Introduction: Coronary Atherosclerosis is the leading cause of death and disability worldwide. Atherosclerosis could be detected noninvasively by coronary calcification, measured by calcium score in CT angiography. Dietary factors are influential in the evolution of coronary plaques, and one of the most prevalent drinks is black tea. We aimed to evaluate the effects of black tea on coronary calcium scores.Methods: This cross-sectional analytical descriptive study was conducted on 200 candidates for CT angiography referred by their physician because their symptoms were suggestive of ischemia. A questionnaire was filled out for every participant, and the habit of tea drinking was asked and marked as none drinker, 1-3 cups per day and >3 cups per day.Results: 89.5% of the participants consumed tea. The mean calcium score in patients who did not drink tea was 674.9±154.74 in those patients who drank 1-3 glasses per day, 269.5±46.9 and in those who drank more than three glasses of tea and was 261.1±45.2. There was a significant statistical relationship between calcium scores and tea intake, independent to other traditional risk factors (P= 0.001). Significant coronary artery plaques were also less prevalent in those who drank tea  than non-drinkers (67%). Still, the number of involved vessels was not significantly different.Conclusion: Regular black tea consumption could have protective effects on coronary artery calcification. Black tea is fermented before the last stage of heating2. Black and green tea have many health benefits, including healthy bones, antioxidants properties, oral health, better heart rate, healthy digestive tract, decreased stress level, and happiness factor.5 Apart from water, tea is one of the most widely consumed beverages worldwide.8 Drinking tea has been correlated with a low incidence of cardiovascular (CV) diseases and cancer, in which oxidative stress plays a critical role. Catechin and flavin are two bioactive phytochemicals responsible for the antioxidant activity of green and black tea.9 Coronary artery calcification (CAC) scoring provides improved predictive ability over conventional risk factor scoring alone.13 The initiation of CV calcification resembles the process of osteogenesis. Studies have shown that osteoblast- and osteoclast-like cells and bone matrix proteins resulted in hydroxyapatite  deposition in the arterial wall. In the case of more severe calcification, fully formed bone could be observed in arteries and valves.15 There is no specific treatment for arterial calcification, and suggested medications such as statins, vasodilators, and other therapies for Atherosclerosis have negligible effect. Thus, it would be better to apply mechanisms such as anti-oxidation to prevent vascular calcifications.16 There is some evidence that moderate tea drinkers lead to slower progression of CAC deposition with subsequent reduced risk for CV events.17 Due to the controversies, this study aimed to evaluate the association between tea drinking and CAC score. CV calcification is a systemic disease that is considered an independent predictor of CV events and all-cause mortality and or renal diseases.4. It was initially addressed by 20 patients to determine its reliability. The existing deficiencies were solved, and Cronbach’s alpha was calculated as 0.88 in the preliminary survey. This cross-sectional analytical descriptive study was approved by the ethics committee of Isfahan University of Medical Sciences (code: 3964002) and conducted in Al-Zahra hospital in Isfahan city from April 2015 to March 2018. Participants were selected from patients referred for computed tomography (CT) angiography to evaluate coronary artery disease (CAD). The patients without contraindication for CT angiography were included in this study. However, patients with a medical history of coronary artery interventions, cardiac surgery, congenital heart disease, inherited metabolic diseases, and renal failure were excluded from this study. Two hundred patients were enrolled in this study after getting signed informed consent. Demographic characteristics of the patient, including age, gender, and occupation, were recorded. Blood pressure, height, and weight of the patients were measured, too. Medical history of CV diseases, hypertension, diabetes, and some of its risk factors such as smoking and hyperlipidemia were checked. Patients were evaluated for tea-drinking habits using a questionnaire developed at the University of Arizona followed by translation and validation CT angiography was done with a standard protocol with GE system, and analysis was done using ADW4.4 software. Each patient’s calcium scores (CS) were determined and recorded in each coronary artery. CS was divided into 5 groups including normal group (CS = 0), minimal (CS = 1-10), mild (CS = 11- 100), moderate (CS = 101-400) and severe (CS> 400). Atherosclerotic plaques were determined and the number of arteries with significant and non-significant stenosis were also determined and recorded.1-α/2 = 1.96), the prevalence of coronary calcification in tea consumers due to the lack of a similar study. Internal was considered a rate of 0.5, and the acceptance of an error rate of 0.075 was estimated at 170 people. The sample size was calculated by the following formula:  The required sample size for this study was derived from the formula considering the 95% confidence level  and frequency (percentage). Kolmogorov-Smirnov test before analysis was done for determining data normality. The Chi-square test compared qualitative data between the case and control groups. Comparison of quantitative variables was made by independent sample T-test and One-way ANOVA. Considering the abnormality of CA distribution, the data were presented in median (25-75) (IQR) and analyzed by non-parametric Mann Whitney and Kruskal Wallis tests. Logistic regression test using backward conditional method was used to determine the effect (odds ratio) of variables affecting calcium score, eliminate the effect of confounding variables, and adjust the effect of variables. Statistically significant data was considered at  In this study, two hundred patients who underwent CT angiography aged 58.3 ± 11.5 (ranging from 30 to 83) participated. 108 (54%) were male, and 92 (46%) were female. Zero, 1, 2, 3, and 4 traditional risk factors for CAD were detected in 32, 70, 58, 30, and 10 patients, respectively. The percentage of CV risk factors is shown in P = 0.036). According to the findings of CT angiography, CA status in 111(55.5%) was normal (CA≤100), and in 89(44.5%) was abnormal (CA>100). The mean CAC score was 674.9 ± 154.74, 269.5 ± 46.9, and 261.1 ± 45.2 in 0, 1-3, and >3 glasses tea drinkers, respectively. The median calcium score among participants was 67 (12-356). The percentage of CS in patients is shown in P< 0.001). The distribution of demographic variables, CV risk factors, and tea intake based on CAC scores are shown in  This result indicated a significant relationship between CAC scores and tea intake (P < 0.001). The median, range, and percentile of 25- 75% CS are shown in terms of the severity of CAD  were normal, 56 (28%) had non-significant plaques, 22 (11%) had stenosis in one vessel, 16 (8%) had stenosis in two vessels, and 45 patients (22.5%) had stenosis in three coronary arteries. Mean scores of calcium in the five groups were 9.9 ± 1.6, 167.7 ± 21.17, 278.1 ± 107.66, 849.9 ± 158, and 708.98 ± 88.5, respectively, and there was a significant relationship between CAD and calcium score (y of CAD . In termP = 0.004). Indeed, age more than 50 years old was associated with an enhanced chance of abnormal CA score to 3.73 times, but other variables had no significant impact on alteration of CA score. On the other way, data analysis using conditional logistic regression through backward conditional method showed that the two mentioned variables had a significant impact on having the chance of normal CA score. In contrast, other variables had no significant confounding effect using both applied methods. Thus, considering the unchanged odds ratio measures (OR), there was no significant confounding impact from other variables such as age and tea consumption  as their principal constituent and a well-characterized antioxidant agent. The protective impacts of flavonoids are mostly due to their anti-oxidative activity. Quercetin can inhibit the oxidative changes of low-density lipoproteins that lead to the formation of fatty plaques.22 Black tea possesses mainly theaflavins and thearubigins which are known as products of tea catechins. There are various pharmacological properties attributed to these catechins including antioxidative, anti-inflammatory and hypolipidemic effects.20 Oxidative stress has been implicated in the development of vascular injury and atherogenesis.25 Green tea extracts generally showed stronger antioxidant activity than the semi-fermented and black tea extracts.18 Polyphenols in green tea have a cardioprotective effect for acute myocardial infraction through free radical-scavenging and antioxidant properties. In vitro studies have shown that polyphenols act as metal ions chelator, ROS or RNS scavenger, ROS generating enzymes inhibitor, and antioxidant enzymes activator.26 These compounds in green tea also promote inhibition of NFκb and activation of PI3K/Akt signaling, decrease in expression of Fas receptor, and modulate lipid metabolism, mitochondrial function. All of these and some others like reduction of cytosolic Ca2+ overload and sensitivity of myofilament to Ca2+ are possible cardioprotective mechanisms of these compounds.29 In an experimental animal study on a hamster model of Atherosclerosis, both teas were equally effective in inhibiting the development of Atherosclerosis through prevention of hyperlipidemia, antioxidant and anti-fibrinolytic properties.19 Endothelial cell dysfunction is involved in the pathogenesis of Atherosclerosis and could be reversed by antioxidant treatment using short- and long-term black tea consumption.31 This finding may partly explain the association between tea intake and decreased CV events.32 In addition, studies have shown that daily intake of black tea for one month improves platelet function and reduces platelet activation with subsequent reduction of the risk of atherosclerosis.34 Reducing the total homocysteine concentration with regular tea consumption is another mechanism for CV protection.35 Some studies have also shown that catechins have effects in preventing and treating cancers. Tea catechins have several effects on cell growth signals. Green tea polyphenols can alter angiogenesis-related miRNA expression in cancers.36 The limitations of this study are the lack of prospective data from tea consumers about clinical events, limited population size . There was also a lack of information from dietary habits that could affect the CAD prevalence. However, this study did not show a relationship between tea consumption and the number of obstructed coronary arteries and atherosclerotic lesions. It can be argued that many factors contribute to the development of coronary artery disease. Indeed, an enhanced CA score does not necessarily indicate increased stenosis of coronary arteries. Black or green tea may positively impact the damage induced by coronary artery calcification as a marker of Atherosclerosis. Possible mechanisms for this effect include reducing platelet activation and direct prevention of calcification. The authors thank Al-Zahra Hospital staff for their cooperation in implementing the study. No financial support has been received from any organization for this work. The implementation process of this study was reviewed by the ethics committee of Isfahan University of Medical Sciences and approved by code: IR.MUI.REC.1396.3.004. The authors state that there is no conflict or competing interest.


