Melissa officinalis extracts (MOEs), this study was aimed at investigating the potential of ethanolic MOE to prevent the development of myocarditis and its ability to ameliorate the severity of experimental autoimmune myocarditis (EAM) by investigating MOE effects on in vivo cardiac function, structure, morphology, and oxidative stress parameters. A total of 50 7-week-old male Dark Agouti rats were enrolled in the study and randomly allocated into the following groups: CTRL, nontreated healthy rats; EAM, nontreated rats with EAM; MOE50, MOE100, and MOE200, rats with EAM treated with either 50, 100, or 200 mg/kg of MOE for 3 weeks per os. Myocarditis was induced by immunization of the rats with porcine myocardial myosin (0.5 mg) emulsion on day 0. Cardiac function and dimensions of the left ventricle (LV) were assessed via echocardiography. Additionally, the blood pressure and heart rate were measured. On day 21, rats were sacrificed and the hearts were isolated for further histopathological analyses (H/E and Picrosirius red staining). The blood samples were collected to determine oxidative stress parameters. The EAM group characteristically showed greater LV wall thickness and lower ejection fraction (50.33 ± 7.94% vs. 84.81 ± 7.74%) and fractional shortening compared to CTRL (p < 0.05). MOE significantly improved echocardiographic parameters (EF in MOE200 81.44 ± 5.51%) and also reduced inflammatory infiltrate  and collagen content  in the heart tissues, especially in the MOE200 group compared to the EAM group. In addition, MOEs induced a significant decrease of prooxidants production  and improved antioxidant defense system via increase in GSH, SOD, and CAT compared to EAM, with medium and high dose being more effective than low dose (p < 0.05). The present study suggests that ethanolic MOEs, especially in a 200 mg/kg dose, improve cardiac function and myocardial architecture, possibly via oxidative stress mitigation, thus preventing heart remodeling, development of dilated cardiomyopathy, and subsequent heart failure connected with EAM. MOEs might be considered as a potentially helpful adjuvant therapy in patients with autoimmune myocarditis.Due to existing evidence regarding antioxidant and anti-inflammatory effects of  It is also estimated that one-third of autoimmune myocarditis cases develop heart failure, and almost 40% of all heart failures in the population under 40 is actually associated with autoimmune myocarditis. In addition, autoimmune myocarditis tends to be unrecognized in patients with DCM, until post mortem histopathological analyses, as its clinical presentation varies widely [Myocarditis is an inflammatory heart disease characterized by nonischemic inflammatory infiltrates in the heart tissue associated with necrosis and/or degeneration of cardiomyocytes. Several entities have been identified as the causes of myocarditis, such as viral or bacterial infections, drugs or toxin usage, and autoimmune processes. Acute myocardial inflammation may progress to subacute and chronic phases and ultimately lead to tissue remodeling, fibrosis, myocardium architecture damage, and depressed contractile function . Autoimms widely .giant cell autoimmune myocarditis is EAM on rodents, which allows investigation of the mechanisms involved in this pathology, as well as testing of novel treatments [Taking into account that current therapeutic options for myocarditis are limited to symptomatic treatment for arrhythmias and heart failure and that no effective therapeutic strategy has been developed yet , the seaeatments –4. The ueatments , 6.Melissa officinalis L. (Lamiaceae), also known as lemon balm, is a perennial herb belonging to lemon scent, mint family of plants. It has been widely used in traditional medicine for centuries, mainly because of its beneficial effects on the nervous system, including anxiety symptoms and palpitation relieving, mild sedative, and hypnotic effects. In addition, a plethora of pharmacological activities of lemon balm have been described: hypoglycemic, hepatoprotective, antibacterial, anti-inflammatory, antioxidant, antiviral, antispasmodic, neuroprotective, and cytotoxic effects. Literature data also suggests its beneficial effects on the cardiovascular system such as antiarrhythmic and vasorelaxant properties and protective effects in myocardial