced liver injury in the laboratory rats model.Although several studies have been conducted on different Clerodendrum wallichii (Merr) were collected from the hill forest of Jiangsu province of China in June 2019. Then, the plant was then identified by a Taxonomist and a voucher of plant specimen has been preserved in the Institute of Botany, Jiangsu province, China.Fresh whole plants of Clerodendrum wallichii (MECW) was prepared as a concentrated gummy mass which was then transferred to a clean beaker and evaporated the methanol residue in the extract.The extract was prepared by maceration techniques , a type Methanol and ethanol were purchased from Merck (Germany). Silymarin (Milk Thistle) was purchased from a local pharmacy which was used as a standard hepatoprotective drug for experimental purposes. Ketamine HCl and xylazine HCl were purchased from Sigma-Aldrich . All other analytical grade reagents were locally procured and purchased.ad libitum. Environmental changes were strictly controlled, and prior to any experiment, all the animals were kept for 1 week to adjust to the new housing environment.Sprague-Dawley (SD) male rats weighing between 120 and 160 g, aged 6–8 weeks old, were purchased from Shanghai Laboratory Animal Center . The rats were housed six per plastic cage provided with wood chip bedding, maintained with a 12/12 h light-dark cycle, and allowed free access to standard rodent diet and water The ethical approval was obtained from the Ethics Committee of the 904 Hospital of PLA Joint Logistic Support Force, Jiangsu, China . All the experiments were conducted according to the approved Animal Use Protocol by the Ethics Committee and in accordance with the Guidelines for Care and Use of Laboratory Animals published by the US National Institutes of Health. The Federation of European Laboratory Animal Science Associations (FELASA) guidelines and recommendations were followed to reduce the pain and stress of the experimental animals. At the end of the experiments, the rats were sacrificed with anesthesia overdose: Ketamine HCl (100 mg/kg) and xylazine (10 mg/kg) through intraperitoneal route .Group 1: nonalcoholic control group Group 2: alcoholic control group Group 3: ethanol (1 ml/100 gm b.w.) + MECW (200 mg/kg b.w.)Group 4: ethanol (1 ml/100 gm b.w.) + MECW (400 mg/kg b.w.)Group 5: ethanol (1 ml/100 gm b.w.) + MECW (600 mg/kg b.w.)Group 6: ethanol (1 ml/100 gm b.w.) + MECW (800 mg/kg b.w.)Group 7: ethanol (1 ml/100 gm b.w.) + MECW (1000 mg/kg b.w.)Group 8: ethanol (1 ml/100 gm b.w.) + Silymarin (25 mg/kg b.w.)Forty SD male rats were randomly divided into eight groups, each group contained 5 rats:C. wallichii (MECW) as mentioned above for 4 weeks along with the administration of ethanol every day. Silymarin was used as a positive control (standard hepatoprotective agent).The animals were treated with different doses of methanol extract of 50 determination) of methanol extract of C. wallichii (MECW) was performed following the OECD (Organization for Economic Cooperation and Development) guideline by Fixed Dose Procedure (OECD protocol no. 420) as followed by Kifayatullah et al., 2015 [An oral acute toxicity study . Briefly, the frozen liver tissue samples were defrosted and homogenized in ice-cold phosphate-buffered saline. The homogenate was centrifuged at 3,000 rpm at 4°C for 10 min. Then, the supernatants were collected and assayed for the determination of CAT, GST, SOD, MDA, and H2O2 by using commercially available kits  as per the procedure followed by Maimaitimin et al., 2018 [The experimental rats were treated with different doses of thanasia . The ratl., 2018 .For the evaluation of the functions of the liver in the treated and untreated rats, the levels of serum glutamic pyruvate transaminase (SGPT), serum glutamic oxaloacetic transaminase (SGOT), and alkaline phosphatase (ALP) were measured using commercial diagnostic kits  as per the procedure described by Bera et al. .pH 7.4) and embedded in paraffin. A portion of the liver was cut (4-5 μm), stained with hematoxylin-eosin (H&E), and the sections were examined with a computer-aided microscope for the determination of morphological and/or pathological changes (×600 magnification). The histological scores were assessed as mentioned by Ishak et al. (1995) [E staining in the livers was scored using a scale of 0 to 4 . Each tissue was evaluated for the sum of three parameters and by the degree of liver injury using a qualitative score that ranged from 0 to 4. A score of 0 was categorized as no damage, scores between 1 and 2 were categorized as light injury, and scores of 3 and 4 were categorized as serious injury.Histopathological experiments were performed as described by Song et al. (2018) . After s. (1995) . The scop value, p < 0.05 was considered statistically significant.The data obtained from the investigation were analyzed by one-way analysis of variance (ANOVA) using Statistical Package SPSS (version 25.0) software of IBM  Corporation, USA, followed by Dunnett's-T3 test to determine statistical significance between groups. The data are means ± SEM (standard error mean) of five animals. The ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001) reduced both the body weight and liver weights in alcoholic rats. The highest reduction of body weight and liver weight was observed by MECW at the dose of 800 mg/kg (4.76 ± 0.372 vs. 16.92 ± 0.846 for body weight and 3.06 ± 0.128 vs 5.55 ± 0.311 for liver weight) compared to the untreated alcoholic control group. This significant reduction has almost changed the body weight and liver weight to the normal value when compared with nonalcoholic control.Administration of ethanol to SD rats significantly increased both the body weight and liver weight compared to the nonalcoholic control group . TreatmeThe level of hepatic enzymes was measured to evaluate the protective effect of MECW against ethanol-induced liver damage in SD rats. Our experiment resulted in the fact that the treatment of the rats with ethanol significantly elevated the levels of important hepatic enzymes SGPT, SGOT, and ALP in serum compared to the control group Figures . However##p < 0.01) compared to the control group (89.6 IU/L vs. 40 IU/L). However, further treatment of the alcohol-induced rats with MECW at the doses of 200, 400, 600, 800, and 1000 mg/kg significantly (∗∗p < 0.01) reduced the level of SGPT compared to the alcoholic control group. This reduction was so potential that the level of SGPT reduced to the normal level which was observed in the serum of nonalcohol-treated rats. The highest reduction of the level of SGPT was observed by MECW at the dose of 800 mg/kg.As we can see in ###p < 0.001, 105.4 vs. 268 IU/L) and ALP  was significantly increased in alcohol-treated rats , CAT , and SOD  in the tissues of the liver when compared to the nonalcoholic control group. However, treatment of rats with all the doses of MECW significantly increased the activities and levels of GSH and SOD compared to the alcoholic control group . However, further treatment of the alcoholic rats with MECW significantly diminished the levels of MDA; the maximum reduction of MDA level was observed by the MECW (600 mg/kg)   .Histopathological observation of liver sections of the nonalcoholic control group showed normal cellular architecture, distinct hepatic cells, sinusoidal spaces, central vein, and no accumulation of fat . In the 50 for oral administration of MECW is higher than 3000 mg/kg BW Therefore, the used doses of MECW (200–1000 mg/kg) were well tolerated by the animals (data not shown).The oral acute toxicity study of MECW was carried out following OECD guideline-Fixed Dose Procedure (OECD protocol no. 420) as mentioned in the Methodology section. No case of mortality was observed during the 14 days of treatment with a limit dose of 3000 mg/kg BW of MECW. All the treated animals could tolerate the MECW doses, and there was no statistically significant difference in body weight between the treated and untreated groups. The animals did not exhibit any abnormalities or major behavioral changes such as respiratory distress, abnormal locomotion, tremors, salivation, diarrhea, sleep, walking backward, reaction to handling, catalepsy, coma, or any toxic symptoms either immediately or during the posttreatment observational period of 14 days. Thus, we can say that the LDClerodendrum wallichii Merr against chronic administration of ethanol-induced liver injury in SD rats. The liver is impaired or damaged or injured by several factors such as chemicals including alcohol [The present study evaluated the hepatoprotective activity of methanol extract of leaves of  alcohol , drugs [ alcohol , viruses alcohol , and str alcohol . In our ALT (previously known as SGPT) and AST (known as SGOT) are aminotransferases that catalyze the interconversion of amino acids and alpha-oxo-acids by transfer of amino groups. AST is present in the cytoplasm as well as in the mitochondria of the hepatic cells whereas ALT is present in the cytoplasm of hepatic cells . Both thThe enzyme ALP is present in the cell membranes of organs that exhibit high excretory or absorptive capacity including cells lining liver sinusoids, epithelial cells of renal tubules, brush border cells of the small intestine, salivary epithelial cells, etc. The ALP is bound to the cell membranes or microsomes . ALP facClerodendrum wallichii has not been performed in this study. Previous studies reported that C. wallichii contains phenolic and flavonoid compounds [Clerodendrum paniculatum flowers (another Clerodendrum species) against carbon tetrachloride-induced hepatotoxicity in rats [in vitro. GC-MS and HPTLC analysis of the flavonoids/phenolic rich fraction of the extract identified 5 major compounds that were supposed to be responsible for its hepatoprotective activity which include gallic acid, quercetin, glyceric acid, pangamic acid, and pilocarpine [Clerodendrum wallichii (MECW) against liver injury by modulating antioxidant enzymes are assumed to be polyphenols and/or flavonoid compounds which may include gallic acid, quercetin, and kaempferol. However, the actual bioactive phytocompound(s) responsible for hepatoprotective action would be determined by extensive phytochemical analysis for the isolation of phytocompounds and further evaluation of isolated compounds for hepatoprotective activities which may lead to bioassay-guided hepatoprotective drug discovery.The phytochemical analysis of the methanol extract of ompounds , 33. Recompounds  reported in rats . They alocarpine . Quercetocarpine . TherefoOur study clearly demonstrated that MECW has a strong hepatoprotective ability against ethanol-induced liver injury in experimental rats. The hepatoprotective activity of MECW was exhibited by controlling the levels of oversecretion of hepatic enzymes and promoting the antioxidants of hepatocytes restoring them to the normal level. However, further investigations are warranted.Clerodendrum wallichii Merr. methanol extract in an animal model. Due to lack of time, we could not isolate the bioactive phytochemicals and evaluation of the isolated compounds for their hepatoprotective activity. However, we will next study the identification and isolation of bioactive compounds responsible for the potential hepatoprotective activity and its toxicological profile as well following bioassay-guided drug discovery guidelines.In this study, we have evaluated the hepatoprotective activity of