Low temperatures are known to limit the growth and geographical distribution of poplars. Although some transcriptomic studies have been conducted to explore the response of poplar leaves to cold stress, only a few have comprehensively analyzed the effects of low temperature on the transcriptome of poplars and identified genes related to cold stress response and repair of freeze–thaw injury.2+ signaling pathway, starch–sucrose metabolism pathway, abscisic acid signaling pathway, and DNA repair. They were functionally annotated; glucan endo-1,3-beta-glucosidase and UDP-glucuronosyltransferase genes, for instance, showed a close relationship with cold resistance. The expression of 11 differentially expressed genes was verified by qRT-PCR; RNA-Seq and qRT-PCR data were found to be consistent, which validated the robustness of our RNA-Seq findings. Finally, multiple sequence alignment and evolutionary analysis were performed, the results of which suggested a close association between several novel genes and cold resistance in Zhongliao1.We exposed the Euramerican poplar Zhongliao1 to low temperatures; after stems were exposed to − 40℃, 4℃, and 20℃, the mixture of phloem and cambium was collected for transcriptome sequencing and bioinformatics analysis. A total of 29,060 genes were detected, including 28,739 known genes and 321 novel genes. Several differentially expressed genes (n = 36) were found to be involved in the CaWe believe that the cold resistance and freeze–thaw injury repair genes identified in this study are of great significance for cold tolerance breeding. Temperature is the main limiting factor that determines the geographical distribution of plants. The global economic losses caused upon exposure of plants to low temperatures are substantial each year. Yang et al.  investig2+ channels, and phospholipase appear to play the role of secondary temperature receptors .Of the 36 DEGs identified in this study, 12 were involved in Ca2+-binding protein), an important component of the second messenger system, plays a key role in Ca2+ signal transduction. CML proteins are the main Ca2+ receptor in cells [2+. CAM was highly induced at low temperatures.CAM , an endoplasmic reticulum membrane protein, plays a catalytic role in the transfer of glucuronic acid from UDP-glucuronic acid to hydrophobic molecules. Most UGTs are located in the cytoplasm , 36; in In plants, there are numerous substrates for glycosidation, which is catalyzed by UGT, including plant hormones and secondary metabolites . Under tOne of the basic requirements of poplar, a perennial woody plant, is to survive cold temperatures. Its capability to reversibly repair freeze–thaw injury is the key to its survival in repeated freeze–thaw environments. In poplar, the key genes involved in DNA repair play an important role in the repair of freeze–thaw injury. The global genome repair process mainly includes damage recognition, DNA unwinding, excision, and DNA synthesis. Figure Arabidopsis thaliana, AtXPB1 and AtXPB2. Results [Nucleotide excision repair is one of the repair pathways employed by plants to protect their genome. Xeroderma pigmentosum group B (XPB) helicase  is a core subunit of the eukaryotic basal transcription factor complex TFIIH which plays a dual role in transcription and DNA repair . XPB was Results  suggest  Results  verifiedhttps://blast.ncbi.nlm.nih.gov/Blast.cgi), it was found that protein Potri.001G101300.v3.0 had the highest similarity with protein PdeHOE87 . The online subcellular positioning tool Cell-PLoc-2 (www.csbio.sjtu.edu.cn/bioinf/Cell-PLoc-2) predicted that this protein is located in the nucleus. DNA repair genes will be the focus of molecular mechanism analysis of poplar resistance to freeze–thaw stress in the future.Potri.014G050200.v3.0 from the pathway of DNA repair and recombination protein RAD54 and RAD54-like protein  and Potri.001G101300.v3.0 from the pathway of DNA excision repair protein ERCC-3  were significantly induced at − 40 °C, suggesting their importance in DNA repair. By analysis using the NCBI BLAST tool (2+ and ABA signaling pathways, starch–sucrose metabolism pathway, and DNA repair. Our findings enhance our understanding of the gene expression pattern of Zhongliao1 at normal and low temperatures, and also lay a foundation for cold tolerance breeding.Poplar is an economically and ecologically important plant species. Low temperatures are known to restrict its growth and geographical distribution. Herein we analyzed the transcriptome of the Euramerican poplar Zhongliao1 to elucidate molecular mechanisms underlying cold stress response. A total of 29,060 genes were detected; furthermore, several key genes related to low temperature stress were identified, which were involved in diverse pathways, including Ca


Four isonitrogenous and isoenergetic diets were formulated including one commercial control  and three experimental diets with gradient FK of 3% , 6%  and 9% , respectively. FK-fed groups showed significantly higher SR than that of C group. The WGR and SGR of fish fed FK3 and FK6 were significantly higher than those of FK9, but not statistical different from the C group. FK-fed groups showed higher apparent digestibility coefficients of dry matter and nutrients. Further, FK-fed groups increased the ratio of SOD/MDA in the plasma and liver, and the upregulation of intestinal Keap1 and downregulation of HIF1α was found in FK3. Furthermore, FK-fed groups showed higher microbial richness and diversity. Pearson correlation analysis found that antioxidant relevant biomarkers were negatively correlated with the relative abundance of certain potential beneficial bacteria. In conclusion, supplemented up to 3–6% FK replacing FM in a low FM diet of largemouth bass could increase growth, survival rate, antioxidant capacity, and improve gut microbiota.A 10-week growth study was conducted to evaluate the effect of a natural gas fermentation bacterial meal (FeedKind The expansion of aquaculture has led to the increase in demand for fishmeal (FM), due to its high protein content, balanced amino acid profiles, high digestibility and good palatability ,2. It is® (FK)  is produced by the continuous gas fermentation using methane as a single carbon source, and ammonia as a nitrogen source by Methylococcus capsulatus Bath, Cupriavidus sp., Aneurinibacillus danicus, and Brevibacillus agri. [® and initially tested in fish feed by Skrede et al. in 1998 [M. capsulatus per kg of feed could prevent the development of dietary soybean meal induced intestinal inflammation in Atlantic salmon [Under the dual demand for food security and carbon neutrality, the SCP obtained from bacteria, fungal and microalgae by recycling waste materials  have the great potential to supply the protein sources with high digestibility, balanced nutrient profile and along with some functional components, such as nucleotide and water-soluble metabolites . Methaneus agri. . It is aus agri. ,14,15. I in 1998 . In gene in 1998 ,21,22. Ic salmon ,24,25.Micropterus salmoides) is an important freshwater carnivorous fish species native to lakes and small rivers in North America [https://www.fishbase.se, accessed on 20 July 2022). Nowadays, it has become an important economic farmed freshwater species in China [Clostridium autoethanogenum protein (CAP) and soy protein concentrated (SPC) with minimized 28–30% FM [Largemouth bass  was supplied by Calysta, Inc  and consisted of inactivated and dried biomass of the aerobic bacteria Methylococcus capsulatus (Bath), Cupriavidus sp., Aneurinibacillus danicus, and Brevibacillus agri, utilizing natural gas as the carbon and energy source. Methylococcus capsulatus represents around 92–94% of the bacterial biomass with the other bacteria making up the rest of the product. Steam dried FM (Prime level) and fish oil was supplied by Tecnológica de Alimentos S.A., Ltd. . Soy protein concentrated (SPC), soybean meal and soy oil were supplied by Bohi Industry Co., Ltd., Shandong, China. Other ingredients were bought from a local market.FeedKindFour isonitrogenous and isoenergetic diets were formulated including one control , and 3 low FM diets with 25% of SPC and gradient FK of 3%, 6% and 9% to further replace FM, respectively , with balanced essential amino acids, fatty acids and available phosphorus. Each diet was processed into 3 mm diameter floating pellets under extrusion condition as previous description . All dieJuvenile largemouth bass were obtained from a single-size-graded population . The experiment was conducted in the indoor circulating water system at the National Aquafeed Safety Assessment Center of IFR, CAAS . All fish were acclimated and fed the commercial diet  for 2 weeks before the trial. Fish with initial body weight of 48.0 ± 0.03 g, were selected and distributed into 16 tanks (256 L) with 25 fish/tank and four replicates each treatment. Largemouth bass were fed twice daily at 08:00 and 17:00 to apparent satiation for 70 days. The water temperature was maintained at 25–27 °C, pH = 7.5–8.5, dissolved oxygen (DO) > 7.0 mg/L and NH4-N < 0.3 mg/L. The photoperiod was 12L: 12D and the light intensity was 400 lx.g for 10 min at 4 °C to obtain plasma and then stored at −80 °C for analysis of hematological parameters. The distal intestine tissues were collected and separated as two parts for histology examination , and RNA isolation, respectively. All samples were stored at −80 °C until analysis. Three fish per tank were collected for the assay of whole-body compositions. The liver tissue of each tank was pooled and freeze-dried for hepatic lipid content determination.At the end of the trial, fish of each tank were batch weighted. The chyme from the distal intestine of 6 individual fish/dietary treatment was randomly collected at 3 h postprandial, and then snap-frozen in liquid nitrogen before stored at −80 °C for microbiota analysis. In this case, 12 fish of each treatment (3 fish from each tank) were randomly selected and anesthetized with chlorobutanol (300 mg/mL) for sampling at postprandial 24 h. The body weight, body length, viscera weight, liver weight, and abdominal adipose weight were recorded individually to calculate fish condition factor (CF), viscera somatic index (VSI), visceral adipose index (VAI), and hepatosomatic index (HSI). Blood was collected from the caudal vein and centrifuged at 4000× 2O3 as an inert marker. Feces collections were initiated 1-week after the formal feeding trail, which allowed sufficient time for all the gut content from any previous diet to be purged as described by Wang et.al. (2012) [Nutrient apparent digestibility of the diets was assessed using the indirect method using 0.1% Y. (2012) . Feces w2O3) in diets and feces was analyzed using inductively coupled plasma atomic emission spectrometry. The amino acid contents of diets and feces were analyzed at Amino Lab, Evonik Industries (Beijing).The macro nutrient composition analyses of the diets and feces were carried out in duplicate according to AOAC, 2019 . The dryThe plasma alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bile acid (TBA), alkaline phosphatase (AKP), glucose, total protein content (TP), Triglyceride (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), malondialdehyde (MDA), and superoxide dismutase (SOD) were determined by assay kits  following the protocols given by the supplier. The content of plasma non-esterified fatty acid (NEFA) was determined by the assay kit . The plasma reactive oxygen species (ROS), lipopolysaccharide (LPS), complement C3 (C3), immunoglobulin M (IgM), and distal intestinal interleukin 1β (IL1β), tumor necrosis factor α (TNFα), interleukin 10 (IL10) were detected by enzyme-linked immunosorbent assay .The fixed distal intestine samples were dehydrated by the standard procedures, and the samples were embedded in paraffin and cut to 6μm sections. The distal intestine was stained following the protocols of hematoxylin and eosin staining (H&E) and Alcian blue staining, Images of the stained sections were acquired with TissueFAXS Histo S, TissueGnostics GmbH, Vienna, Austria).Total RNA isolation, quantification, reverse transcription and mRNA quantification of distal intestine were conducted following the protocols as previously described . EF1α  using pre-filled plates (12–230 kDa) according to the manufacturer’s standard instructions.Distal intestine was homogenized in RIPA buffer  with an added phosphatase inhibitor cocktail . The protein concentration was measured using a BCA Protein Quantification Kit . The primary antibodies against HIF1α  and β-tubulin  were infused to probe target proteins prior to protein visualization using a horseradish peroxidase secondary antibody . Automated Western blots were performed on a Jess ® soil DNA Kit . Purity and quality of the genomic DNA were checked on 1% agarose gels. The PCR amplification of