ischemia-reperfusion injury [n injury –9. Thesen injury , 11. It Up until now, the beneficial effects of bioactive plant compounds such as curcumin, quercetin, apigenin, berberine, resveratrol, oleanolic acid, catechin, and epigallocatechin have been confirmed in EAM model –14. Sevein vivo cardiac function, structure, morphology, and oxidative stress parameters.Considering proven antioxidant and anti-inflammatory effects of MOEs, we aimed to investigate the potential of ethanolic MOE to prevent the development of myocarditis and its ability to ameliorate the severity of EAM by investigating MOEs effects on All experimental procedures involving laboratory animals used in this research were approved by Ethics Committee for experimental animal well-being of the Faculty of Medical Sciences, University of Kragujevac  No. 01-10171. Furthermore, all the experimental procedures were performed according to European Directive 2010/63/EU for the welfare of laboratory animals, number and principles of Good Laboratory Practice (GLP) (86/609/EEC). Additionally, experiments were carried out following the European Union Directive 86/609/EES for the Protection of the Vertebrate Animals used for Experimental and other Scientific Purposes and the principles of ethics.Melissa officinalis L. (Lamiaceae) purchased from Bilje Borca, LLC  were used. The dried plant material was pulverized with a mill  and stored in well-sealed paper bags at room temperature until the extract was made. The ethanolic MOE was obtained under the reflux of the solvent. This method involves extraction at the boiling point of the solvent (70% ethanol). The extraction was performed for 2.5 hours, after which the mixture was filtered through gauze and left at room temperature to spontaneously precipitate ballast substances. Finally, the obtained liquid extract was filtered , while we used a rotary vacuum evaporator  at 40°C, 90 rpm, and 250 mbar vacuum to obtain dry extract, which was stored in dark glass vials at +4°C until administration [For the purposes of this research, dried leaves of stration .Dark Agouti (DA) rats, weighing 150 ± 20 g at the beginning of the experiment, purchased from the Military Medical Academy Animal House, Belgrade. Firstly, animals were acclimatized for two weeks and kept in polyethylene cages (4 per cage) under standardized controlled environmental conditions (22 ± 2°C and a 12 h light/dark cycle). Free access to standard food  and water (ad libitum) was provided for all animals.The study involved a total of 50 seven-week-old male Sigma-Aldrich, Munich, Germany) was dissolved in 0.01 M phosphate-buffered saline (PBS) in one tube and emulsified with an equal volume of complete Freund's adjuvant (FCA) supplemented with Mycobacterium tuberculosis  at a concentration of 10 mg/ml mixed in a separate tube. The suspensions from both tubes were then mixed, vortexed, and transferred to a syringe. The suspension was then homogenized by moving the content back and forth between the two syringes for 60 min. The final volume of the suspension was drawn into a 1 ml sterile syringe with Luer-Lock tip and connected to a 26G needle. The suspension was prepared ex tempore on the day of immunization. On day 0, the rats were injected subcutaneously into both rear hind footpads with 0.1 ml of final emulsion (0.05 ml per footpad), yielding an immunizing dose of 0.25 mg/body of cardiac myosin per rat. The CFA emulsified with PBS was applied to the control group [Calcium-activated myosin from the porcine heart (ol group .n = 50) were randomly allocated into five different groups: CTRL, healthy nontreated rats; EAM, nontreated rats with myocarditis; and MOE50, MOE100, and MOE200, rats with myocarditis treated with three different doses  of ethanolic MOEs. Treatment involved daily per os application (every day at the same time) of MOE dissolved in distilled water, ex tempore (volume of 300 μl approximately). All animals were weighed during the protocol to adjust the MOE dose according to the rats' body weight.The rats  and spleen weight (Sw) to body weight (Hw/Bw and Sw/Bw) ratios.The rat's body weight was measured directly before Rat Tail Cuff Method Blood Pressure Systems (MRBP-R), IITC Life Science Inc., Los Angeles, CA, USA) twice, first at the beginning of the experimental protocol (day 0) in order to check the homogeneity