Control of glucose, blood pressure, cholesterol, and smoking improves the prognosis of individuals with diabetes mellitus. Our objective was to assess the level of control of these risk factors in Brazilian adults with known diabetes and evaluate correlates of target achievement.Cross-sectional sample of the Brazilian Longitudinal Study of Adult Health, composed of participants reporting a previous diagnosis of diabetes or the use oof antidiabetic medication. We measured glycated hemoglobin (HbA1c) and LDL-cholesterol at a central laboratory and blood pressure following standardized protocols. We defined HbA1c  < 7% as glucose control (target A); blood pressure  < 140/90 mmHg (or  < 130/80 mmHg in high cardiovascular risk) as blood pressure control (target B), and LDL-c  < 100 mg/dl (or  < 70 mg/dl in high risk) as lipid control (target C), according to the 2022 American Diabetes Association guidelines.Among 2062 individuals with diabetes, 1364 (66.1%) reached target A, 1596 (77.4%) target B, and 1086 (52.7%) target C; only 590 (28.6%) achieved all three targets. When also considering a non-smoking target, those achieving all targets dropped to 555 (26.9%). Women , those aged  ≥ 74 , and those with greater per capita income  were more likely to reach glucose control. Those black  or with a longer duration of diabetes  were less likely. Women  and those with private health insurance  were more likely to achieve two or more ABC targets; and those black  and with a longer duration of diabetes  less likely.Control of ABC targets was poor, notably for LDL-c and especially when considering combined control. Indicators of a disadvantaged social situation were associated with less frequent control. Diabetes mellitus is a chronic and complex disease that requires continuous medical care. In addition to adequate glycemic control, multifactorial risk reduction is indicated . In clinRecommended therapeutic targets, also called the ABC goals, usually include (A) glycated hemoglobin (HbA1c) less than 7%, (B) a blood pressure < 140/90 mmHg, and (C) LDL-cholesterol (LDL-c) < 100 mg/dl. Tighter control has also been suggested for those with cardiovascular disease or at high risk of developing it . AdditioStudies demonstrating the control of multiple risk factors in diabetes are scarce in low- and middle-income countries. Based on a small subsample of a probabilistic national Brazilian survey, we demonstrated that target achievement is usually poor, except for non-smoking . HoweverWe conducted a cross-sectional study based on the third clinic visit 2017–2019) of ELSA-Brasil. The study was approved by the research ethics committees of participating institutions, and all participants gave their written informed consent. The ELSA-Brasil cohort enrolled 15,105 in-service or retired civil servants aged 35 to 74 at six public institutions of higher education located in capital cities of the states of Bahia, Espírito Santo, Minas Gerais, Rio de Janeiro, São Paulo, and Rio Grande do Sul [–2019 of Centrally trained and certified teams conducted standardized interviews and clinical assessments and collected samples for biochemical tests . We obta2 (kg/m2).Medication use was confirmed by packaging or prescriptions brought to the clinic. Blood pressure was measured three times, and systolic and diastolic blood pressure each was ascertained as the mean of the last two measurements. We assessed weight and height using a standardized protocol and calculated body mass index (BMI) as weight/height®, Roche Diagnostics), HbA1c by high-pressure chromatography , total cholesterol by enzymatic colorimetric method, and triglycerides by glycerol-phosphate peroxidase . Low-density cholesterol (LDL-c) was estimated by the Friedewald equation when total triglycerides were  < 400 mg/dl and measured directly when they were  ≥ 400 mg/dl.We obtained blood samples after an overnight (> 8 h) fast and froze and shipped them to a central laboratory for determination. Plasma glucose was measured using the hexokinase method  was estimated based on age, sex, diabetes, smoking, systolic blood pressure, and total cholesterol according to the WHO Risk Chart Working Group chart for the Tropical Latin America region . We cateIn line with the 2022 American Diabetes Association (ADA) guidelines for therapeutic targets , we cons©2009–2020 RStudio Inc.).We described categorical variables as frequencies and percentages, and continuous ones as means and standard deviations (SD). We performed unadjusted statistical testing with the chi-square test for categorical and ANOVA for continuous variables. We analyzed the adjusted associations of socio-demographic and clinical factors with the level of control using Poisson regression with robust variance. We undertook all analyses with R software  participants at visit 3, 2385 (18.9%) had a known diagnosis of diabetes. After excluding 323 participants with missing data on risk factor control or covariates, 2062 remained.2, and 1812 (87.9%) individuals related use of an antidiabetic drug.  were men, 1217 (59.0%) aged 45–64, 961 (46.6%) with self-declared white race/skin color, and 1068 (51.8%) with a complete university education. Additionally, 1380 (66.9%) reported having private health insurance. Mean BMI was 29.6 (4.97) kg/mFigure As further seen in Table As seen in crude comparisons in Table As seen in Table In this free-living sample of 2063 Brazilian adults with known diabetes, HbA1c was controlled in more than half of the sample (66.1%), as were blood pressure (77.4%) and LDL-c (52.6%). However, only 28.6% of participants had all three factors controlled. Some indicators of greater social privilege  are associated with meeting targets.The fraction of individuals reaching glycemic, blood pressure, and LDL-c goals in the ELSA-Brasil cohort was greater than that seen in the 2013 Brazilian National Health Survey : 66.1% iIn studies in diverse countries, attainment of all ABC goals was always low. In the US NHANES, 22.2% of individuals simultaneously achieved all three targets  . In a stWe found several characteristics that identified those not reaching targets for hyperglycemia and the ABC goals. As expected, a greater duration of diabetes was one. A higher BMI was marginally associated with a lesser frequency of control. Women were more frequently in glycemic control and achieved greater ABC control. The additional factors associated with worse control—being non-white, with lower income, and not having private health insurance, all point to better control being in part the result of social privilege.Similarly, a representative survey of adults with diabetes showed that private health insurance led to their receiving better quality primary care, as measured by the cardinal attributes of quality primary care, especially access. Greater access to care provides a logical pathway linking this insurance to better control . A multiOur study has limitations, principally that our sample is composed of active or retired civil servants, a socially privileged sample when compared to the general Brazilian population in terms of educational achievement, income, and job stability among other factors. That we found major socioeconomic determinants of control in this more privileged population only emphasizes the likelihood of greater health disparities in achieving ABC targets in the general diabetic population.Strengths of our study include its free-living sample of participants obtained in multiple cities across Brazil, different from many other studies which investigated less representative inpatient or outpatient samples which will have both more comorbidities and, by their entry criteria, better access to care. Additional strengths include ELSA´s careful and extensive collection of factors examined, its standardized and centralized laboratory measurements, and its sample size permitting adequate investigation of epidemiologically relevant associations.As has been shown for health outcomes in general , the corThese findings are particularly relevant now, as actions aimed to achieve greater control at the health system level, supported by greater tracking and feedback of care, are now feasible given advances in information technology. The implementation and expansion of a diabetes registry orienting patient care in several Asian countries produced improvement in control of all the ABCs. In Hong Kong, a setting for which longer follow-up is available, the implementation of the registry was accompanied by a 40% decrease in CVD or microvascular complications and a 66% decrease in all-cause mortality,  and was Additionally, issues of relaxed control, especially of HbA1c, in older patients and those with greater morbidity and thus greater difficulty in managing multiple medications, are also important . The AmeIn conclusion, control of ABC targets was poor, notably for LDL-c and especially when considering combined control. Our findings reinforce that much room exists for improvement in controlling these modifiable prognostic factors, notably LDL-c and especially when considering combined control. Less frequent control among those black, with lower income, and without health insurance reinforces the role of social factors in the multicausal context of control of risk factors for complications in diabetes. With due attention to social determinants and focusing on better integration of health system data to evaluate and orient patient care, health systems and clinicians can and should strive to implement better care for people with diabetes.