bacterial 16S rRNA gene (V3-V4 hypervariable region) were amplified using the universal primers 336F (5′-GTACTCCTACGGGAGGCAGCA-3′) and 806R (5′-GTGGACTACHVGGGTWTCTAAT-3′). The PCR amplification of 16S rRNA gene were run in triplicate. After the PCR amplification, the quality of PCR products was evaluated for library preparation on 1.5% agarose gels and the samples with a bright band of size between 300 bp and 350 bp were used for further analysis. Before library preparation, the PCR products were purified according to manufacturer’s instructions of AxyPrep DNA Gel Extraction Kit , and then quantified using Quantus ™ Fluorometer . The Illumina MiSeq PE300 platform  was used for the library preparation and sequencing based on the standard protocols.About 100 mg distal intestinal chyme from each fish were used for DNA extraction based on the manufacturer’s instructions from the E.Z.N.A. The raw 16S rRNA gene sequencing reads were demultiplexed, quality-filtered by fast version 0.20.0, and merged by FLASH version 1.2.7 . Operatip < 0.05. Permutation multivariate analysis of variance (PERMANOVA) was performed with 999 permutations to the unweighted and weighted UniFrac distance matrix results from the beta diversity analysis of QIIME 2. Group differences in relative abundance of microbiota at the phylum and genus taxonomic level were tested using the nonparametric Kruskal-Wallis test followed by Dunn’s multiple comparisons. All the graphics were drawn by GraphPad Prism 8.0 . The Pearson correlation between the gut microbiota and macro biomarkers from the same tank was analyzed using GraphPad Prism 8.0. Values marked with symbol “*” are significant correlations (p < 0.05), and “**” are very significant correlations (p < 0.01).All data were reported as the mean value with the standard errors of the mean (mean ± SEM). The data except microbiota data were evaluated for normal distribution and homogeneity of variance using Shapiro-Wilk test and the Barlett’s, respectively. A one-way analysis of variance (ANOVA), followed by Duncan’s multiple comparisons was conducted by SPSS 25.0 . Differences were regarded as significant when p < 0.05). The weight gain rate (WGR) and specific growth rate . The α-MSH, hormones that suppress feed intake in the central nervous system, gradually increased with replacement levels (p < 0.05) (p < 0.05), but no significant difference from the C group  C. The fe C group .p > 0.05, in p < 0.05, in There were no significant differences in whole body moisture, crude protein, and crude lipid content among treatment groups (p) and energy (ADCe). Furthermore, the FK groups showed much higher Cu digestibility than that of the C group . There were no differences in plasma AST and AKP among groups.The liver function and metabolites in plasma of largemouth bass are presented in p < 0.05). The metabolites of lipid metabolism, including metabolites of fatty acids, triglyceride and cholesterol were generally at homeostasis status among groups at postprandial 24 h.Higher plasma glucose and lower TP in the FK6 group was observed . Additionally, the plasma and hepatic MDA were significantly reduced, and hepatic SOD increased in FK groups. Both in plasma and liver tissue, SOD/MDA ratio were significantly increased in FK groups compared to that of the fish fed control diet . The immunity parameters, including plasma LPS, complement C3 and IgM of FK6 and FK9 groups were significantly higher than those in the control and FK3 group (p < 0.05).With the exception of LPS and complement C3 in the FK3 group, pro- and anti-inflammatory and immune responses were elevated in the FK groups. In particular, the proinflammatory cytokines IL1β, TNFα, and the anti-inflammatory cytokine factors IL10 in the distal intestinal of the FK groups were significantly increased compared to the C group (CREB mRNA level was not significantly different among groups (p < 0.05).The CREB and cAMP were energy metabolism biomarkers. The g groups A, but thp < 0.05). Further, FK-fed groups tended to have higher microbial diversity (Shannon and Simpson index) compared to the C group, but no statistically significant differences were found between dietary treatments (p > 0.05).The alpha diversity of microbiota is displayed in p < 0.05, Results from the permutation multivariate analysis of variance (PERMANOVA) analysis of weighted and unweighted UniFrac showed significant differences in gut micro-biota between dietary treatments , while lower relative abundance of genera Rhodococcus, Bu.Ca.Paraburkholderia, Paracoccus and Corynebacterium were observed in the FK-fed diets compare to the C group. Fish showed an increased relative abundance of genera Enterococcus with dietary the increasing levels of FK  (family)  (family), Bifidobacterium and Enterococcus. Additionally, the liver SOD levels showed a clear positive correlation with the relative abundance of Carnobacterium, Megasphaera, Megamonas and Enterococcus, but negatively correlated with Bu.Ca.Paraburkholderia and Corynebacterium.For the results of Pearson correlation analysis, SGR and FI showed a strong negative relationship with the relative abundance of Clostridium autoethanogenum protein [Methylococcus capsulatus (FeedKind®) [p < 0.05). It should be noted that there is precedent for SCP sources, and in particular M. capsulatus, to increase survival [Over the last decade, the farming of largemouth bass has progressed from feeding animals with trash fish to feeding them with artificial diets containing high levels of FM and protein, but low starch ,43. As w protein ,44,45, c protein , and MetedKind®) , have beedKind®) ,32, the edKind®) . In the survival  and to rsurvival ,46,47,48The digestibility of the feed is one of the important components in development of feeds as it affects the amount of nutrients absorbed by animals per quantity of feed ingested. The higher the digestibility for any dietary component, the more efficient the diet can be considered. The results of the current study indicate that the apparent digestibility of gross energy, protein, as well asl dry matter of the FM replacement groups were significantly higher than the C group. In contrast, it has been reported that the FM protein was replaced up to 30% by bacterial protein meal in the diet without significant effect on the digestibility in Japanese yellowtail . SimilarM. capsulatus levels were increased to 18% in Atlantic halibut (Hippoglossus hippoglossus) diets, feed intake and growth was reduced in fish compared to the control group [Palatability could be the main reason for the low growth performance in fish fed with alternative diets ,53. Geneol group . In the ol group ,59. Introl group . AlthougCirrhinus mrigala Hamilton) when exposed to 0.02, 0.10, 0.15 and 0.23 mg L−1 waterborne copper [Cyprinus carpio) exposed to high copper (290 μg·g−1 dry weight) in freshwater reservoirs [4) and organic (copper-ethanolamine) showed no significant differences in the growth performance in red drum (Sciaenops ocellatus) with no detrimental effects being observed in red drum fed diets supplemented with 20 mg Cu/kg, which mainly excreted via the feces [Compared to fish meal, FK contains high levels of nucleic acids and copper, which may elicit negative responses on feed intake and utilization of the diets. High dietary levels of brewer yeast (≥500 g/kg), which contained 2.5–10% ribose nucleic acid, could result in reduced palatability and growth in rainbow trout . Since ce copper , no appaservoirs . Anotherservoirs . Althougservoirs . The sighe feces . Highesthe feces .Ctenopharyngodon idella) [Acipenser schrenckii), with a significant downregulation of Keap1 [Danio rerio) [Keap1 at mRNA levels and a significant downregulation of the HIF1α protein expression in distal intestine tissue.A positive effect of Cu is to improve the antioxidant capacity of the largemouth bass. Dietary Cu could enhance antioxidant defense in the hepatopancreas and intestine, improve growth, digestive and absorptive capacity, as well as decrease lipid peroxidation and protein oxidation in grass Carp ( idella) . SOD is  idella)  whilst M idella) . Compare idella) . Plant pof Keap1 . HIF1α, of Keap1 . The slco rerio) . Keap1-No rerio) . In thisMethylococcus capsulatus. Methylococcus capsulatus is a Gram-negative bacterial, and bacterial LPS is the main component of the outer membrane of Gram-negative bacteria and represents a “double-edged sword”, which can act as bacterial endotoxin to release endogenous active factors [Liver function and metabolites in plasma are important tools for the indication of the health condition of fish ,75. The  factors , also kn factors . The res factors . The incCarnobacterium, was negatively correlated with growth rate, feed intake, as well as the levels of MDA in plasma and liver. The negative correlation with growth and higher abundance of Carnobacterium was noted to the FK9 group, which showed unfavorable growth rate compared to the other groups. Regarding the negative relation between Carnobacterium and the levels of plasma and liver MDA, this suggests that dietary FK might increase Carnobacterium abundance, and then regulate antioxidant capacity. Additionally, the relative abundance of Bifidobacteria was negatively related to the levels of liver MDA. The Bifidobacteria is one of best-known probiotic strains, has been used because of its antioxidant properties and because its role in preventing oxidative stress has been reported [Pseudomonas and Psychrobacter [Regarding gut microbiota, the substantially higher microbial richness was observed in fish fed diets with FK compared to the C group. Microbial diversity showed a similar picture, although this was not statistically significant. Our findings suggest that dietary FK may imply a more functional gut, since a high microbial diversity is commonly taken as an indicator of normal and healthy physiological intestinal functions, whereas low diverse microbiota is generally connected with dysbiosis . A similreported ,87. Our robacter . Unforturobacter ,88,89 anrobacter , which m® in the diet of largemouth bass improves growth, nutrients digestibility and liver functions via the regulation of antioxidant capacity and microbiota and efforts should be made to further refine inclusion rates and to test diets under field conditions. Moreover, FeedKind® protein is produced with both biogas and renewable electricity and has minimal land use and water requirements compared to terrestrial based feed ingredients, which has a carbon footprint that is comparable to or better than many other feed sources. Hence, the development of nutrient-balanced and economical sustainability FeedKind® provided huge potential as fishmeal alternatives in aquaculture.In conclusion, the results indicate that high FM diet  is not best choice for largemouth bass compared to the FK3  and FK6 . Although inclusion of 9% FK in the diet significantly reduced feed intake and thus growth performance the use of attractants to negate the effect of high FK inclusions on feed intake allowed an increase in the amount of FK that could be included. This would, however, need to be balanced with the potential negative effects of increased copper and LPS in the diet. Replacing fishmeal with 3–6% FeedKind


Acute and transient psychotic disorders (ATPDs) are a group of diagnoses with acute onset and polymorphic psychotic symptoms, divided into 6 subtypes, which may or may not associate with acute stress. Suicide is a serious public health problem, having long-lasting effects on social well-being and economics. The prevalence of suicide attempts (SA) in Latvia remains unknown, several studies are currently in progress.To determine the prevalence of SA among patients with ATPDs and features of this subgroup of patients.Retrospective study based on socio-demographic data and additional diagnoses of all patients with ATPDs provided by the National Center of Statistics of Diseases.There were 1779 patients with ATDPs in 2014-2019, 44 people were admitted with a SA . There was a significant difference between sexes in the mean age – 39.64±14.66 for women, 31.94±11.88 for men (p<.001), as well as in case of associated acute stress – 37.61±12.95 with and 35.33±13.15 without it (p=.01). There were no differences in age or ATPDs subtypes between patients with SA and without one. Men with SA abused alcohol in everyday life more than women and men without SA , women with SA had acculturational difficulties more often than men and women without SA . Intentional self-harm by sharp objects was more common among men (p<.001), intentional self-poisoning among women (p<.001).The prevalence of SA among patients with ATPDs was 2.47%. Factors, which were more commonly observed in patients with SA were alcohol consumption and acculturational difficulties. Types of self-harming differ between sexes.No significant relationships.