of the animals, when no difference was found, and then after accomplishing the 3-week protocol before sacrificing the animals (day 21) [The systolic and diastolic blood pressures (SBP and DBP) and heart rate (HR) were measured by a tail-cuff noninvasive method BP system ((day 21) .in vivo cardiac function and the development of autoimmune myocarditis. The procedure was repeated twice, first at the beginning (day 0) to check the homogeneity of the animals and their health, when no difference was found, and then at the end of the experimental protocol (day 21) before sacrificing the animals. The animals were anesthetized with mixture of ketamine (75 mg/kg) and xylazine (5 mg/kg) intraperitoneally. Echocardiograms were performed using a Hewlett-Packard Sonos 5500  sector scanner equipped with a 15.0 MHz phased-array transducer as in our previous research [Teichholz formula [Transthoracic echocardiography was performed to assess the effects of MOE on research . From thocol day  before sShimadzu UV 1800 spectrophotometer, Kyoto, Japan). The blood samples were centrifuged in order to separate the plasma and obtain red blood cell (RBC) lysate suspension by washing isolated separated RBCs 3 times with ice cold saline. The following prooxidant parameters were determined from plasma samples: superoxide anion radical (O2−), hydrogen peroxide (H2O2), nitrites (NO2−), and index of lipid peroxidation measured as thiobarbituric acid reactive substances (TBARS). Antioxidant protection parameters were determined from erythrocyte lysate samples: the activity of catalase (CAT) and superoxide-dismutase (SOD) and the level of reduced glutathione (GSH).After completing the 3-week protocol, all animals were anesthetized by short ketamine and xylazine narcosis and sacrificed by decapitation. The blood samples were collected to determine oxidative stress parameters spectrophotometrically (2− with nitro blue tetrazolium (NBT). The protocol included mixing of 50 μl of plasma samples and 950 μl of assay mixture, followed by measuring on 550 nm in triplicate every 60 s [The quantification of superoxide anion radical was based on the reaction of Oery 60 s .2O2) determination method was based on the oxidation of phenol red with horseradish peroxidase enzyme. 200 μl of plasma sample was mixed with 800 μl of PRS (phenol red solution) and 10 μl POD (horseradish peroxidase (1 : 20)). Measuring was performed at 610 nm [The hydrogen peroxide (Ht 610 nm .μl of PCA (perchloride acid), 400 μl of 20 mM ethylenediaminetetraacetic acid (EDTA), and 200 μl of the plasma sample were mixed, put on the ice for 15 min, and centrifuged for 15 min at 6000 rpm. After separating the supernatant, 220 μl K2CO3 was added. Measuring was performed at 550 nm [Nitric oxide (NO) level was assessed indirectly by measuring nitrite concentration, since NO decomposes rapidly forming an equal amount of nitrite products. First, 100 μl sample and 400 μl trichloroacetic acid (TCA), which was then put on ice for 10 min and centrifuged for 15 min at 6000 rpm. Next, 1% TBA (thiobarbituric acid) in 0.05 NaOH was incubated with the obtained sample at 100°C for 15 min and after 10 min measured at wavelength of 530 nm [Index of lipid peroxidation in the plasma samples was estimated indirectly by measuring TBARS. First, TBA extract was made by mixing 800 f 530 nm .2O2 were used for CAT determination. Detection was performed at 360 nm [μl 0.1% EDTA, 400 μl lysate, and 750 μl precipitation solution . This was followed by mixing in the vortex machine and extraction on cold ice (15 min) and centrifugation at 4000 rpm (10 min). Distilled water was used as a blank probe. The level of GSH was measured at 420 nm and expressed as nanomoles per milliliter of RBCs [Obtained lysates containing about 50 g Hb/l were used to determine antioxidant enzyme activity. CAT buffer, sample, and 10 mM Ht 360 nm . SOD actt 360 nm , 24. The of RBCs .