Physical activity (PA) is a basic and initiative conservative management for people with knee osteoarthritis (KOA). This study aimed to explore the potential indicators of PA levels in people with KOA.  We designed a cross-sectional study where people with KOA were consecutively approached by the Orthopedic Outpatient Department in a hospital in southern Taiwan. People older than 50 years that could communicate and consent to the present study were enrolled. As a dependent variable, the Chinese version of the Physical Activity Scale for the Elderly (PASE-C) was used to assess the participant's PA levels. Considering differences in sex, a PASE-C score cut-off point of 140 for men and 120 for women was used. Participants were then divided into “active” and “inactive” groups. We measured independent variables consisting of the demographic and clinical characteristics, such as comorbidities measured by the Charlson Comorbidity Index (CCI), depression status measured by the Geriatric Depression Scale-5, body mass index, KOA history , knee pain , the severity of symptoms measured by the Western Ontario and McMaster Universities Osteoarthritis Index, and 6-meter preferred walking speed. Multiple logistic regression was performed to identify significant relationships between PA among people with KOA. n = 137) were categorized as “inactive PA,” while 27.1% (n = 51) of participants were categorized as “active PA” . Multiple logistic regression showed a positive association of 6-meter preferred walking speed with active PA , whereas advanced age and comorbidity (CCI≥1 vs. CCI<1) were negatively associated with active PA with an OR (95% CI) score of 0.91 (0.86–0.97) and 0.37 (0.15–0.87), respectively.  We analyzed a total of 188 people with KOA (56 men and 132 women) with a mean age of 69.4 ± 7.9 (range: 51 to 90 years). Approximately 72.9% ( People with KOA require appropriate lifestyle management to increase PA. Walking speed may be an effective factor for predicting PA among people with KOA. Healthcare providers treating KOA patients should be aware of their PA levels, especially those at risk. Knee osteoarthritis (KOA) is a very common, age-related degenerative joint disease characterized by the breakdown and loss of joint cartilage, subchondral bone degeneration, and synovitis. , 2 The ePhysical activity (PA) is a basic initiative and conservative choice in promoting health and is considered a priority during KOA management, 6. PA iStudies show that advanced age–23, femaWe conducted a cross-sectional study with purposive sampling from July to August 2020. People with KOA who attended the orthopedic outpatient clinic of a hospital in southern Taiwan were recruited. Participation required people to (1) be clinically diagnosed with KOA without a replaced knee and (2) provide informed consent or, if unable to, proxy-informed consent from their substitute decision-maker. Patients were excluded if diagnosed with the inflammatory rheumatologic disease , other severe diseases , or were unable to complete assessments due to other issues.Using in-person interviews, all participants were asked to answer questions regarding their PA, demographic and clinical characteristics, such as the severity of KOA symptoms, and 6-meter preferred walking speed. The data collection process was noninvasive and performed by researchers and two trained research assistants. Ethical approval was obtained from the institutional review committee of the selected hospital (No. KSVGH20-CT6-09).Self-reporting is the most commonly used method to measure PA in large observational studies. PA level, as the primary outcome of the present study, was assessed using the Chinese version of the Physical Activity Scale for the Elderly (PASE-C), which w2 were considered obese, according to the WHO criteria. The severity of KOA symptoms was measured using the Western Ontario and McMaster Universities Osteoarthritis (WOMAC) Index. [We collected data regarding age, gender, an education level (year), marital status (living with or without a spouse), and clinical characteristics regarding comorbidities, depression, body mass index (BMI), self-reported KOA history , site of knee pain , and severity of KOA symptoms. Comorbidities were measured using the Charlson Comorbidity Index (CCI) with a self-reported doctors' diagnosis method.  Depressi) Index.  The WOMAIn our study, the 6-meter preferred walking speed was specifically chosen to assess PA levels. Participants were instructed to walk at their usual pace without running or jogging for a distance of 6 meters. Walking time and speed were calculated by dividing distance by time (in minutes).t-tests were used to compare mean values for continuous variables, and χ2 -tests were used to compare proportions of categorical variables. Any significant demographic and clinical characteristics and 6-m preferred walking speed according to bivariate analyses (Student's t-tests or χ2 tests) were used in multiple logistic regression using the enter method. This allowed us to adjust for known factors affecting active PA and identify significant independent factors among people with KOA. The results were reported as odds ratios (ORs) in 95% confidence intervals (CIs). For all statistical tests, SPSS for Windows 23.0  was used and a P value of less than 0.05 was considered statistically significant.Descriptive statistics are reported as a number  for categorical variables and as mean ± standard deviation (SD) for continuous variables. When comparing inactive and active PA groups, Student's A total of 188 people with KOA , consisting of 132 women and 56 men, were enrolled in this study. The mean PASE-C score was 93.0 ± 65.1 (range: 0–398.5). We used a score cut-off point of 140 for men and 120 for women  to distiP < 0.05). No significant differences between groups were found for the other variables. P < 0.05).χ2 = 9.39, df = 8, P = 0.311), which did not reach significance, the regression model showed ideal goodness of fit. Cox and Snell and Nagelkerke R2 values were 0.21 and 0.30, implying that approximately 21.0%–30.0% of the variance in PA was accounted for by this model. Tests on the Wald values for age, CCI score, and 6-meter preferred walking speed resulted in P values ≤ 0.05, indicating that these three variables are independent factors.All variables with significant differences between groups in Tables Our study assessed PA levels using the PASE-C questionnaire. The mean PASE-C (SD) score was 93.0 (±65.1), with a range of 0 to 398.5. A previous study using the same PASE-C questionnaire found a mean ± SD of 104.4 ± SD 47.1 in a community-dwelling of older people.  This resAfter controlling all variables, we found that faster walking speed still showed a strong relationship with active PA. Walking speed has been termed “the sixth vital sign”  due to iP=0.002). Each added year of life was associated with a 9% decrease in OR of PA in this study sample. This outcome was congruent with previous studies[As expected, advanced age was negatively associated with active PA  had cardiovascular disease and 19.1% (n = 36) had diabetes, both of which showed significant differences between active and inactive groups according to bivariate analyses(People with osteoarthritis (OA) commonly show various comorbidities that are likely to influence PA behavior. In the  analyses. This ouFor variables relating to KOA, such as self-reported KOA history, pain site  or the WOMAC score, only functioned as significant factors in the bivariate analyses but not in the multiple logistic regression analyses. Empirical studies have demonstrated that increasing PA or exercise can reduce pain and improve mobility in people with KOA–9. HowevIn previous studies, spousal support for PA was a key factor in initiating and maintaining PA in the general population,46. SpouOur study sheds light on factors affecting PA in people with KOA, however, it was subject to some limitations. First, there exists no comparable standard to use as supporting evidence for the validity of PASE-C score outcomes in older adults. This self-reported questionnaire may also be prone to recall bias, misclassification, and over- or underestimation of PA levels. Further research using objective PA measures, such as accelerometers cached in wearable devices, should allow for more accurate outcomes. Second, since the cross-sectional nature of our study prevents inferring causation from our results, any relationships should be carefully interpreted before implying causation. Further longitudinal studies may be required to explore these relationships. Third, our sample size was relatively small at 188 KOA patients, all of whom were enrolled from the OPD of the same hospital. Caution is therefore needed when generalizing our results to other populations or patients in different hospitals. Finally, the fact that only 21–30% of the variance can be explained by the multiple logistic regression model, implies that there are some variables that can potentially explain the remaining variance. Further studies regarding advancing possible variables might be needed. Despite these limitations, our study provides useful information for policymakers, health planners, and health care providers by highlighting potential factors influencing PA levels among people with KOA. By revealing these significant relationships, this study advances our present understanding of PA status among people with KOA.Increasing PA is an important goal for people with KOA, but patients included in this study showed low PA levels. Our findings suggest that interventions aimed at increasing PA in people with KOA are essential, especially for people of advanced age or with comorbidities or lower preferred 6-meter walking speed.The funding organization had no role in the conduct of the study, collection, interpretation of data, or decision to submit the manuscript.