Cassia fistula Linn, generally recognized as Indian laburnum, is one of the ancient trees in the Indian subcontinent used for its ornamental and diverse medicinal properties. It is known for its ethnic medicinal uses in inflammatory and infectious pathologies such as antihelmintic, purgative, carminative, antipyretic, expectorant, analgesic, laxative, antiseptic, and antidote against snake poison. The Cassia bark is rich in anthraquinones, flavanols glycosides, and sitosterols, which renders it cardioprotective properties. The existing experiments were designed to assess the potential of Cassia fistula bark against isoproterenol (ISP)-induced cardiotoxicity in rats, which has not been validated yet. The bark was successively extracted with five different solvents, and each extract was subjected to in vitro antioxidant studies. Further acute oral toxicity assays were carried out preceding in vivo myocardial studies. Cardiotoxicity-inducing agent, ISP, was administrated to the rats for two consecutive days (8th and 9th). Based on in vitro studies, the Cassia fistula methanolic extract (CFME) was administered in two doses: CFME-LD (lower dose 250 mg/kg) and CFME-HD (high dose 500 mg/kg) separately. It was found that CFME produced a substantial decrease in lipid peroxidation and an increase in antioxidants in myocardial tissues. CFME abrogated the levels of triglyceride and total cholesterol with a decrease in alanine transaminase (ALT) and aspartate transaminase (AST) activity in serum at both doses. 2,3,5-Triphenyltetrazolium chloride (TTC) staining and histopathology also revealed the protective effects of CFME against ISP-induced myocardial infarction. The study showed the significant role of the CFME as a strong antioxidant and cardioprotective action in ISP-induced toxicity. Cardiovascular toxicity embraces damage to the heart by means of oxidative stress, inflammation, and toxin-induced functional abnormality in electrophysiology and muscle damage. Environmental factors and our lifestyle play a key role in determining our cardiovascular health. There is a close and well-established link between pulmonary disease and cardiotoxicity. Drug-induced cardiotoxicity is the single most frequent and major adverse effect aligned with most of the clinically used different classes of drugs like anticancer, antiretroviral, sympathomimetic, female hormones, and nonsteroidal anti-inflammatory drugs. Drug-induced cardiotoxicity initially causes cardiac muscle dysfunction that progressively leads to myocardial infarction and heart failure .β1 adrenergic receptors [β-adrenoceptors (cardiac β1 and β2-adrenoceptors), accumulation of calcium in cardiac cells, and chronic discharges of pro-inflammatory cytokine storm  by ISP causes irreversible alternations in myocardial structure, function, and biochemical markers. It is very difficult to manage, suppress, and reverse ISP-induced cardiac toxicity.The mechanism of drug-induced cardiotoxicity is very complex and may vary among the drugs, but in the general ground with the generation of free radical oxygen species and oxidative stress through various phases of drug metabolism. Isoproterenol (ISP)-induced cardiotoxicity is pathologically similar to acute myocardial infarction, hence, commonly used in experimental settings for acute myocardial infarction. Among the different animal models of myocardial infarction, the ISP-induced myocardial necrosis model is considered the most authentic one –3. The peceptors , 5. The Subcutaneous administration of ISP leads to myocardial necrosis in the endocardium of the left ventricles and interventricular septum. Multiple mechanisms for ISP-induced myocardial necrosis have been described. One of the acknowledged pathways amid these is an upsurge in oxidative strain as a result of ISP metabolic products and the generation of redundant free radicals .Cassia fistula Linn, also recognized as Bactyrilobium fistula Willd, commonly known as Indian Laburnum or Amaltas or Golden Shower Tree, is an evergreen, fast-growing medium-size tree belonging to the family, Caesalpinioideae. It is indigenous to India and South Asia. Its flowers appear in a pendulous fashion and look highly attractive and beautiful and, hence, are usually cultivated with the ornamental objective. Cassia fistula is ethnomedicinally used to treat anorexia, dermal contaminations, jaundice, ulcers, and inflammatory circumstances like rheumatism [Mankind always seeks nature to rescue themselves from diseases. The remedial use of medicinal plants is as antique as mankind itself. eumatism . It is aeumatism –10.Cassia fistula bark decoction is employed in the treatment of chronic diseases, namely, leprosy, syphilis, amenorrhea, and heart diseases. This plant is also documented in British Pharmacopoeia [In the Ayurvedic classification of treatment, it is known as Aragvadha meaning “disease killer” plant. In the Unani system of medicine, acopoeia  because Cassia fistula Linn. Tree, namely, bark, heartwood, flowers, leaves, and seed kernels are attributed with medicinal properties. The high medicinal prominence of Cassia fistula is due to the presence of many bioactive phytocomponents particularly anthraquinone glycosides, lignans, flavonoids, alkaloids, tannins, resins, sesquiterpenes, and fatty acids [Cassia fistula contains β-sitosterol, fistucacidin , leucocyanidin, lupeol, oxyanthraquinone, dihydroxyanthraquinone, and flavonol glycosides  and kits for the analysis of aspartate aminotransferase (AST), alanine aminotransferase (ALT), triglycerides (TG), and total cholesterol (TC) were acquired from Arkray Healthcare Pvt., Ltd., Mumbai, India (AutoSpan®) and Reckon Diagnostics P. Ltd., Vadodara, India. Colorimetric kits were used to estimate malondialdehyde (MDA) (Cell Biolabs Inc.), reduced glutathione (GSH) (G-Biosciences), and superoxide dismutase (SOD) (BioVision) activity.Cassia fistula (CF) was air-dried in shade and coarsely ground. 300 g of the powdered drug was consecutively extracted in the Soxhlet apparatus using five different solvents successively, that is, petroleum ether (PE), chloroform (CE), ethyl acetate (EE), methanol (ME), and aqueous extracts (AE). All the obtained extracts were dried using a rotary evaporator at 45°C and stored in vacuum desiccators.The bark of the stem of C. fistula bark was carried out, and the results were expressed in IC50  with respect to butylated hydroxy toluene (BHT) [The DPPH free radical scavenging activity of various extracts of ne (BHT) .μg/ml) with respect to ascorbic acid (AA) [FRAPSs of all of the above extracts were tested at five diverse strengths  .b.w.), were chosen at random for the animal activity and were quarantined for one week when first received in the institutional animal house facility, and health of animals was checked regularly. Animals were housed in standard-sized polypropylene cages fitted with a filter top using envelope bedding under controlled temperature conditions (23 ± 2°C), humidity (40 ± 10%), and 12-hour artificial dark and light cycles (7 : 00 AM to 7 : 00 PM). Animals were fed with a standard feed based on pellets, and purified water was given ad libitum throughout the experimental period as per Committee for the Purpose of Control And Supervision of Experiments on Animals (CPCSEA) guidelines. The research study was conducted postclearance from the Institutional Animal Ethics Committee (IAEC) of Amar Shaheed Baba Ajit Singh Jujhar Singh Memorial College of Pharmacy, BELA, Ropar vide approval no. ASCB/IAEC/03/11/053. Five rats per cage were allowed to acclimate for one week in advance of the experiments.Both male and female albino Wistar rats, weighing 180 ± 20 g body weight  guidelines 423.b.w.). The albino rats were randomly assorted into six groups of seven animals each using single blind mode. Group I (control) rats received normal saline solution through intragastric intubation and served as a control. Group II (ISP) rats were administered ISP  intraperitoneally (i.p.) twice at 24-hour intervals on days 8 and 9. The rats in Group III (CFME-LD per se) received the methanolic extract of Cassia fistula stem bark at lower dose, that is, 250 mg/kg post-oral (p.o.) daily for up to 9 days. Group IV (CFME-HD per se) rats received the methanolic extract of Cassia fistula stem bark at higher dose, that is, 500 mg/kg p.o.  for 9 days. Group V (CFME-LD + ISP) received the methanolic extract of Cassia fistula stem bark  daily for 9 days and ISP on days 8 and 9. Group VI (CHME-HD + SP) rats received the methanolic extract of Cassia fistula stem bark  for 9 days and ISP on days 8 and 9. The blood sampling was carried out on the last day for biochemical estimation, and hemodynamic parameters were evaluated. The experiment ended after 9 days, and all animals were killed with cervical decapitation after a one-night fast. The myocardial infarction was triggered using isoproterenol hydrochloride (ISP) injection (dose 5.25 and 8.5 mg/kg (1) Blood and Tissue Sampling. The rats were anesthetized using urethane injection . 2 ml of blood was collected in heparinized vials through retro-orbital plexus. The vials were held at room temperature for 15 minutes and then centrifuged at 3000 revolutions per minute for 10 minutes. The collected serum was stored at −80°C for future analysis.(2) Assessment of Oxidative Stress Biomarkers in Myocardial Tissue. The whole heart was harvested, pulverized (1 cm3), homogenized  in 0.05 M ice-cold phosphate-buffered saline (PBS), and centrifuged (3000 × g) for 10 minutes at 4°C. The accumulated supernatant was kept for the assessment of the antioxidant activities such as lipid peroxidation [xidation  and reduxidation  by the s(3) Assessment of Hemodynamic Parameters. Rats were anesthetized at 37°C with 25% urethane . To execute tracheotomy, a ventral midline incision was made in the neck. A polyethylene tube with an internal diameter of 0.30 mm and an outside diameter of 0.40 mm, coupled to a three-way cannula, was used to cannulate the left carotid artery. The cannula was heparinized (300 IU/ml) and linked to an AD Instruments POWER LAB 4/30 system  via a pressure transducer to monitor systolic arterial pressure (SAP), diastolic arterial pressure (DAP), mean arterial pressure (MAP), and heart rate (HR).(4) Assessment of Myocardial Infarction. The hearts of the experimental rats were removed after cervical dislocation using anaesthesia. On 10th day of the study, a 2,3,5-triphenyl tetrazolium chloride (TTC) test was carried out as per the procedure of Fishbein et al. [n et al. . Cold wan et al. .