μm thick serial sections were stained by the H/E (hematoxylin/eosin) method for the purpose of morphometric analysis of cells and verification of morphological changes and by the Picrosirius red staining for collagen detection. Images of heart tissue sections were taken on an Olympus BX51 light microscope. Morphometric analysis of cardiomyocytes  was performed in the Axiovision image analysis program , where 100-120 cells per animal were analyzed [Image Pro-Plus programs . The analysis of cell infiltrate density and the collagen content was performed on 10 sections, of the total number of serial sections of the heart, with every 20th section of heart tissue analyzed, i.e., the distance between the analyzed plates was 100 μm. The results are presented as percentages. It is important to emphasize that no cell infiltrate was verified in the control group, and the value for infiltrate density was presented as 0%.The isolated hearts were measured and then cut into two halves so that the left and right halves of the heart were available for further histological analysis. The hearts were then fixed in 4% neutral paraformaldehyde, dehydrated in increasing alcohol concentrations , cleared in xylene, immersed in paraffin, and prepared for further analysis. 5 analyzed . The celIBM SPSS 20.0 was used for statistical data processing for Windows. The Kolmogorov-Smirnov and Shapiro-Wilk tests, histogram, and normal QQ plot tests were used to examine the normality of the distribution. Data are expressed as mean value (X) ± standard deviation (SD) and analyzed by one-way analysis of variance (ANOVA), followed by the Bonferroni test. A value of p < 0.05 was considered significant.p < 0.01). Additionally, medium and high doses of MOE (groups MOE100 and MOE200) significantly lowered heart weights and Hw/Bw ratio compared to both EAM and MOE50 groups (p < 0.05). The EAM group also showed a significantly increased Sw and Sw/Bw ratio compared to CTRL, while MOE100 and MOE200 significantly lowered these two parameters. The Sw/Bw ratio reduction was the most prominent in the MOE200 group .Immunized nontreated EAM rats and rats treated with a low dose of MOE (MOE50) were shown to have significantly increased heart weights and Hw/Bw ratio compared to the healthy CTRL group (00 group .p < 0.05), while no significant differences in this parameter were noticed between other groups. Also, no significant changes in DBP were observed between groups , while treatment with MOE100 and MOE200 (72.47% and 81.44%) markedly improved EF after 3-week supplementation compared to both the EAM and MOE50 groups. Only the highest dose MOE200 succeeded in normalizing EF to levels similar to CTRL values. A similar trend was observed in the FS parameter, which was significantly lower in EAM rats relative to CTRL, while all three doses of MOE led to an increment of FS compared to the EAM group (p < 0.05). The EAM group was also associated with LVPWd and LVPWs thickening compared to healthy rat hearts, all three doses of MOE led to reduction of these parameters, while MOE200 seemed to normalize these values to levels similar to control ones  was observed in EAM rats compared to the CTRL group (p < 0.05). However, treatment with MOE in all three doses succeeded in significantly decreasing the level of TBARS and NO2− (p < 0.05) compared to EAM, with no effect on the level of hydrogen peroxide. The level of superoxide anion radical was significantly lowered only by medium and high dose MOE100 and MOE200 compared to EAM. In addition, the highest and medium dose of the applied extract showed a more dominant effect on O2−, NO2−, and TBARS compared to MOE50 (p < 0.05), while MOE treatment improved antioxidant protection via an increase in all three parameters compared to EAM (p < 0.05). Medium and high doses of MOE significantly improved all three parameters compared to EAM and MOE50. Additionally, medium and high dose of MOE significantly improved SOD and GSH compared to the CTRL group (p < 0.05). No differences were observed between the MOE200 and MOE100 groups in all 3 measured parameters (p > 0.05). Low-dose MOE50 improved SOD and CAT compared to EAM to a lesser extent than MOE100 and MOE200 . Additionally, significant differences between different doses of MOE were observed, and a dose-dependent effect was noticed (p < 0.01).The preserved myocardial structure was observed in the CTRL group of rats. H-/E-stained sections of the EAM group of rats confirmed the presence of severe myocarditis characterized by massive inflammatory cell infiltration, destruction of myocardial fibers, swelling of cardiomyocytes, interstitial edema, and increased sarcoplasmic eosinophilia. However, MOE treatment especially MOE200 improved cardiac structure after 3-week treatment. EAM