Dittrichia viscosa extract) and a beneficial microorganism . A combination of transcriptomics and metabolomics approaches were chosen in order to study grapevine gene expression and to identify gene marker candidates, as well as, to determine differentially concentrated grapevine metabolites in response to biocontrol product treatments. Grapevine plants were cultivated in greenhouse under controlled conditions and submitted to the treatments. Thereafter, leaves were sampled 24h after treatment to carry out the gene expression study by RT-qPCR for the three cultivars and by RNA-sequencing for ‘Garnacha Blanca’. Differentially expressed genes (DEGs) were investigated for both treatments and highly influenced DEGs were selected to be tested in the three cultivars as treatment gene markers. In addition, the extraction of leaf components was performed to quantify metabolites, such as phytohormones, organic acids, and phenols. Considering the upregulated and downregulated genes and the enhanced metabolites concentrations, the treatments had an effect on jasmonic acid, ethylene, and phenylpropanoids defense pathways. In addition, several DEG markers were identified presenting a stable overexpression after the treatments in the three grapevine cultivars. These gene markers could be used to monitor the activity of the products in field treatments. Further research will be necessary to confirm these primary results under field conditions.The present study aims to evaluate the response of the three Mediterranean local grapevines ‘Garnacha Blanca’, ‘Garnacha Tinta’, and ‘Macabeo’ to treatments with biocontrol products, namely a botanical extract (Akivi,  If the first infections are not controlled, the diseases spread quickly in the vineyard and mildews can infect berries as well. These diseases can cause severe crop losses depending on the season and the cultivation area, reducing the harvest quality and yield, plant vigor and photosynthesis  . Vineyarviticola . These cynthesis .The main grapevine cultivars are susceptible to these diseases and vineyard protection requires intensive treatments with plant protection products (PPPs), such as chemical fungicides from bud burst until ripening . The freVitis can present direct activity in grapevine against downy mildew  direct action against the pathogen  or (ii) y mildew . Some bey mildew  or to shy mildew  . Moreovy mildew . Some bey mildew . It is rray mold  and a Say mildew . Bioconty mildew .P. viticola and E. necator, whereas JA/ET pathway against necrotrophic pathogens, such as B. cinerea. However, the two pathways can be activated simultaneously  PAMP-triggered immunity (PTI) or effector-triggered immunity (ETI). Both PTI and ETI stimulate plant systemic acquired resistance (SAR) . Beneficaneously . Direct aneously . DespiteDittrichia viscosa extract) and a beneficial bacterial strain . Both products are still in development and the combination of a whole genome transcriptomics approach and a targeted metabolomics approach was chosen to elucidate the grapevine response to the treatments. The objectives of this work are: (i) to study grapevine gene expression response after biocontrol product treatment using transcriptomics; (ii) to identify robust gene marker candidates presenting stable differential expression after treatment within the three grapevine cultivars; and (iii) to determine grapevine metabolites variations after biocontrol product treatment using targeted metabolomics.This study aims to evaluate the response of three appreciated and autochthonous grapevine cultivars of the Mediterranean zone, concretely of Catalonia (Spain): Garnacha Blanca, Garnacha Tinta, and Macabeo to biocontrol product treatments. Two biocontrol products from different origins were investigated: a botanical extract , a fermentation process was done in a pilot-scale bioreactor  with a working volume of 30 L of production medium for 48 h at 28°C, pH7 and agitation ramp from 50 to 500 rpm. The production medium consisted of a modification of the original recipe of -1 instead of 1 g L-1 of KH2PO4, 1 g L-1 instead of 4 g L-1 of L-monosodium glutamate, 5 g L-1 of molasses and 1 g L-1 of soy flour instead of 342 g L-1 of saccharose, 1 mL L-1 instead of 5 mL L-1 of ferric citrate solution, and 1 mL L-1 of oligoelement solution at 0.1 mg mL-1 instead of at 0.1 mg L-1. After fermentation, the cells were harvested by centrifugation  at 10,000 rpm and the concentrated cell suspension was mixed with skimmed milk . The bacterial suspension was frozen at −70°C and lyophilized in a laboratory scale freeze-dryer . Dried samples were stored in vacuum sealed plastic-coated aluminum bags.For lyophilized Bacillus (BF), a fermentation process was carried out in a 2-L Erlenmeyer flask for 48 h at 28°C and shaking at 150 rpm with 500 mL of the original recipe of production medium (modification: oligoelement solution was used at 0.1 mg mL-1 instead of at 0.1 mg L-1). After fermentation, the cells were harvested by centrifugation at 13,200 g for 10 min  and concentrated 10X with the corresponding volume of supernatant.For fresh Dittrichia viscosa composed of a high content of polyphenols and terpenes (The plant extract Akivi was provided by S.A.S. AkiNaO (France). It is a formulated botanical extract prototype from terpenes .2.2Vitis vinifera L.), namely Garnacha Blanca, Garnacha Tinta and Macabeo, grafted on rootstock 110R, were obtained from commercial nurseries . One-year-old bench-grafted grapevine rootlings were planted in a 2 L pot with 80% of the growing media , 20% of perlite , and 4 g of the fertilizer . Bench-grafted grapevines were grown in a greenhouse at 25 ± 2°C, 60 ± 10% relative humidity and a 16:8 h light:dark photoperiod. Young stocks with at least about 4 to 6 expanded leaves were used for the experiments.Three grapevine cultivars (-1 (Aki), and Bacillus UdG at 108 CFU mL-1 lyophilized (BL) and fresh (BF). Water was used as the solvent to prepare each treatment. The BF treatment was only used in the experiment with cv. Garnacha Blanca. A non-treated control (NTC) using water was included in all the experiments. The products were sprayed on adaxial and abaxial leaf surfaces using an airbrush until near run-off.The treatments consisted of Akivi at 0.521 g LThe experimental design for cv. Garnacha Blanca stocks included 4 randomized blocks corresponding to the different treatment modalities , while for cvs. Garnacha Tinta and Macabeo included 3 blocks . Each block was composed of 4 biological replicates of 5 plants.2.3Sampling was carried out 24 h after spraying plants with the products. At that time transcriptomics response, as well as, phytormone signalization and metabolomics responses can be evaluated. Four biological replicates were sampled for each treatment for RNA-sequencing analysis (RNA-seq), and three biological replicates for reverse transcription quantitative PCR (RT-qPCR) analysis. Two leaves from each plant  were harvested, grounded, and soaked in liquid nitrogen. Each ground leaf sample was added to 2 mL tubes containing two borosilicate glass beads in order to obtain a fine powder using Tissuelyzer II system  for 1 min at 30 Hz.For total RNA isolation from grapevine leaves, the commercial kit Spectrum™ Plant Total RNA Kit  was used . RNA quality was evaluated using electrophoresis on 1.0% agarose gels.Prior to RNA-seq analysis, a R.I.N. measurement was carried out using an Agilent 2100 Bioanalyzer  to check RNA integrity from cv. Garnacha Blanca samples and RNA extracted in each sample was quantified by using the Qubit 2.0 Fluorometer .2.4The plant response to treatments using transcriptomics was studied on cv. Garnacha Blanca grapevine leaves after spray application with Aki, BL, BF or water (NTC). A total of 16 samples were used for the library construction.The RNA-seq transcriptome library was prepared using the TruSeq Stranded mRNA Sample Prep kit  following the manufacturer’s instructions using 1-2 µg of good quality RNA (R.I.N. > 7) as input. The RNA was fragmented by 3 minutes at 94°C and each purification step was performed by using 0.81X Agencourt AMPure XP beads. Final libraries were quantified by using the Qubit 2.0 Fluorometer  and quality tested by Agilent 2100 Bioanalyzer RNA Nano assay . Libraries were then processed with Illumina cBot for cluster generation on the flowcell, following the manufacturer’s instructions and sequenced on paired-end  at the multiplexing level requested on NovaSeq6000 (Illumina). The CASAVA 1.8.2 version of the Illumina pipeline was used to process raw data for both format conversion and de-multiplexing.https://www.bioinformatics.babraham.ac.uk/projects/fastqc/) before trimming and removal of adapters with BBDuk (https://jgi.doe.gov/data-and-tools/bbtools/) setting a minimum base quality of 25 and a minimum read length of 35 bp. Reads were then mapped against the V. vinifera L. genome (V. vinifera cv. Pinot noir var. PN40024) (version 12X Ensembl) with STAR v2.6 (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3530905/). FeatureCounts v1.6.1 (https://academic.oup.com/bioinformatics/article/30/7/923/232889) was then used to obtain raw expression counts for each annotated gene using only uniquely mapping reads (MAPQ>=30). The differential gene expression (DGE) analysis was conducted with the R package edgeR (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2796818/) using the Trimmed mean of M-values (TMM) normalization method and considering as significant the genes with a False Discovery Rate (FDR) ≤ 0.05. Fragments Per Kilobase Million (FPKM) were obtained with edgeR. Gene Ontology (GO) Enrichment Analysis was performed using in-house scripts based on the AgriGO publication (https://academic.oup.com/nar/article/45/W1/W122/3796337). GO enrichment analysis was carried out using a threshold value  < 0.05. The main biological functions were selected considering the Gene Ontology (GO) terms that showed at least 4 affected DEGs. Then the selected GO terms were analyzed using REVIGO web platform (http://revigo.irb.hr/) in order to summarize GO terms by removing redundancies. For each biological function category, different GO terms clusters (representative groups) that presented semantic