μm segments (using a microtome cutter) and tainted with hematoxylin and eosin (H&E) stains, enclosed using transparent concealment slips and artificial resin DPX . Under a light microscope at 100 magnifications, the pathologic structural alterations were studied [The cardiac tissue samples were washed in PBS solution and secured in 10% neutral buffered formalin for 24 hours at room temperature for histopathological investigation. The samples were then dehydrated using a gradient of ethanol concentrations (70–100%), cleaned in xylene, and entrenched in paraffin wax. Permanent slides were equipped with 5- studied .p < 0.05 was considered to be significant. The data were expressed as mean ± standard error of mean (S.E.M.), respectively.The data were analyzed using a one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test, using GraphPad Prism 5.0 software package. A value of Percentage yield of 4.12%, 4.88%, 8.87%, 17.56%, and 9.45% (w/w) was obtained from various extracts of PE, CE, EE, ME, and AE, respectively.in vitro DPPH free radical scavenging activity of diverse extracts revealed that the reducing power of the methanolic extract of Cassia fistula (CFME) stem bark is the highest with an IC50 value of 3.07 μg/ml as compared to standard BHT (2.91 μg/ml). The radical scavenging activity in the various extracts decreased in the following order ME > EE > CE > AE > PE as shown in μg/ml). The methanolic extract of Cassia fistula (CFME) exhibited a good reducing power at 100 μg/ml concentrations as shown in in vivo studies.The Cassia fistula stem bark was revealed to be safer where no animal mortality is seen; therefore, the one-fourth and one-eighth of the maximum tolerated dose was chosen for further study.On the basis of 14-day observations, the 2000 mg/kg of methanolic extract of the b.w. administered in Groups III and IV for a duration of 9 days did not show considerable changes in biochemical , hemodynamic, assessment of myocardium, and histopathological markers as equated to the normal control collection.Due to the high polyphenolic content and strong antioxidant potential, CFME was explored for cardioprotective activity. CFME at 250 mg/kg and 500 mg/kg of p < 0.001) amplified the serum levels AST, ALT, TG, and TC in rats as compared to the administration of saline in control groups. However, Groups V and VI showed a significantly reduced AST  and ALT  activity, and TG  and TC  concentrations as compared to Group II (The acquaintance of ISP significantly (Group II .p < 0.001) decreased GSH and SOD activity, whereas MDA levels were significantly (p < 0.001) increased as associated with saline control Group I. However, in this study, Groups V and VI displayed a substantial intensification in the GSH  and SOD  activity, whereas MDA levels were significantly  declined when juxtaposed with Group II (Group II showed a significantly (Group II .p < 0.001) decrease in systolic arterial pressure (SAP), diastolic arterial pressure (DAP), and mean arterial pressure (MAP) as compared to Group I. Despite the fact that the Group II rats had a higher heart rate (HR), the difference was not statistically significant. In comparison with Group II rats, there was no substantial variance in AP, SAP, MAP, and DAP levels in Group V rats. Group VI showed that the levels of AP (p < 0.01), SAP (p < 0.05), MAP (p < 0.05), and DAP (p < 0.05) were significantly increased as compared to Group II rats, whereas there was no significant alteration in HR between both Groups V and VI  and CFME-HD (500 mg/kg) demonstrated a well-preserved normal morphology of cardiac muscle with little necrosis. The histo-architecture of the ISP-treated group clearly showed signs of cell infiltration, myocardial phagocytosis, and extravasations of RBCs (red blood cells) followed by characterized by inflammation, oedema, and necrosis , whereasitecture .Heart is one of the most imperative organs of the human organ system pumping blood throughout the body. Even minor imbalances in heart functioning leads to the arousal of acute and chronic complications. As per one of the WHO  reports, heart-related diseases including inflammations and strokes will become the prime cause of mortality and disability globally by 2020 . MyocardCassia fistula for cardioprotective effect using the chemical-based ISP-induced myocardial necrosis model in rats. Cassia species are known for their antioxidative and cardioprotective potential enlisted in various treaties, traditional medicine books, and pharmacopoeias [Cassia siamea Lamk. (leaves) and Cassia fistula  has been already reported for cardiac potential via doxorubicin-induced cardiotoxicity model [This study investigated stem bark extracts of copoeias . Cassia ty model , 21. Howty model –39.in vitro antioxidant assays were carried out for all the five extracts using the DPPH free radical scavenging method and reducing power assay. In both the assays, the methanol extract showed a significant antioxidant activity as compared to EE > CE > AE > PE, respectively. Based on the in vitro results and documented literature of Cassia fistula bark, the methanol extract (CFME) was further accounted for in vivo studies.In the present research, polarity-based successive extractions were carried out, of which the highest yield was found in the methanol extract (ME) followed by AE > EE > CE > PE. Further p < 0.001) as compared to saline-treated control groups (Group I) as well as Groups III and IV (CFME per se groups). The changes brought in serum biomarker concentrations were due to ISP-induced myocardial injury, which alters cardiac cells' membrane permeability, thus releasing marker enzymes in the blood stream [It was found that on the induction of myocardial infarction by ISP in rats given on the 8th and 9th days, the level of all serum biomarkers was significantly increased .Also, an increase in the levels of total cholesterol (39.78%) and triglycerides was observed in ISP-treated rats. It may be due to cyclic AMP (adenosine monophosphate) alterations in cardiac cells, which further leads to myocardial infarction making it a vicious circle for other cardiovascular diseases , 45. HowAs discussed above that ISP induces oxidative stress chemically in the heart that in turn leads to the decline of both SOD activity and GSH level, while plasma TBARS levels shoot up –48. AlsoPrevious literature research reported the effect of ISP-induced myocardial necrosis in rats by alterations in systolic and diastolic functions in the heart that were similar to the present study as a substantial decrease in their AP (15.4%), SAP (25.5%); DAP (19.8%), and MAP (18.5%) was observed in ISP-induced rats. CFME at higher dose demonstrated a significant improvement in AP, SAP, DAP, and MAP by 7.6%, 6.8%, 8.6%, and 6.8%, respectively, while no significant difference in heart pressure was found on the administration of CFME at lower dose. According to a report by Rona and Zhou, an increased heart rate give rise to more oxygen demand and consumption in the body, which further aggravates myocardial necrosis , 48. HowCassia fistula bark methanolic extract in ISP-induced chemical model of cardiotoxicity. CFME showed a significant reduction in myocardial infarction in rats without producing any serious side effects. The present ISP model was found better in comparison with the earlier used doxorubicin model in evaluating the cardioprotective properties of Cassia fistula methanolic stem bark extract. Hence, natural plant-based products containing secondary metabolites such as glycosides, flavonoids, and carotenoids [The present research justifies the cardioprotective effect of otenoids  can playCassia fistula is a bioremedial plant known for the abolition of a number of diseases. The extract of its stem bark is reported to contain fistucacidin , β-sitosterol, lupeol leucocyanidin, hexacosanol oxyanthraquinone, dihydroxyanthraquinone, and flavonol glycosides. Numerous bioactive constituents present in the plant contributed to enhance its medicinal value. The methanolic extract of Cassia fistula in our study revealed that it possesses marked antioxidant and antihyperlipidemic activity. The extract had no significant effect on the hemodynamic parameters; that is, the heart rate remains stable. Altogether, it can be concluded that the methanolic extract of Cassia fistula has a potent cardioprotective effect via adrenergic inhibition. The phytoconstituents responsible for the cardioprotective effect might be the area of research in future.The


Meningioma is the most common intracranial neoplasm, yet there is no effective therapy for recurrent/refractory meningiomas after surgery and radiation. Prostate-specific membrane antigen (PSMA) is an enzyme upregulated on endothelial cells of multiple neoplasms and is being investigated as a theranostic target. Until now, PSMA has not been studied in meningiomas. We aimed to verify PSMA endothelial expression in meningiomas, detect tumor grade variability, and investigate the relationship of PSMA signal with tumor recurrence. We analyzed 96 archival meningiomas including 58 de novo and 38 recurrent specimens. All specimens were stained routinely and immunostained for CD31 and PSMA. Slides were scanned and analyzed producing raw data for images of PSMA, CD31, PSMA/CD31, and PSMA/vasculature. PSMA expression was seen within 98.9% of meningioma samples. In the total cohort, higher-grade tumors had increased expression of raw PSMA and PSMA/CD31, and PSMA/vasculature ratios compared to grade 1 tumors. PSMA expression and PSMA/vasculature ratios (p = 0.0015) were higher in recurrent versus de novo tumors among paired samples. ROC curves demonstrated PSMA/CD31, PSMA/vasculature, and raw CD31 as indicators of tumor recurrence. Thus, PSMA is expressed within endothelial cells of meningiomas, is increased with tumor grade and recurrence, and persists with prior irradiation. Meningioma is the most common central nervous system tumor, accounting for 39.0% of all intracranial neoplasms . AlthougProstate-specific membrane antigen (PSMA) is a 100-kDa transmembrane peptidase expressed in prostate epithelium that is upregulated in prostate adenocarcinoma and has a positive association with tumor aggressiveness markers such as the Gleason score , 5. AddiHaemels et al  in 2020 As recent studies correlate tumor angiogenesis with PSMA staining and the ability to utilize molecular imaging to monitor PSMA-avid tumors and their response to therapy, targeting PSMA-expressing endothelial cells of brain tumors may prove to be therapeutically useful , 15. To By assessing PSMA endothelial staining in meningiomas grades 1–3, we aimed to demonstrate the variability of PSMA expression in meningiomas in the context of WHO-defined atypical features , and the Ki-67 proliferation index. Importantly, we evaluated PSMA signal and tumor recurrence in paired patient samples.After institutional board review and approval at Lifespan Hospital with the Warren Alpert Medical School of Brown University, Providence, Rhode Island, formalin-fixed, paraffin-embedded tissues from 69 individual patients who underwent surgical resection between 1996 and 2022 for meningioma, WHO grades 1–3 were obtained. We analyzed 96 archival meningioma cases, selecting 58 de novo  and 38 recurrent  specimens. All cases were stained with hematoxylin and eosin (H&E), with subsequent examination by a neuropathologist who selected samples with the most viable tumor to perform immunohistochemical analysis. The selected tissue samples were stained with anti-CD31 to highlight tumor vasculature as a control. Immunohistochemical staining for PSMA was performed to assess PSMA expression within the tumor vasculature.The primary antibodies used were anti-CD31 Endothelial Cell (concentrate) Clone JC70A purchased from Cell Marque,  and Prostate-Specific Membrane Antigen (concentrate) Clone 3E6, #M362001-2 purchased from DakoCytomation Inc. .Four-micrometer-thick sections were cut and baked in an Isotemp oven for 30 minutes at 60°C. Slides were put onto the Dako Link48 for antigen retrieval, and the Flex kit was used for antigen detection. Antigen retrieval was performed for 45 minutes at pH 6 (Citrate). The primary antibody to CD31 was used at a dilution of 1:150 and was automatically titrated onto the slides and subsequently incubated for 30 minutes. The universal secondary antibody was automatically dispensed and incubated