heart tissue sections showed a significant presence of inflammatory infiltrate compared to CTRL healthy hearts. Inflammatory infiltrate consisted of different leukocytes including mononuclear cells, polymorphonuclear neutrophils and multinucleated giant cells, which mainly infiltrated the epicardium of the ventricular wall. Namely, all three doses of MOE  significantly reduced inflammatory infiltrate density  compared to the EAM group . Additiop < 0.01), while 3-week MOE treatment succeeded to diminish these changes. All three doses of MOE significantly reduced the elevated collagen content compared to EAM rats  with the most prominent effect noticed in the MOE200 group which normalized the collagen content to CTRL values. Also, a dose-dependent effect of MOE treatment on collagen content was noticed (p < 0.05)  induced significant almost threefold increase (by 261.31%) in collagen content compared to the CTRL group of healthy rat hearts ( < 0.05) .p < 0.01). However, MOE treatment significantly decreased these two parameters compared to the EAM group. Medium and high dose of MOE showed superior effects compared to a low dose of MOE (MOE50 group) (p < 0.01). No differences in the measured parameters were noticed between MOE100 and MOE200 . The pathophysiology of this disease is very complex and not fully elucidated yet. However, it is known that excessive ROS production and subsequent oxidative stress induce the release of inflammatory cytokines and chemokines included in leukocytes' migration to the heart tissue. Additionally, oxidative stress may cause cardiomyocyte damage by necrosis or apoptosis . DCM inv2 receptors, blockage of Ca2+ and K+ channels, and slowing ventricular conductivity [2+ blockage, nitric oxide pathway, but also prostacyclin and EDHF pathways [Hemodynamic measurements implicated that the EAM group was associated with a severe drop of ejection fraction (EF) and fractional shortening (FS), LV wall thickening, and increased heart rate. With morphometric changes, heart enlargement by increased Hw/Bw ratio, and cardiomyocyte cross section area and longitudinal diameter increment in EAM rats, all of the above-mentioned indicates disturbed heart function and the beginning of characteristic myocarditis induced DCM. Similar findings are presented in other studies using the EAM model , 28. MOEuctivity , 30, 31.uctivity . Neverthpathways , 24.in vivo in the carrageenan-induced paw edema model [γ and downregulating NF-κB-mediated pathways [α and IL-17 and upregulation of anti-inflammatory cytokine IL-10 [Dark Agouti strain of rats, making it more comparable to our study. Other authors suggest that even in 10 mg/kg quercetin can protect the heart from EAM-induced damage via modulation of MAPK signaling cascade, more precisely by suppressing the myocardial endothelin-1 and also the mitogen-activated protein kinases (MAPK) [κB and ICAM-1 [In our study, induction of EAM was confirmed histopathologically by characteristic severe inflammatory infiltration and fibrosis of the heart tissues which is in line with other studies using the EAM rat model , 27, 32.ma model , but alsma model , 33. Evema model . Also, ipathways . Quercetne IL-10 . This sts (MAPK) . Potent s (MAPK) , 38. Ano2−, H2O2, NO2−, and TBARS and impaired antioxidant defense system . This is consistent with earlier studies that reported various oxidative stress marker elevations in this disease, such as superoxide anion [2−, TBARS, and NO2−, with strong antioxidant effect and free scavenging properties of this plant and its phenolic compounds being the most responsible for this effect. Various studies showed MOEs' ability to decrease oxidative stress in different cardiovascular models, even when using shorter time of exposition (7 or 14 days) than in this study [in vitro investigations on MOEs confirmed its strong free radical scavenging properties on DPPH, ABTS, O2−, and NO2− radicals, but also iron (II) chelating activity of that potentiates its antioxidant properties [An important aspect in the pathophysiology of autoimmune myocarditis is the link between inflammation and excessive ROS production, otherwise oxidative stress, which we also evaluated in this study. Three weeks post-immunization, the EAM group was associated with a significant release of prooxidants Ode anion , lipid pde anion , 40. MOEis