similarity were obtained. The affected DEGs corresponding to all the GO terms of each cluster were added. In addition, GO terms that presented a background number over 1000 genes (BG-Item) were discarded since they are general GO terms. Clusters that showed less than 10 DEGs were joined under the term “other” considering the total number of genes. In addition, metabolic pathways influenced by the treatments were defined using Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation  in comparison with the NTC. The two Gene expression levels were assessed on the basis of unique mapped genes and were calculated using the FPKM method. FPKM values were used to analyze the differences in gene expression between treatments  and NTC, by calculating a Fold-Change (FC) value.2(FC) > |1.4| and good repeatability among the three biological replicates.Due to the high biological variability, the DEGs screening was conducted on the three biological replicates that presented less variability between each other, in order to avoid hiding a part of the treatment impact on the plant. DEGs exclusively altered by each treatment were targeted. The criteria of selection during the screening were based on DEGs presenting high differential expression value, specifically Log2.62(FC) > |1.4| and good repeatability among the three biological replicates) and their expression level was validated by RT-qPCR  corresponding to copy numbers ranging between 102 and 107. Ct values in each dilution were measured in triplicate and a negative non-template control was included in each run. Real-time PCR reactions included 10 μL SYBR® Green PCR Master Mix (Applied Biosystems), 6 μL RNase-free water, 1 μL of each forward and reverse primer  -1) x100, where “a” is the slope of the curve.Standard curves for DEGs and the endogenous gene were obtained using decimal dilutions of extracted recombinant plasmid DNA  as explained above. First-strand of cDNA was synthetized from RNA using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) according to the manufacturer’s instructions. The absence of chromosomal DNA contamination was confirmed by minus-reverse transcriptase control in qPCR. Quantitative PCR was carried out in a QuantStudio™ 5 Real-Time PCR System (Applied Biosystems) to assess the transcriptional level of 27 DEGs. All the information of the selected genes and primers designed by Primer-BLAST tool from the Nacional Centre for Biotechnology Information (NCBI) are shown in 1 or 2−1) higher or less than the calibrator condition  method was used to assess the relative quantification of gene expression. Similar amplification efficiencies of all gene primer pairs were checked  as explained above.For phytohormone extraction, 250 mg of fresh grapevine leaves were grounded in an ice-cold mortar with 750 μL of extraction solution  . Then, t-1 and 10 µl injected volume. The elution gradient was carried out with a binary solvent system consisting of 0,1% of formic acid in methanol (solvent A) and 0,1% formic acid in milli-Q H2O (solvent B) with the following proportions (v/v) of solvent A (t (min), %A):  , , ,  and . MS/MS experiments were performed on an ABI 4000 Qtrap mass spectrometer (Sciex). All the analyses were performed using the Turbo Ionspray source in negative ion mode except for MeJA and ACC.Plant hormones were analyzed by LC-ESI-MS/MS system in multiple reaction monitoring mode (MRM) according to Quantification was made by injection of extracted and spiked samples in multiple reaction monitoring (MRM) mode. Identification of phytohormones was based on retention time and presence of peak in the MRM trace compared with those of the standards.Organic acids (OA) were extracted with a classical extraction protocol. Briefly, 250 mg of fresh grapevine leave powder was grounded in an ice-cold mortar with 2 mL of hydrochloric acid (0.025N). Then, the supernatant was collected after centrifugation at 1000 g for 15 min at 4°C. Meanwhile, Sep-Pack C18 cartridges  were activated with (i) 1.4 mL of methanol, (ii) 0.7 mL of milli-Q water, and (iii) 1.4 mL of hydrochloric acid . Supernatant (1.4 mL) were passed through the cartridge to recover 0.7 mL of clean extract. Finally, samples were filtered at 0.22 μm just prior to injection into an HPLC system.-1 and 10 µl injected volume. The injection method ran 15 min with an isocratic flow of 50 nM Potassium dihydrogen phosphate (KH2PO4) adjusted at a pH of 2.8 using Phosphoric acid (H3PO4).Organic acids were analyzed by HPLC-UV system  in the following conditions : YMC-PacThe following standards were used for OA measurements: acetic acid, cis-aconitic acid, trans-aconitic acid, ascorbic acid, citric acid, isocitric acid, formic acid, fumaric acid, galacturonic acid, gluconic acid, glucuronic acid, glutamic acid, glycine, glycolic acid, glyoxylic acid, lactic acid, maleic acid, malic acid, malonic acid, oxalic acid, oxoglutaric acid, pyruvic acid, quinic acid, succinic acid, tannic acid, and tartaric acid.2 = 0.9967), oxalic acid , oxoglutaric acid , tartaric acid .Four peaks corresponding to OA were detected on samples HPLC-UV chromatograms. Then, these peaks’ retention times were compared with the retention times of 26 standards injected in the same conditions, and the identification was confirmed by standard enrichment injection within the grapevine samples. The four OA were identified  and quantified thanks to calibration curves. Calibration curves: malic acid  was applied to the RNA-seq data comparing the biological replicates of each treatment modality with the NTC. The statistical analysis of the RT-qPCR data was done using REST2009 Software . DEGs stFor metabolite measurements, to identify significant differences between treated (Aki and BL) and NTC leaves, several statistical tests were performed. All tests were performed on R software (R version 3.5.2) with a significant level of p-Value < 0.05. First, each of the metabolite datasets were tested (Shapiro-Wilk and Bartlett tests) to determine the suitability of parametric or non-parametric tests. For parametric tests, one-way analysis of variance (ANOVA) was carried out followed by a Tukey’s multiple comparisons test. For non-parametric tests, Kruskal-Wallis test followed by Dunn test were carried out.33.1The 16 sequencing samples produced around 39 million of total sequencing raw reads for NTC and Aki treatments, while around 36 million for BL and BF treatments  and 15.8 (NTC and Aki) million reads from each treatment could be mapped,   from each treatment. The PCA analysis revealed that one out of four biological replicates of each treatment  did not cluster as expected from the experimental design , 84.21% (BL) and 85.43% (BF) of the variance. In addition, the PC1 explained 63.33% (Aki), 60.5% (BL) and 49.38% (BF) of the variability in gene expression between each treatment and the NTC.The RNA-seq raw transcriptomic data were submitted to the GEO repository of the National Center for Biotechnology Information (NCBI) (GSE211268).3.22(FC)) and the statistical significance of the differential expression test (-Log10(FDR)) is displayed.Gene transcription in cv. Garnacha Blanca grapevine leaves was triggered by Aki, BL and BF treatments to varying degrees. The volcano plots show the degree of variation of the Differential Expression of Genes (DEGs) based on red and green dots (Bacillus treatments (BL and BF) were observed since the most of genes were distributed between Log2(FC) values of -4 and 4 and with significance values (-Log10(FDR)) up to 75 (downregulated genes) and 50 (upregulated genes). However, Akivi plot differed from Bacillus ones since the main of genes were distributed between Log2(FC) values of -3 and 3 and with lower significance values of 20 (downregulated genes) and 60 (upregulated genes).Plot similarities within Additionally, heatmaps of these DEGs for each treatment effect are shown in Bacillus treatments (BL and BF) altered the expression level of a higher number of genes than the botanical extract Akivi treatment (Aki). BL and BF treatments showed 438 and 396 upregulated DEGs, respectively, and 481 and 313 downregulated DEGs, respectively, whereas Aki treatment showed a total of 278 upregulated and 225 downregulated DEGs. In addition, the plant response towards Bacillus (both BL and BF) and Akivi (Aki) treatments was fairly specific since only 31 upregulated and 68 downregulated genes were common to all three treatments.As shown in Venn diagrams Figure 1Bacillus treatments, both lyophilized and fresh, shared a high number of up- (43.1%) and downregulated (41.3%) genes. These genes were altered by the Bacillus treatments, independently of being the product lyophilized or not. Therefore, these shared genes were used for the following validation of RNA-seq results by RT-qPCR. From the 583 upregulated genes after either BL or BF treatments, 251 genes were shared. From the rest of genes, 187 and 145 were only upregulated after BL and BF treatments, respectively. Whereas from the 562 downregulated genes after BL or BF treatments, 232 genes were shared. From the remaining genes, 249 and 81 were only upregulated after BL and BF, respectively.However, the 3.33.3.1GO enrichment analysis was carried out to evaluate the major biological functions of DEGs influenced by the Aki, BL, and BF treatments. The biological functions are classified into three categories: biological process (BP), cellular component (CC), and molecular function (MF). Upregulated GO terms were identified in 34.4, 25.8 and 25.0% of DEGs after the Aki, BL and BF treatments, respectively, whereas 35.3, 32.9 and 41.4% were downregulated  treatments. Five GO terms were shared by the two Bacillus treatments (BF and BL), while eight, three, and eight GO terms were unique for Aki, BF and BL, respectively.Some of the upregulated GO terms from BP category that were arranged in two well-defined clusters are related to plant defense response, namely “stress response” and “regulation of defense response” Figure 4Bacillus (BF and BL), while the GO terms immune response and plant-type hypersensitive response, and response to wounding, biotic stimulus and ethylene were unique for Aki.From the cluster named “regulation of defense response”, the