for 20 minutes. Slides were removed from the Link48 and put into deionized water mixture, counterstained with hematoxylin, 0.2% ammonia water for 1 minute, and then run under deionized water for 5 minutes. Slides were then dehydrated and coverslipped.Four-micrometer-thick sections were cut and baked in an Isotemp oven for 30 minutes at 60°C. Slides were put onto the Dako Link48 for antigen retrieval, and the Flex kit with mouse linker was used for antigen detection. Antigen retrieval was performed for 45 minutes at pH 9 (EDTA). The primary antibody for PSMA was used at a dilution of 1:10 and was automatically titrated onto the slides and subsequently incubated for 10 minutes. The universal secondary antibody was automatically dispensed and incubated for 15 minutes. Slides were removed from the Link48 and put into a deionized water mixture, counterstained with hematoxylin, 0.2% ammonia water for 1 minute, and then run under deionized water for 5 minutes. Slides were then dehydrated and coverslipped.Both H&E and immunostained sections were scanned on a Leica Aperio CS2 WSI slide scanner at 20× magnification objective lens (0.5 μm/pixel). All of the digital slides were then downloaded from the Aperio vendor agnostic whole slide image (WSI) viewer in the standard visualization system image format with variable degrees of JPEG image compression. WSIs were deidentified and labeled with a sticker and numbered 1–96. Data transfer and storage were undertaken in a Health Insurance Portability and Accountability Act of 1996 (HIPPA)-compliant manner.QuPath (version 0.2)  was utilVascular density was calculated from H&E-stained slides, while PSMA and CD31 were calculated from IHC-stained slides. Ground truth for vasculature as defined on H&E-stained slides included 3 shared layers of the vessel wall for arteries and veins , with small arteries and arterioles usually containing 1 or 2 layers of smooth muscle cells, and often absence of the external elastic membrane in arterioles. Additionally, continuous capillaries are present in the central nervous system , rangingAn overview biomarker quantification from WSIs is shown in The dependent variables  were examined in International Business Machines Corporation Statistical Package for the Social Sciences (IBM SPSS) 27 as a function of dichotomized categorical values  seen in In total, 96 specimens (both de novo tumors and recurrent tumors) were included on the WSI open access solution to machine learning. These were derived from de novo (primary tumors) and recurrent  from 69 patients, of which 37 cases comprised paired patient samples (both de novo and recurrent specimens). A total of 58 de novo  specimens and 38 recurrent  specimens were evaluated and are represented in The entire cohort consists of 96 patient samples. Overall, patients ≥50 years old had increased raw PSMA (p = 0.010), PSMA/CD31 ratio (p = 0.010), and PSMA/vasculature ratio (p = 0.002) compared with patients <50 years of age. Patients with tumor size ≥4 cm had increased raw CD31 (p = 0.006) and an increased PSMA/CD31 ratio (p = 0.038) compared to patients with tumor sizes <4 cm in greatest dimension grossly. Higher-grade tumors (grades 2–3) had increased expression of raw PSMA (p = 0.039), PSMA/CD31 ratio (p = 0.034), and PSMA/vasculature ratio (p = 0.015) compared to grade 1 tumors. Biomarker expression among the total cohort with regard to increased raw PSMA and PSMA/CD31 ratios is shown in Patients with de novo grade 1 meningiomas had no significant differences in raw PSMA expression (p = 0.821), raw CD31 (p = 0.754), PSMA/CD31 ratio (p = 0.958), or PSMA/vasculature ratio (p = 0.651) compared to de novo grade 2–3 tumors represented in NF2 mutations), while skull base meningiomas demonstrate different mutations . Accordingly, we analyzed 2 groups: one combining lobar, parasagittal, and spine into one group (57 cases), and sphenoid, optic nerve/intraorbital, posterior fossa, temporal fossa, and frontal skull base together in a second (“skull base”) group (16 cases).  There were no significant differences in raw PSMA expression (p = 0.286), raw CD31 expression (p = 0.714), PSMA/CD31 ratio (p = 0.191), and PSMA/vasculature ratio (p = 0.739).The tumors in this cohort localize as follows: lobar , intraventricular, sphenoid, intraorbital/optic nerve, parasagittal, midline, posterior fossa, temporal fossa, frontal skull base, spine, and unspecified. According to the 2021 WHO brain tumor classification scheme (18), convexity and the majority of spinal meningiomas have similar molecular findings . Raw PSMA had an AUC of 0.5642 with a SE of 0.0734 and a 95% confidence interval of 04202 to 0.7082 (p = 0.394). PSMA/CD31 AUC of 0.7843 with a SE of 0.05651 and a 95% confidence interval of 0.6736 to 0.8951 (p < 0.001). PSMA/vasculature had an AUC of 0.6456 with a SE of 0.06483 with a 95% confidence interval of 0.5185 to 0.7726 (p = 0.033).The median raw CD31 expression was 1.675 in the initial and 1.40 in the paired recurrence specimens with a median difference of 0.1500 (p = 0.999). The median raw PSMA expression was 0.66 in the initial and 1.17 in the paired recurrence specimens with a median difference of 0.2215 (p = 0.2099). The median PSMA/CD31 ratio expression was 0.734 in the initial and 1.048 in the paired recurrence specimens with a median difference of 0.03019 (p = 0.7024). The raw PSMA and CD31 colocalization in an individual paired patient sample is shown in The present study measured the expression of PSMA in meningiomas to explore the feasibility of targeting PSMA in initial diagnostic, subsequent PET/CT monitoring, and in the selective delivery of PSMA-targeted therapeutic agents. We found that 98.9% of meningioma specimens expressed PSMA within their endothelial cells. Additionally, across the total cohort PSMA expression was higher in tumors grades 2–3 when compared to grade 1 and in recurrent tumors compared to de novo tumors. As the vast majority of meningiomas are benign, the mainstay of therapy for grade 1 tumors is gross total resection. In tumors grades 2–3, patients are additionally treated with radiation therapy subsequent to surgical resection. Unfortunately, higher-grade tumors have increased aggressiveness, rates of recurrence, and refractoriness to therapy, including the presence of atypical features and a Ki-67 index of proliferation ≥4% . In tumoDiagnostic imaging has made great changes to the early diagnosis and management of patients with PSMA-expressing tumors, namely those with a history of prostatic adenocarcinoma. 68GA-PSMA-11 was one the first positron emitting radionucleotides to be used in diagnostic imaging . CurrentNumerous studies have shown that PSMA has not been reported in normal vasculature and is the only specific neovascular target currently known . AdditioOur findings confirm that PSMA is expressed within the neovasculature of meningiomas. While de novo meningiomas had no significant expression of raw PSMA when comparing de novo grade 1 versus grades 2–3 tumors, the raw PSMA expression relative to grade was increased in recurrent tumors. This finding suggests that the angiogenesis of tumor neovascularization may increase with recurrence and may function as a driver of increased angiogenesis. As PSMA expression is increased in recurrent tumors, irrespective of prior radiation therapy, it is likely that PSMA is a driver of the neovascularization rather than as a tumor response to radiation therapy. Our findings show that in tumors that are recurrent, of higher grade, and have undergone radiation therapy, PSMA expression remains intact, and the raw expression is increased. Thus, as PSMA is unregulated in patients with recurrent tumor, the neovasculature is potentially targetable by PSMA directed therapies.Our observed variability of PSMA staining between grade 1 tumors of differing subtypes needs to be more comprehensively studied. Of the de novo grade 1 tumors investigated, there were extremes of PSMA staining in relation to the variability of tumor neovasculature; namely, angiomatous samples were at the higher end of raw PSMA expression, while fibrous subtypes were at the lower end of the spectrum. Additionally, heterogeneity of tumor staining in the tissue sample should be studied more comprehensively. As the majority of de novo grades 1–3 tumors were acquired before the patients underwent radiation therapy, the intratumoral heterogeneity is most likely due to a combination of the proliferation of angiogenic vessels per subtype, and in cases of recurrence, to grade. As identified in prostate cancer, expression of PSMA is highest in higher-grade tumors .The cutoff for patient age of 50 was instituted as the mean age of our entire cohort was 58 years old. Other variables, including the cutoff of <4 and ≥4 for both mitoses per 10 high-power fields and the rate of proliferation as determined by estimation or quantification of immunolabeling with Ki-67, were based on the 2021 CNS WHO criteria for evaluation of Ki-67 as a marker of tumor aggressiveness and recurrence and the number of mitoses as diagnostic criteria involved in the grading of meningiomas . AdditioOur data follow the studies of expression of PSMA in primary glial tumors , 7. WernOur study has a few limitations. Although studies have correlated mitotic index with the mitosis marker phosphohistone H3 (pHH3) , 26, we Machine learning was utilized in this study to completely objectify the results and eliminate human variance in light microscopic interpretation. The goal is to perform reproducible scoring of PSMA in meningioma along with other biomarkers from WSI. However, further research and validation of computational biomarkers is required before coming up with such a system for routine clinical pathologic use. One such project would be to correlate molecular imaging for PSMA with tissue expression to see if the PSMA positivity has relevance to the patient, particularly regarding anti-PSMA therapy. Determining cutoffs for PSMA expression could be coupled with more advanced machine learning and multi-omics approaches to predict treatment response to PSMA radioligand therapy.177Lu), favored for use in prostate cancer secondary to minimal tissue penetration and a long half-life, which facilitates a higher degree of radiation. Radioligand therapy with 177Lu-PSMA-617 has been shown to prolong imaging-based survival when used in conjunction with standard of care for patients with PSMA-positive, advanced metastatic castration-resistant prostate cancer (PSMA radioligand therapy has emerged as a significant therapeutic in prostate cancer , where se cancer .Lastly, since the current study focused mainly on banked tumor tissue, we did not look into the relationship between PSMA expression in meningioma and neuroimaging features such as peritumoral edema. Quantitative and qualitative information derived from perfusion MRI analyzing peritumoral edema may suggest the histological grade of meningioma preoperatively and potentially predict likely patient outcomes . This imIn conclusion, the present study suggests that PSMA expression in endothelial cells of meningiomas increases with recurrence and grade. These results provide a rationale for meningioma treatment in accordance with PSMA expression and call for clinical trial testing utilizing PSMA-targeted therapies as alternative diagnostic, therapeutic, and maintenance approaches for recurrent/refractory meningiomas.nlac089_Supplementary_DataClick here for additional data file.