study , 39. We operties , 42. Besoperties . The antoperties .Thus, oxidative stress ameliorating effect of the applied MOE treatment may also be one of the main mechanisms of autoimmune myocarditis improvement achieved by MOEs.M. officinalis effects in cardiac autoimmunity. However, additional experiments and future investigations are necessary and should help in revealing the exact mechanism of action of MOEs in EAM pathology. MOEs should be considered as a potentially helpful adjuvant therapy in patients with autoimmune myocarditis.In the light of these findings, the present study suggests that ethanolic MOEs improve cardiac function and myocardial architecture and mitigate oxidative stress, thus preventing heart remodeling, DCM, and subsequent heart failure connected with human giant cell myocarditis and EAM. However, the most prominent reduction of cardiac inflammatory infiltration, fibrosis, and oxidative stress with preservation of ejection fraction has been observed with the highest dose of MOE, 200 mg/kg. This study is the first to provide evidence on


Protein digests were analyzed by liquid chromatography–mass spectrometry. Differential abundance (DA) of proteins between groups was evaluated by Fisher's exact test with p < 0.0004 significance level after correction for multiple comparisons. DA was found between all groups (p < 0.0001): GF vs. normal (n = 130 proteins), coughing vs. normal (n = 22 proteins), and vomiting/regurgitating vs. normal (n = 20 proteins). Protein abundance was highly variable between dogs. Gastrointestinal-specific proteins were found in OP swabs from vomiting/regurgitating and coughing dogs but not from healthy dogs. In conclusion, the proteomic composition of the OP varies between health and disease. The presence of gastrointestinal-specific proteins in OP of coughing dogs may suggest reflux and/or aspiration as contributing factors. The variable protein abundance warrants investigation into biomarker panels.Reflux and aspiration in people cause and exacerbate respiratory diseases in the absence of gastrointestinal signs. Protein biomarkers in humans detect extraesophageal reflux (EER) from oropharyngeal (OP) and bronchoalveloar lavage samples. Reflux likely contributes to respiratory disease in dogs. The objectives of this study were to analyze the canine gastric fluid (GF) proteome and compare this to the OP proteome in normal, vomiting/regurgitating, and coughing dogs to identify biomarkers for EER/aspiration. Twenty-three client-owned dogs were enrolled. Canine GF samples ( Biomarkers diagnose, monitor, and identify populations at risk for disease. Biomarkers also have the potential to advance the understanding of disease pathogenesis by allowing an objective investigation of the efficacy of novel therapeutics –5. In reReflux of gastrointestinal contents is a source of acute and chronic pulmonary disease, with a prevalence of 50% in humans with chronic cough, asthma, chronic obstructive pulmonary disease, and idiopathic pulmonary fibrosis –10. ReflThe two major types of reflux implicated in the development of respiratory disease are gastroesophageal reflux (GER) and extraesophageal reflux (EER). Gastroesophageal reflux refers to reflux of gastric contents into the esophagus, while EER refers to refluxate reaching structures beyond the esophagus, including the oro- and nasopharynx, larynx, and airways . Reflux Human gastric fluid (GF) has been extensively characterized, allowing for the identification of reflux biomarkers outside the gut –18. GastThe objectives of this pilot study were to characterize the protein composition of canine GF using liquid chromatography–mass spectrometry (LC–MS) and compare the GF proteomic profile to that of the OP in healthy, vomiting/regurgitating, and coughing dogs to detect potential biomarkers of EER. We hypothesized that by using a high-coverage proteome analysis we could identify proteins relevant as potential biomarkers of canine extraesophageal reflux disease (EERD).via nasogastric or orogastric tubes in patients for whom these interventions were deemed medically necessary by the attending clinician. Sampling of gastric fluid from healthy dogs could not be ethically justified due to risk to the patient. Gastric fluid samples were collected prior to feeding (≥6 h). Oropharyngeal swabs were collected from