GO term regulation of jasmonic acid mediated signalling pathway was shared by all treatments. Whereas the GO term regulation of defense response was shared by Aki and BF, regulation of systemic acquired resistance was unique for Aki. From the cluster named “stress response”, the GO terms response to osmotic stress, response to karrikin, and response to hydrogen peroxide were unique for 2(FC) > 1.4) related to the GO terms included in “regulation of defense response” and “stress response” clusters are shown in Bacillus). After Bacillus treatment, one gene related to the regulation of jasmonic acid, and several genes related to transcription factors, chaperones, enzymes as catalase, PR protein with antimicrobial activity, abscisic acid receptor, and cold induced protein were upregulated. However, after Akivi treatment, two genes related to the regulation of SAR, one defense response related gene, three genes related to the response to chitin, and one gene related to PR protein with antimicrobial activity were upregulated.The upregulated genes  < 1.4) related to the GO terms included in “stress-related response” cluster are shown in Bacillus treatments, some DEGs related to cellular cycle were downregulated. In particular, six cyclin proteins and one annexin protein were downregulated for Aki treatment. While four cyclin proteins were downregulated for Bacillus treatments.The downregulated genes  that intervene in systemic acquired resistance SAR were downregulated after Bacillus treatments.Considering different stress responses, after Aki treatment two genes connected with receptor like kinases that intervene in plant innate immunity were downregulated, while transcription factors to several stresses and abiotic stresses were downregulated after 3.3.2Bacillus treatments .KEGG pathway analysis was performed to evaluate the biological mechanisms influenced by the Aki, BL, and BF treatments. Few pathways were associated with DEGs affected by the treatments and none was shared between Akivi and The following pathways “Glutathione metabolism”, “Linoleic acid metabolism”, “alpha-Linolenic acid metabolism”, “Terpenoid backbone biosynthesis”, “beta-Alanine metabolism”, and “Fatty acid degradation” were triggered after Aki treatment. Whereas “Starch and sucrose metabolism” was triggered by both BL and BF treatments, “Ribosome biogenesis in eukaryotes” was exclusively triggered by BF treatment.The pathway “Porphyrin and chlorophyll metabolism” was reduced after Aki treatment, while “DNA replication” was reduced after both BL and BF treatments. “Cysteine and methionine metabolism” was reduced after BL treatment; while “Fructose and mannose metabolism”, “Phenylpropanoid biosynthesis”, and “Starch and sucrose metabolism” were reduced after BF treatment.3.43.4.1A total of 27 DEGs were selected since their expression level was modified due to Aki, BL and BF treatments according to the results of RNA-seq analysis Table 5.From the 12 DEGs highly triggered by Aki treatment, eight genes are related to defense response . Specifically, two genes are involved in detoxification of reactive oxidative species (A5 and A6); two genes are related to hormone signalling pathway , one gene is involved in biosynthesis of secondary metabolites (A7), and one gene is a marker of SAR response (A11). From the 15 DEGs highly triggered by BL and BF treatments, four genes are involved in defense response .3.4.2Standard curves of the 27 DEGs showed R-squared values above 0.99 and, in general, amplification efficiencies above 90%, except for three DEGs  that showed slightly lower efficiencies above 80% Table 6.Moreover, the relative expression levels of the 27 DEGs on cv. Garnacha Blanca obtained by RT-qPCR and RNA-seq analysis were highly consistent for both Aki and BL treatments Figure 63.4.3The expression levels of the 27 DEGs were subjected to RT-qPCR using samples from experiments performed with two other grapevine cultivars, namely Garnacha Tinta and Macabeo. Within the NTC samples of the ‘Garnacha Tinta’ and ‘Macabeo’ experiments, the expression levels of the 27 DEGs were stable showing fold change values close to 1 Table 6.Concerning the 12 selected DEGs by Akivi treatment, nine  and six genes  in ‘Garnacha Tinta’ and ‘Macabeo’, respectively, showed differential expression levels with statistical significance compared to the NTC  Table 6.In relation to the 15 selected DEGs by BL treatment, twelve genes showed differential expression levels with statistical significance compared to the NTC within ‘Garnacha Tinta’  and ‘Macabeo’   Table 6.3.5Foliar Aki and BL treatments slightly influenced some of the mineral nutrient concentrations in the grapevine leaves, but not enough to affect plant development in any of the three cultivars  and total phenolic compounds concentrations were compared for each treatment (Aki and BL) with the NTC Table 7.Regarding Aki treatment, no phytohormones were significantly influenced in the same way among the three grapevine cultivars. The SA tended to present higher levels after Aki treatment in ‘Garnacha Blanca’ and ‘Macabeo’ and was significantly enhanced in ‘Garnacha Tinta’. The GAs showed an opposite pattern being their levels significantly increased in ‘Garnacha Blanca’, but reduced in ‘Macabeo’. The MeJA in ‘Garnacha Blanca’ was significantly enhanced after Aki treatment. Although all the studied phytohormones, with the exception of JA, tended to be enhanced after Aki treatment in ‘Garnacha Blanca’; JA, ACC, and ABA were not significantly influenced by Aki treatment in any of the three cultivars.BL treatment significantly enhanced JA in ‘Garnacha Tinta’ and MeJA in ‘Garnacha Blanca’. Oppositely, GAs were significantly reduced after BL treatment in ‘Macabeo’. However, neither SA, ACC, nor ABA were significantly influenced by BL treatment in any of the three cultivars.After BL or Aki treatment, phytohormone content changed without a clear pattern and the establishment of a defense signalling triggering mechanism was not possible to infer from our data.It is worth to mention that ABA global values detected in ‘Macabeo’ are higher than the values detected in the two ‘Garnacha’ varieties.Four OA were identified in the leaves of the three grapevine cultivars: oxalic, tartaric, malic, and oxoglutaric Table 7.Total phenolic compounds concentration was significantly enhanced after BL treatment in ‘Macabeo’ and in ‘Garnacha Blanca’. Aki treatment only tend to increase the level of total phenolic compounds.4Bacillus UdG  was investigated. Among the environmentally friendly compounds considered as PPPs by the European Union legislation, beneficial microorganisms and natural substances (i.e. plant extracts) are included. Bacillus UdG and Akivi were used in this study representing each modality. Previous studies have reported the features and potential of both biocontrol products  whose expression levels were influenced by the treatments. Interestingly, the two Bacillus UdG treatments, both BF and BL triggered the same pathways. However, BF and BL did not always trigger the same genes of the above-mentioned pathways. These results underline the importance of the product formulation since it may determine its efficacy and mode of action. Only Aki and BL treatments will be discussed hereafter.The response of grapevines to foliar treatments with the botanical extract Akivi (Aki) and the beneficial microorganism products . Figure2(FC) values higher than 1.4. However, phytohormone concentrations related to JA defense pathway were affected in cvs. Garnacha Blanca and Tinta. Specifically, MeJA concentration was significantly higher after Aki and BL treatments in ‘Garnacha Blanca’ and JA concentration was doubled after BL treatment in ‘Garnacha Tinta’. In agreement with metabolite contents, several genes regulated by JA pathway were upregulated by the treatments. Namely, the expression level of genes related to non-inducible immunity 1 (NIM1) were upregulated by Aki treatment and the expression level of a gene related to enhanced disease susceptibility (EDS1) and to nonexpressor of pathogenesis-related genes 1 (NPR1) was slightly upregulated by both treatments. Moreover, Aki and BL treatments upregulated the expression of several genes involved in different transcription factors with WRKYs domain (WRKYs TFs) and pathogenesis related proteins (PR). Interestingly, one of the PR related genes (VIT_03s0088g00810) was highly influenced by Aki treatment. Therefore, JA defensive pathway seemed to be triggered by both treatments. This is in agreement with the essential role of JA as a phytohormone involved in the regulation of defense gene expression (Gene transcription related to JA biosynthesis was slightly influenced by either Aki and BL treatments since lipoxygenases (LOX) related genes did not show overexpression with Logpression , such aspression . These rpression .The expression level of genes related to SA pathway were not clearly affected by neither Aki nor BL treatments. However, the measured concentration of SA phytohormone in grapevine leaves treated with Aki tended to be higher than in leaves treated with NTC or BL on all three cultivars, especially in ‘Garnacha Tinta’ in which significant differences were observed. It could be explained by the upregulation of the expression of some genes related to EDS1, NIM1, and NPR1 already commented above in JA paragraph. Actually the mentioned genes are described as modulators that intervene in SA accumulation and they are produced in crosstalk between SA and JA pathways . InteresThe biosynthesis of ET seemed to be triggered by both treatments through the upregulation of the expression level of 1-aminocyclopropane-1-carboxylate oxidase (ACO) related genes. Moreover, the concentration of the ET precursor 1-aminocyclopropane-1-carboxylic acid (ACC) tended to present higher levels in ‘Garnacha Blanca’ leaves after Aki and BL treatments. These results were in agreement with the upregulation of expression of genes related to response factors regulated by ET /ERF TF, AP2 TF) by both treatments. In addition, the expression level of one of the genes related to ERF TF was highly influenced by Aki treatment (VIT_17s0000g00200). Therefore, ET defensive pathway