We show that maternal social isolation induces distinct and robust metabolic profiles in the blood plasma of adult F3 offspring, which are characterized by critical switches in energy metabolism, such as upregulated formate and creatine phosphate metabolisms and downregulated glucose metabolism. Both trans- and multigenerational stress altered plasma metabolomic profiles in adult offspring when compared to controls. Social isolation stress increasingly affected pathways involved in energy metabolism and protein biosynthesis, particularly in branched-chain amino acid synthesis, the tricarboxylic acid cycle , muscle performance , and immunoregulation . Levels of creatine phosphate, leucine, and isoleucine were associated with changes in anxiety-like behaviours in open field exploration. The findings reveal the metabolic underpinnings of epigenetically heritable diseases and suggest that even remote maternal social stress may become a risk factor for metabolic diseases, such as diabetes, and adverse mental health outcomes. Metabolomic signatures of transgenerational stress may aid in the risk prediction and early diagnosis of non-communicable diseases in precision medicine approaches.Metabolic risk factors are among the most common causes of noncommunicable diseases, and stress critically contributes to metabolic risk. In particular, social isolation during pregnancy may represent a salient stressor that affects offspring metabolic health, with potentially adverse consequences for future generations. Here, we used proton nuclear magnetic resonance ( The global rates of metabolic diseases have increased sharply over the past few decades . MetabolMaternal stress affects offspring physiology and neurodevelopment by programming the fetal hypothalamic–pituitary–adrenal (HPA) axis ,8,9,10. Prenatal stress has been linked to altered DNA methylation marks  and micr1H NMR) spectroscopy and supervised and unsupervised machine learning approaches to probe robust metabolic signatures in rat blood plasma generated by transgenerational and multigenerational maternal stress in offspring from the third filial generation (F3). The models allow differentiation between the metabolomic impact of transgenerational epigenetic inheritance versus cumulative generational prenatal stress on adverse health outcomes. Both stress models were previously linked to preterm birth and metabolic disease [Because upstream epigenetic regulation driven by prenatal or remote ancestral stress is reflected in downstream cellular metabolic functions, the metabolome, i.e., the sum of all metabolites in an organism, arguably represents a direct result of transgenerational programming ,40,41 an disease , anxiety disease ,32,35, i disease ,43, and  disease ,31. Thisp < 0.05) or VIAVC analysis. Metabolite profiles of the transgenerational prenatal stress (TPS) and multigenerational prenatal stress (MPS) groups were clearly separated from CONT  and degradation (p < 0.01). In addition, the glucose-alanine cycle (p < 0.01) and alanine metabolism were altered in the MSEA. Pathway topology supported the effects on branched-chain amino acid synthesis (p < 0.01) and degradation (p < 0.01) pathways but did not reveal significant pathway hits in the glucose-alanine cycle or alanine metabolic pathways (p < 0.01), however, was also shown to be affected by TPS.Metabolite set enrichment analysis (MSEA) of the TPS compared to the CONT group A indicatpathways B. The amp = 0.013) in the MPS group when compared to CONT .MSEA revealed a significant impact on glycine, serine, and threonine metabolism ( to CONT A. The pap < 0.01), methionine metabolism (p < 0.01), glycine, serine, threonine metabolism (p < 0.01), and alanine metabolism (p = 0.05) to be significantly affected. The TPS versus MPS topology analysis (p < 0.01) to be most significantly affected, followed by the TCA cycle (p < 0.01), the alanine, aspartate, and glutamate metabolic pathway (p < 0.01), the cyanoamino acid pathway (p = 0.03), the methane metabolic pathway (p = 0.05), and the aminoacyl tRNA pathway (p = 0.05).When the impacts of trans- versus multigenerational stress were compared , the metanalysis B showed p = 0.007) and MPS rats . In the MPS group, creatine phosphate was also significantly correlated with the total distance traveled , the number of vertical moves such as rears , and the distance traveled in the center  of the open field arena (ld arena . In genep = 0.02), vertical time , and central distance  in the open field task  cycle, and in muscular ATP regeneration. If lactate levels are low in TPS animals, then metabolic acidosis and muscular fatigue may occur more quickly . SimilarTPS rats also revealed significantly downregulated choline and serine levels compared to the MPS group. Choline and its metabolites are required for several physiological functions, such as cell signaling and cholinergic transmission , and mayOverall, ancestral stress had the largest impact on protein biosynthesis, energy generation, and immunity. Thus, the formation and breakdown of amino acids, as well as energy metabolism, are likely involved in the physiological and behavioural response to both TPS and MPS. These results are reinforced by the correlational analysis demonstrating associations between particular metabolites with anxiety-like behaviours. Previous studies have associated reduced exploratory behaviour, such as vertical rearing, with higher levels of anxiety . FurtherBCAAs such as leucine and isoleucine provided further insight into the metabolic underpinning of anxiety-like behaviours. More time spent rearing was linked to greater leucine and isoleucine concentrations in rats from the TPS group. Greater levels of leucine and isoleucine were associated with more distance travelled in the center of the open field in the MPS group. This change was mirrored in the TPS lineage with greater levels of leucine and isoleucine translating to greater total and central distance travelled in the open field task, linking remote ancestral stress to altered cellular energy metabolism and behavioural change. Thus, higher levels of leucine and isoleucine may be linked to higher stress resilience, whereas lower levels of these BCAAs may serve as markers of anxiety-like behaviours. Lower levels of BCAAs have also been linked to adverse metabolic and inflammatory functions ,57 in adThe present downstream cellular metabolic changes may reflect differential underlying epigenetic changes that are potentially heritable. The TPS generation of the maternal lineage in particular excludes direct exposure effects and unambiguously suggests heritable epigenetic transgenerational mechanisms for offspring three generations removed from maternal stress ,77 to reThe present study used a novel approach to quantify outcomes of ancestral stress and HPA axis dysregulation by taking a metabolomic perspective and providing possible mechanisms for health outcomes in an animal model. The metabolomic biomarkers identified in this study may provide potential biomarkers for the risk prediction and early diagnosis of non-genetically heritable stress-related diseases. The identification of predictive and diagnostic metabolic biomarkers that identify adverse health trajectories linked to maternal stress is critical for precision medicine approaches, enabling the identification of the most vulnerable individuals who are at the highest risk. Because the orchestrated regulation of metabolic pathways is essential for stress regulation, development, and successful aging, its failure may lead to cell and tissue dysfunction and result in chronic and non-communicable diseases. On the other hand, metabolic profiles are often cell-type specific , thus prn = 8), transgenerational prenatal stress , and multigenerational prenatal stress . Four samples from the control group did not yield enough plasma for metabolomics analysis, resulting in a reduced control group size . TPS rats were the third filial (F3) generation of a lineage in which only the first filial (F1) generation was stressed prenatally; subsequent generations remained unstressed . One male offspring per litter was randomly selected for this metabolomics study. Bystander effects of stress were avoided by using designated testing and housing spaces. All housing, handling, testing, and tissue sampling conditions were harmonized and carefully controlled across generations.The rats were housed in polycarbonate shoebox cages on corn cob bedding under a 12 h light/dark cycle with lights on at 7:30 AM. The room temperature was maintained at 20 °C with relative humidity at 30%. All procedures were performed in accordance with the guidelines of the Canadian Council on Animal Care and approved by the University of Lethbridge Animal Welfare Committee.Anxiety-like behaviours towards a novel environment and risk taking were assessed in an open field test at P90. Rats were individually placed into an Accuscan activity monitoring box  and recorded for 10 min. VersaMax™ software  monitored the animals’ horizontal and vertical activity to be analyzed using VersaDat™ software . Vertic1H NMR spectroscopy [g for 15 min at 4 °C. Samples were stored at −80 °C until further processing.At the age of 140 days (P140), 600 μL of blood was collected from the lateral tail vein using a 23-gauge butterfly needle coated with lithium heparin, while rats were anaesthetized using 4% isoflurane. All blood samples were collected at the same time of day, between 8:00 and 9:00 AM in the morning. EDTA and citrate collection tubes were avoided, as they give additional spectral signals in troscopy . Blood wg for 30 min at 4 °C. Supernatant was decanted to fresh tubes and allowed to dry under a gentle stream of nitrogen gas until all liquid had evaporated. Dried pellets were then re-suspended in 600 μL of phosphate buffer calibrated to pH 7.4 and vortexed for 10 s, or until the pellets were completely dissolved. The phosphate buffer was prepared as a 4:1 ratio of KH2PO4:K2HPO4 in a 4:1 H2O:D2O solution to obtain a final concentration of 0.5 M. The D2O contained 0.03% w/v trimethylsilyl propanoic acid (TSP) as a chemical shift reference for 1H NMR spectroscopy. To maintain metabolite integrity, 0.03% w/v of sodium azide (NaN3) was also added to the buffer as an antimicrobial agent. Finally, samples were centrifuged again at 12,000× g for 5 min at 4 °C and 550 μL of re-suspended supernatant was added to 5 mm NMR tubes for data acquisition.Water soluble metabolites were extracted from plasma using a methanol precipitation protocol . MethanoNMR spectra were collected on a 700 MHz Bruker Avance III HD spectrometer . A Shapiro–Wilk test for data normality was applied and all data were found to be non-parametric. A Mann–Whitney U test was subsequently used to determine which spectral bins were significantly altered in comparison groups . Bonferr® , respectively. The variable importance analysis based on random variable combination (VIAVC) algorithm [PCA and Mann–Whitney U tests were carried out using the web-based Metaboanalyst software  and MATLlgorithm  was utillgorithm . PCA scolgorithm ,90. The p-values equal to or less than 0.05 were considered significant.Spearman’s rank correlations were carried out using SPSS 26 for Windows  to determine the relationship between normalized metabolite concentrations and behavioural data. This analysis concerned only metabolites determined to be significantly altered between groups, based on the multivariate and univariate statistical tests outlined above. Samples with A spectral database of pure metabolites was generated and used to identify most metabolites found in the NMR spectra. The Human Metabolome Database ,92,93 wa