seemed to be triggered by both treatments. ET response factors are key regulators of JA, ET, and ABA pathways in response to biotic and abiotic stresses, activating PR genes, such as osmotins (PR-5), chitinases (PR-3) and β-1,3-glucanases (PR-2) , which wThe ABA biosynthesis was triggered by BL treatment through the upregulation of 9-cis-epoxycarotenoid dioxygenase (NCED) related gene expression. In fact, ABA biosynthesis starts with carotenoids and involves NCED enzyme that is strongly upregulated by stress . ABA is This study also underlined that the expression of a Phenylalanine Ammonia Lyase (PAL) and a Chalcone Synthase (CHS) related genes were upregulated after BL treatment, whereas the expression of one Leucoanthocyanidin dioxygenase (LAR) related gene was upregulated after both Aki and BL treatments. PAL, CHS, LAR, and flavonol synthase (FS) are key enzymes for biosynthesis of several secondary metabolites, such as phenylpropanoids, or phytoalexins isoflavonoids . These e2+) signaling pathways were slightly upregulated after Aki treatment and some of them after BL treatment as well, such as Ca2+ Dependent Kinases (CDPKs), Calmodulin (CaM), Respiratory Burst Oxydase Protein (RBOHF) and Heat Shock Transcription Factors (HS TFs) with an upregulation lower than Log2(FC) > 1.4. In addition, one CaM and several peroxidases (PO) related genes were clearly upregulated after Aki treatment, and two of them were highly upregulated by Aki treatment  and involved in hypersensitive response (HR). As no phytotoxicity was observed after the treatments, Aki treatment may prime HR to be faster in case of pathogen infection. A crosstalk is described between MAPKs, JA, SA, and ET pathways  and Calcium ion  that showed higher concentration in ‘Garnacha Blanca’ leaves after BL treatment and lower concentration in ‘Macabeo’ leaves after Aki and BL treatments. The 2-OG is involved in gibberellin (GA), alkaloid and flavonoid biosynthesis . Indeed,The concentration of Tartaric acid in grapevine leaves was also affected since lower concentrations were detected in ‘Garnacha Tinta’ leaves treated with BL. Grapevine presents a high concentration on tartaric acid and its biosynthesis occurs in leaves and berries. Tartaric acid was shown to be involved in various processes and abrupt changes in its biosynthesis were linked with oxidative burst as well as ascorbate/glutathione redox state in berries . More inGrapevine response to Aki and BL treatments at transcripts and metabolic level indicate the ability of these products to trigger plant defense response. In fact, many transcripts related to defense responses were detected by the RNA-seq analysis of leaves. Some of the transcripts did not present differential expression, but others were highly affected by Aki treatment  and were selected as DEGs markers candidates . Considering all the upregulated transcripts in JA, ET, SA, and ABA pathways Figure 8Rheum palmatum plant extract (Trichoderma harzianum T39 (Citrullus lanatus) roots treated with the beneficial microorganism candidate B. velezensis against Fusarium oxysporum fungal pathogen , callose deposition in stomata cells, phenylpropanoid compounds accumulation  and hypersensitive response. Thiamine triggered several genes involved in defense response like PR genes  and lipoxygenases pathway involved in JA biosynthesis . Some of them  are involved in the main pathways related to plant defense response Figure 8ynthesis . The higBacillus treatments and could be used to test different doses and formulations of the biocontrol products in greenhouse-controlled conditions. Actually, defining treatment dose and formulation are crucial steps in product development that highly impact its efficacy. As observed in the present study, grapevine response is variable according to the studied cultivar as described other studies  and BL  treatments in the three grapevine cultivars. Therefore, they could be considered as appropriate markers of Akivi and  studies . Therefo studies .5Grapevine response to the Aki and BL treatments at transcripts and metabolites levels gave insights on modes of action of these biocontrol products that are under development. RNA sequencing analysis showed different gene expression patterns after foliar treatments with the biocontrol products compared with the NTC in ‘Garnacha Blanca’. Furthermore, RT-qPCR enabled the quantification of several selected genes (DEG) in three different cultivars. This information was complemented with metabolic analysis . Considering all the upregulated transcripts and enhanced metabolites concentrations related to JA, ET, and phenylpropanoids pathways, strong indication was found of grapevine defense induction by the treatments. However, further studies are necessary to confirm these first results and a kinetics study could be interesting. In addition, several DEGs markers were identified presenting a stable overexpression after the treatments (Aki or BL) in the three grapevine cultivars. They could be used as markers of activity of the products for further investigations.https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE211268.The original contributions presented in the study are publicly available. This data can be found here: ML, EM, and EB obtained the financial support. MR, ND, ML, EM, and EB conceived and designed the research. MR, ND, ML, RT and EB conducted and performed the experiments. MR, ML, and EB analyzed the data. MR and ND wrote the first draft of the manuscript. ML, EM, and EB revised and edited the manuscript. All authors contributed to the article and approved the submitted version.


This work aimed to examine the function of phosphatase and tensin homologue deleted on chromosome 10 (PTEN) in skull base chordoma (SBC) at the clinical and cellular levels.Totally 65 paraffin-embedded and 86 frozen specimens from 96 patients administered surgery were analyzed. Immunohistochemical staining and quantitative real-time polymerase chain reaction were performed, and the associations of PTEN expression with clinical features were assessed. At the cellular level, PTEN was knocked down by the siRNA approach in the UCH-1 cell line, and cell proliferation and invasion were detected by the CCK-8 and migration assays, respectively.p = 0.025), and elevated in soft SBCs in comparison with hard tumors (p = 0.017). Increased PTEN protein expression was associated with decreased risk of tumor progression . At the gene expression level, the cut-off value was set at 10.5 after ROC curve analysis, and SBC specimens were divided into two groups: PTEN high group, ΔCt value below 10.5; PTEN low group, ΔCt value above 10.5. In multivariate regression analysis of PFS, the risk of tumor progression was increased in PTEN low group tumors in comparison with PTEN high group SBCs (p = 0.006). In the CCK-8 assay, in comparison with control cells, PTEN knockdown cells had increased absorbance, suggesting elevated cell proliferation rate. In the invasion assay, the number of tumor cells penetrating into the lower chamber was significantly increased in the PTEN knockdown group compared with control cells.At the protein level, PTEN expression was increased in non-bone-invasive tumor samples in comparison with bone-invasive specimens (Decreased PTEN expression in SBC, at the protein and gene levels, is associated with reduced PFS. PTEN knockdown in chordoma cells led to enhanced proliferation and invasiveness. Chordoma represents a low-grade malignancy originating from notochord remnants , 2 that Previously, brachyury and additional biomarkers were detected in chordoma , 10; howTumor specimens were obtained from SBC cases surgically treated between January 2008 and November 2015, and diagnosed as chordoma. Cases with paraffin-embedded samples assessable by immunohistochemistry or frozen samples usable for mRNA extraction were included. Exclusion criteria were: clinical data unavailability; other tumors or genetic disorders; other serious diseases that could affect prognosis. This study conformed to the Committee on Publication Ethics (COPE) and the International Committee of Medical Journal Editors (ICMJE) recommendations for ethics and had approval from Beijing Tiantan Hospital's ethics committee. Each patient provided signed informed consent. Data collection and analysis were performed in a blinded fashion.Demographic data and treatment history, including postsurgical radiation therapy, were obtained from medical records. Presurgical Karnofsky Performance Status (KPS) scores, surgical methods, blood supply and tumor texture were retrieved from the surgical records. Tumor blood supply was either abundant or poor, based on which the cases were categorized. Tumor texture was recorded as soft or hard (tumors with both soft and hard constituents were considered hard tumors). Follow-up was mostly performed on an outpatient basis, and phone calls were utilized for those unable to visit the hospital. Tumor progression was considered for recurrence or regrowth of the residual tumor.Histological analysis was performed by 2 or more pathologists with >10 years of experience in chordoma assessment. The tumors were grouped into conventional, chondroid, poorly differentiated and dedifferentiated types, as proposed by the International Agency for Research on Cancer . No poorMagnetic resonance (MR) imaging scans were assessed by two experienced radiologists with the Picture Archiving and Communication System. Tumor diameter was measured separately in three mutually perpendicular dimensions, with the greatest value considered the “diameter”. Invasiveness was considered for bone  or non-bTumor specimens underwent formalin fixation in the 30 min following extraction from patients, paraffin-embedding, sectioning at and 5-µm. Then, the sections were dewaxed and hydrated. After antigen retrieval, the specimens underwent incubation with endogenous peroxidase and blocking. Next, sections were submitted to successive incubations with primary  and secondary . Finally, treatment with diaminobenzidine was followed by dehydration and mounting.Five or more high-power fields were assessed per sample at 400×, and positivity rates for various samples were determined by two pathologists independently. Consensual discussions were applied to resolve any discrepancy.Total RNA was extracted with TRIzol from tumor specimens snap frozen in liquid nitrogen in the initial 30 min following extraction from patients. In brief, 1 ml TRIzol  was mixed with 20 mg of tissue. PrimeScript RT reagent Kit with gDNA Eraser  was utilized for cDNA synthesis, as directed by the manufacturer. qRT-PCR was performed with TaqMan probes for PTEN  and GAPD−ΔΔCT method was applied to examine transfection efficiency; transfection efficiency above 50% was considered to indicate a qualified transfection.In cellar level experiments, TRIzol was used for RNA extraction, followed by reverse transcription with Reverse Transcription Kit . Quantitative polymerase chain reaction (PCR) was carried out with real time-PCR Recording Kit (Takara) by the SYBR Green method. The primers were as follows: PTEN, forward 5′-GCC CTG TAC CAT CCC AAG TC-3′ and reverse 5′-GAT GCT GCC GGT AAA CTC CA-3′; GAPDH, forward 5′-GGA GCG AGA TCC CTC CAA AAT-3′ and reverse 5′-GGC TGT TGT CAT ACT TCT CAT GG-3′. Amplification was performed at 95°C (30 s), followed by 40 cycles of 95°C (5 s) and 60°C (30 s); or 95°C(15 s), 60°C (30 s) and 95°C (15 s). The 2https://www.chordomafoundation.org). Pretreatment of culture plates/flasks was carried out with gelatin  to increase cell adhesion. IMDM and RPMI 1,640 (Thermo Fisher Scientific) were used as the basic medium mixture (4:1), supplemented with nonessential amino acid solution (1×) and penicillin/streptomycin (1×) (Thermo Fisher Scientific) and 10% fetal bovine serum . This cell line was subcultured every three days.The chordoma UCH-1 cell line was acquired from Chordoma Foundation (TM was utilized as the transfection reagent (Gima Genetics), and tumor cells were grown in 6-well plates in antibiotic-free medium to approximately 6  ×  104/well; the medium was changed before transfection. The transfection medium comprised 6 pmol siRNA, 200 µl Opti-MEM medium, and 15 µl siRNA MATE™, and was added to tumor cells and incubated under routine conditions. After 6 h of transfection, the medium was changed, and transfection efficiency was examined after 48 h of incubation.Cell transfection was performed by applying short interfering RNA (siRNA), the target siRNAs  were designed as follows: 5′-UGA ACC UGA UCA UUA UAG A-3′; the negative control siRNA was 5′-ATC TAG ATT AAC GAC ATT G-3′. SiRNA MATECell Counting Kit-8  was utilized for cell proliferation assessment. Cells were inoculated in a 96-well plate, and medium containing 10% CCK-8 was replaced after transfection for 24, 48 and 72 h, respectively. Absorbance at 450 nm was measured on a microtiter plate reader.The 24-well transwell chambers  were applied for cell invasion experiments. The chambers were pretreated witt Matrigel (Corning), followed by addition of serum-free cell suspension and 20% serum-containing medium into the upper and lower chambers, respectively. Staining was performed after 12 h of incubation.p < 0.05 were further assessed by multivariable analysis. P < 0.05 indicated statistical significance.SPSS  was utilized for data analysis. Differences in PTEN expression between the SBC types were assessed by the t test or rank sum test. Receiver operating characteristic (ROC) curve analysis was carried out to set a cutoff value. The Kaplan-Meier (K-M) method was employed for examining survival and progression risks between different PTEN expression groups. In overall survival (OS) and progression-free survival (PFS) Cox regression models, each parameter was firstly included in univariable analysis; parameters with Ninety-eight patients were included in this study. Only cases administered their first surgery in our hospital were assessed. Paraffin-embedded and frozen specimens were obtained from 65 to 86 cases, respectively. Both sample types were available for 53 cases. The patients were 11–64 years old , including 51 women and 47 men. Following the first surgical procedure in our hospital, 33 cases underwent another surgery for tumor progression, 2 had two further surgeries each, 3 had three further surgeries each, and 1 had four more surgeries for multiple postsurgical tumor progression events. Eighteen patients received postsurgical radiation therapy, 3 only had conventional radiation therapy, 12 only underwent stereotactic radiation therapy, 2 only had proton radiation therapy, and 1 had stereotactic and proton radiotherapies. In the present study, 26 cases died and 58 had tumor progression. Three-year OS was 70%. PFS rates at 3 and 5 years were 37% and 18%, respectively, and median PFS was 30.5 months. (More data in p = 0.619) and sex (p = 0.420) were similar between this subgroup and the overall population. Table 1 lists all patient features. PTEN was mostly detected in the matrix, cytosol and nucleus. The positivity rates for all tumors were 20%–100% . PTEN protein expression significantly differed between SBC tumors of divergent invasive types or textures. PTEN was upregulated in non-bone-invasive tumor samples in comparison with bone-invasive tumor specimens (p = 0.025) (p = 0.017) .The 65 patients with paraffin-embedded specimens were 11 to 63 years old , including 38 women and 27 men. Age (= 0.025) , and PTE= 0.017) . In univp = 0.986) and sex (p = 0.846) were comparable between this subgroup and the overall population . In K-M curve analysis, PFS was increased in PTEN high group compared with PTEN low group (p = 0.014) (p = 0.006), consistent with protein level data . In total, 45 women and 41 men were included. Age (= 0.014) . After ivel data .UCH-1 cells were transfected with siRNA to knockdown PTEN, and the knockdown rate was 64.1% after transfection . AbsorbaIn the present study, the protein and mRNA amounts of PTEN were assessed, and its functions and preliminary mechanism were further examined at the cellular level. The results indicated that decreased PTEN expression in SBCs had associations with tumor invasion, hard tumor texture and decreased PFS, and PTEN knockdown in tumor cells led to enhanced cell proliferation and invasion.PTEN is encoded by a tumor suppressor gene commonly deleted in malignant diseases. PTEN protein is located in the cytoplasm and nucleus, and often secreted into the extracellular space . PTEN plPTEN loss is associated with cell growth and tumor progression. Park et al. found the mTOR pathway is activated after PTEN deletion in adult retinal ganglion cells, which robustly induces axon regeneration upon optic nerve damage . PenningPTEN deficiency is associated with tumor invasion and migration. Studies demonstrated PTEN deletion might lead to metastatic invasive prostate cancer , 32. Masp = 0.054, hazard ratio: 1.712, 95% confidence level: 0.974–3.008). Specifically, hard tumors exhibited higher progression rates than soft tumors, indicating that hard tumors grow faster than soft tumors. This difference may be caused by reduced PTEN expression in tumors with hard components. Another explanation is that tumors with reduced PTEN expression are more invasive to surrounding bone, and the bone component makes the tumors harder compared with soft tumors with increased PTEN expression.Although the relationship between PTEN expression and tumor texture is rarely described in the literature, PTEN expression was associated with tumor texture in SBCs. PTEN expression level in soft SBCs was increased compared with hard tumors. We learn from The present study included 96 SBC cases with 86 frozen and 65 paraffin-embedded specimens, with available clinical information. the relationship between PTEN and patients' prognosis and clinical characteristics were investigated at protein and gene level respectively, the function of PTEN in chordoma at the cellular level was further verified. This is the largest study to date about PTEN in skull base chordoma, and this is also the first study to demonstrate the role of PTEN in skull base chordoma at protein, genet, and cellular levels. The results indicated that PTEN in SBCs control tumor invasion and tumor progression. These findings provide novel insights into the development of tools for SBC treatment.in vitro experiments caused by the slow growth of other chordoma cell lines, we will repeat the experiment involved in this study in other cell lines in the future.Nevertheless, this study had shortcomings. Firstly, not all the patients included contributed both frozen and paraffin-embedded specimens. Secondly, the present study was mainly exploratory, and the mechanism was not adequately studied. Additionally, only one cell line was used PTEN expression in SBCs was assessed at the protein and mRNA levels. In addition, the associations of PTEN expression with patient features were analyzed, and its functions and preliminary mechanism were further verified at the cellular level. The results indicated that PTEN protein expression in SBC was correlated with tumor invasiveness and tumor texture, and decreased PTEN protein and gene expression levels in SBC were associated with reduced PFS. Finally, knockdown of PTEN in tumor cells led to higher proliferation and invasion.Skull base chordoma represents a rarely diagnosed low-grade malignancy. With invasive growth, this tumor damages the surrounding bone seriously and is prone to recurrence after surgery; its long-term prognosis is poor, and there is no effective chemotherapy. Relevant factors and mechanisms involved in skull base chordoma are urgently needed. This study focused on PTEN expression in this tumor. In this study, 96 patients with skull base chordoma were included, including 65 with paraffin-embedded specimens and 86 with frozen specimens. Immunohistochemical staining and qRT-PCR were performed to detect PTEN expression at the protein and mRNA levels, respectively. PTEN expression and the clinical characteristics of SBC patients were next analyzed. Further, the PTEN gene in the chordoma cell line UCH-1 was knocked down using an siRNA, and cell function changes after knockdown were analyzed. Finally, our results revealed decreased PTEN expression in SBC was associated with shorter progression free survival, and PTEN silencing in chordoma cells led to higher cell proliferation and invasiveness.