While osteoporosis is characterized by skeletal fragility due to increased bone turnover and low bone mineral density (BMD), subjects with abdominal obesity and type-2 diabetes have increased risk of bone fractures despite low bone turnover and increased BMD. Diets with increased protein content are reported to increase bone turnover in healthy adults and may be a point of interest in preserving bone strength in subjects with abdominal obesity and/or type-2 diabetes.We examined the effect of 12-weeks dietary intervention on bone turnover in 64 adults with abdominal obesity using data from the MERITS trial. The trial was a randomized, controlled, double blinded study in which participants were allocated to receive either 60 g/d of whey protein hydrolysate or maltodextrin in combination with either high (30 g/d) or low dietary fiber intake (10 g/d). Primarily, we assessed changes in plasma markers of bone turnover Procollagen type 1 N-terminal propeptide (p1NP), C-terminal telopeptide type-1 collagen (CTX), and parathyroid hormone (PTH) within the four intervention groups. In addition, we measured u-calcium and u-carbamide excretion, 25(OH)D, and BMD by whole body DXA scans. Finally, we compared changes in insulin resistance  with changes in bone turnover markers.www.clinicaltrials.gov as NCT02931630.The trial was registered at Sixty-four subjects were included in the study. We did not find any effect of twelve weeks of high protein or high fiber intake on plasma levels of P1NP or CTX. There was a nonsignificant positive association between protein intake and PTH levels (p=0.06). U-calcium and u-carbamide increased in both protein groups. There was a positive association between change in HOMA-IR and PTH (p=0.042), while changes in P1NP and CTX did not associate to changes in HOMA-IR.Twelve weeks of increased whey protein intake in subjects with abdominal obesity did not affect markers of bone turnover significantly, although tended to increase PTH levels. Dietary fiber intake did not affect bone turnover. We report a positive association between change in HOMA-IR and PTH supporting a hypothesis of insulin resistance as a potential key factor in the expanding field of bone fragility in T2D subjects. A diet rich in protein may be beneficial to skeletal properties, since bone structure largely consists of proteins integrated within the organic collagen matrix during mineralization. Nevertheless, the effect of high protein diets remains poorly established when it comes to bone health .Most studies on protein diets and bone health are performed on healthy or postmenopausal women, where an increased risk of fractures relates to increased bone turnover with resulting loss of bone mass. However, in dysmetabolic conditions dominated by insulin resistance such as abdominal obesity, metabolic syndrome or prediabetes and type 2 diabetes, the determination of bone properties seems to be quite different. Contrasting to postmenopausal osteoporosis, bone health is dominated by low bone turnover , which mFormerly, obesity was considered a protective feature against fractures due to mechanical protective properties of subcutaneous fat in the hip region and an increased bone density from increased mechanical load. Within the last decades, this assumption has been challenged , 10. Alt2  versus isocaloric maltodextrin induced an increase in CTX and preserved fat-free mass in elderly men and women with BMI from 19 to 32 kg/mStudies on long-term protein supplements and bone are primarily performed on subjects with osteoporosis - a disease in which the increased fracture risk is based on elevated bone turnover and thus very different from obesity and T2D . The effUsing data from the MERITS trial , we examMoreover, we aimed to examine the association between change in bone turnover and insulin resistance.The present study was part of the MERITS trial, described in detail previously . The triWe recruited 73 subjects with age ≥40 years with abdominal circumference ≥94 cm (men) or ≥80 cm (women). Exclusion criteria included osteoporosis, diabetes, severe renal, cardiovascular, or psychiatric illness or medical treatment with hormonal replacement therapy or corticosteroids. We allowed for the continued use of regular medication, including vitamin D and calcium supplements, if no changes occurred during the trial or three months prior to inclusion.By blocks of eight, we randomized the participants by age and sex into one of four groups: WP + high fiber (WP-HiFi), WP + low fiber (WP-LoFi), maltodextrin + high fiber (MD-HiFi), or maltodextrin + low fiber (MD-LoFi).For a period of 12 weeks, the participants received powder supplements twice daily (2 x 30 g of WP or MD). Furthermore, participants were to substitute bread and cereal products of their normal diet with test products  of either high or low fiber content aiming at a fiber intake of 30 g (HiFi) or 10 g (LoFi) fiber per day from test products.® HYDRO.REBUILD) and MD (Glucidex® 19). Lantmännen /Lantmännen Cerealia AB  provided bread and cereal products.Arla Foods Ingredients Group P/S  provided the WP hydrolysate . Data on nutritional composition of the test products are given elsewhere . CalciumThe HiFi bread and cereals consisted of enzyme-treated wheat bran and refined wheat, while LoFi products were based only on refined wheat. Enzyme treatment was performed by DuPont Industrial Biosciences Aps  to increase fiber solubility for improved baking properties.ClinicalTrials.gov (NCT02931630).The study was conducted in accordance with the Declaration of Helsinki of 1975 (as revised in 1983), approved by the Central Denmark Region Committees on Health Research Ethics  and registered at At trial initiation, participants attended the clinic after an overnight fast (minimum 10 h) by means of car or public transportation. Blood was drawn from an antecubital vein between 7.00 and 8.30 AM. A Whole-body Dual-Energy X-ray Absorptiometry (DXA) scan  was performed to assess body composition. Whole body BMD excluding the head region is reported in this manuscript. Participants collected 24-h urine samples using 3L containers in cooling bags, which were analyzed for carbamide and calcium. The procedures were repeated after 12 weeks of dietary intervention.Participants picked up the intervention products at the test site with regular intervals.Dietary adherence was assessed by self-reported 3-day weighed dietary records at the beginning, middle and end of the trial, and by measuring urinary carbamide and plasma alkylresorcinols .g for 15 min at 4 °C, immediately frozen at −20 °C and moved to −80 °C within 8 h. All fasting values were calculated as the mean of three consecutive fasting blood samples.Blood samples were centrifuged at 2000 × Plasma glucose was measured on Cobas c111-system by standard enzymatic colorimetric assays using commercial kits . Intra-/inter-assay precision were between 0.8–1.1% and 0.5–0.6%. Plasma insulin was measured by ELISA technique using commercial kits  with intra-/inter-assay precision of 5.1–7.5% and 4.2–9.3%.Serum CTX, P1NP, and PTH were measured on Cobas 6000 e 601 system using ELISA sandwich immunometric assays method (Roche Diagnostics GmbH). Intra-/inter-assay precision was 1.4–3.2%/1.9–3.4% for P1NP, 1.2–4.7%/1.5–5.7% for CTX, and 1.1–2.0%/1.7–3.4% for PTH.Plasma ionized calcium, phosphate, magnesium, alkaline phosphatase, 25(OH)D, glomerular filtration rate (GFR) and urinary calcium and carbamide were analyzed at the Department of Clinical Biochemistry at Aarhus University Hospital, Denmark (DS/EN ISO 15189:2013 approved).All analyses were pre-specified at the project origin.The final analysis included only participants who completed the trial.Baseline values are displayed as means with SD unless otherwise indicated.Two-factor ANOVA was used to assess any effect or interaction of protein or fiber intake on markers of bone turnover. Any significant effect was followed by a pairwise comparison of groups corrected for multiple comparisons by Tukey-Kramer method. Estimates were adjusted for age and sex.Linear regression analysis was used to determine associations between change in insulin resistance and bone turnover markers in all subjects. Assumptions of linear regression were checked using scatter plots, QQ plots and histograms of the residuals. The regression analyses were not corrected for multiple testing due to the exploratory nature of the study and the intent to generate new hypotheses.Normality and variance across groups was checked by diagnostic plots of the residuals. If these were not met, the dependent variable was log-transformed.Homeostasis model assessment of insulin resistance was calculated by the formula: Fasting plasma glucose (mmol/L) x fasting plasma insulin (mU/L)/22.5.All statistics were performed using STATA/IC 15.1 .Power calculations for sample size were based on expected change in postprandial triglycerides, which was the primary aim of the MERITS trial .Baseline characteristics by randomization group are listed in Study flow chart and dietary intake reports have previously been reported . In brieAs expected, we found an increased u-calcium and u-carbamide in both protein groups.There was a non-significant negative association (p=0.06) between change in dietary fiber intake and change in 25(OH)D.The intervention did not affect insulin resistance within the four groups . Table It is a common belief, that bone health is influenced by dietary habits. In the standard treatment of osteoporosis , a bone Diets of increased protein content are generally advised to preserve bone mass –31, althIn type 2 diabetes, abdominal obesity and metabolic syndrome, bone turnover is low - in contrast to postmenopausal osteoporosis, where bone turnover is increased. Fracture risk is however increased, and the low bone turnover is suggested to accumulate microfractures and make the bone more fragile. Thus, increasing bone turnover in type 2 diabetes, abdominal obesity and metabolic syndrome may be beneficial for bone health. Very little research, if any, explore the long-term effects of diet and especially enhanced protein intake on bone within weight stable subjects with type 2 diabetes, abdominal obesity or metabolic syndrome.We hypothesized that the increase in protein intake would lead to increased bone turnover. This was however not the case. Both bone formation and degradation markers remained unchanged after 12 weeks of whey protein versus placebo. Although, we did observe a negative trend for an association between protein intake and PTH levels (p=0.06). In normal weight subjects, low protein diets are known to lead to secondary increase in PTH which is believed to relate to reduced intestinal calcium absorption and increased bone turnover . LikewisWe report an increase in urinary calcium excretion in the protein group. It is well known that increased protein intake induces increased urinary calcium excretion . IncreasThus, a protein-rich diet in subjects with abdominal obesity appears to affect calcium homeostasis similarly to normal weight individuals.post hoc study.The secondary hypothesis of the study was that insulin resistance and bone turnover are inversely associated. We did however not observe any difference in insulin sensitivity by HOMA-IR or bone markers between groups. In a similar dietary intervention study on type 2 diabetic subjects a high protein diet improved HbA1c but not HOMA-IR, indicating that primarily postprandial insulin sensitivity was affected , which ivice versa, assuming that the regulatory mechanism is reversible. This was not the case, as we did not see any association between change in P1NP or CTX with change in HOMA-IR. We did find an association between increase in PTH and increase in insulin resistance in all subjects independent of the dietary intervention. This finding is interesting as it aligns with previous reports were hyperparathyroidism is associated with diabetes and insulin resistance . We found a nonsignificant increase in 25(OH)D within the LoFi groups (p=0.06), that was most likely due to lower baseline values in these groups. Any seasonable variation during the 13 months the trial ran (from May 2016 to June 2017), was expected similar within groups because of the continuous block randomization.We report the findings of a The current study did not find an effect of long-term supplementation of dietary protein or fiber on bone turnover in subjects with abdominal obesity. PTH tended to associate negatively with protein intake, although bone turnover markers remained unaffected. A high protein intake induced increased urinary calcium excretion, unrelated to increased calcium intake. We did find an association between measures of insulin resistance and levels of circulating PTH levels, which supports the hypothesis that insulin resistance may be key to understand the low bone turnover and increased bone fragility observed in subjects with T2D.More long-term studies on diet and the bone turnover of subjects with type 2 diabetes, abdominal obesity and metabolic syndrome are needed.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Central Denmark Region Committees on Health Research Ethics . The participants provided their written informed consent to participate in this study.RF-N, ER, SG, and KH conceived and designed the study. RF-N and ER conducted the study. RF-N, PV, and JS-L analyzed the data. RF-N wrote the initial manuscript. All authors critically reviewed and approved the final manuscript.The study was supported by a grant from Innovation Fund Denmark - MERITS (4105-00002B) and Steno Collaborative Grant, Novo Nordisk Foundation, Denmark (Grant no. NNF18OC0052064).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.