healthy dogs, dogs with a history of regurgitation/vomiting, and coughing dogs. The healthy dogs had an unremarkable physical examination and no clinical evidence of gastrointestinal (GI) or respiratory disease within the preceding 6 months. The regurgitating/vomiting dogs were enrolled if they had a documented episode of regurgitation or vomiting within 12 h of sample collection. The coughing dogs were enrolled if they had a history of cough for >2 weeks without a clinical evidence of GI disease . Dogs were excluded from the study if they had a mixed clinical evidence of respiratory and GI disease or if they were receiving prokinetics, antacids, antibiotics, or probiotics at least 2 weeks before the sample collection. Dogs with a gag at the end of a cough paroxysm were not considered to have a GI disease and were therefore not excluded.Twenty-three companion dogs presenting to the University of Missouri Veterinary Health Center were prospectively enrolled with informed owner consent (University of Missouri IACUC #9154). Residual canine GF was collected directly via nasogastric or orogastric tubes and placed in a sterile red top blood collection tube. Samples were collected from unsedated dogs using minimum physical restraint needed for sample collection. Oropharyngeal swabs, collected by vigorously rubbing this region in each gently restrained dog meeting our inclusion criteria, were placed in 2 ml of sterile saline in a red top tube as mentioned earlier. The samples were gently vortexed and stored in polypropylene conical bottom Eppendorf tubes  at −20°C. All samples were evaluated within 2 weeks of collection.Two milliliters of GF was collected Proteome analysis was performed by the Gehrke Proteomics Center at the University of Missouri. Protein was extracted using four volumes of ice-cold acetone. Following washing with 80% acetone (in water), the pellet was re-suspended with 6 M urea, 2 M thiourea, and 100 mM ammonium bicarbonate. Protein was quantified using the EZQ assay according to the manufacturer's instructions (Invitrogen/Life Technologies). After quantification, the protein amount was normalized across all samples by dilution in urea buffer. Twenty-five micrograms of protein from each sample was digested with trypsin. The resulting peptides were desalted using C18 pipette tips (Pierce/Thermo), lyophilized, and re-suspended in 25 μl of acetonitrile/formic acid (0.1%). Ten micrograms of the re-suspended peptides was loaded on a C8 trap column  and then separated using a 400-nl/min 70-min gradient on a self-packed C18 column (75 um × 20 cm × 1.7 um particles—Waters BEH C18) at 50°C in the CaptiveSpray nanospray source. The mobile phase consisted of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B), and the LC gradient was for B: initially 2%, followed by 26-min ramp up to 17%, 17–25% over 36 min, 25–37% over 15 min, gradient of 37–80% over 6 min, and then held at 80% for 7 min, with a total run time of 90 min. MS + MS/MS data were acquired on a Proxeon Easy nLC system attached to an LTQ Orbitrap mass spectrometer using the parallel accumulation serial fragmentation (PASEF) (1) method over the 90-min gradient. Capillary voltage was set to 1,600 V, trapped ion mobility spectrometry (tims)-on, PASEF-on , and 100–1,700 m/z mass range. Cycle time was for 1 MS and 15 PASEF = 1.8 s (~120 MS/MS acquired per cycle). Repeated acquisition was threshold × 4 within 0.4 min. Active exclusion involved release after 1 min . MS data were collected over an m/z range of 100–1,700. During MS/MS data collection, each tims cycle included 1 MS + an average of 10 PASEF MS/MS scans.Canis lupis familiaris  was downloaded from NCBI using the search term “dogs.” Data were searched with the following parameters: trypsin (full) as enzyme, two missed cleavages allowed, six- to 150-amino-acid peptide length, 10 ppm mass tolerance on precursor ions, 0.6 Da on fragment ions, carbamidomethyl cysteine as a fixed modification, and oxidized methionine as variable. Spectrum grouper was used with the following parameters: precursor mass criterion, “same measure mass-to-charge”; precursor mass tolerance, 10 ppm; max RT difference (min), 1.5; allow mass analyzer mismatch, false; and allow MS order mismatch, false. The Percolator algorithm was used to automatically search a decoy database with the following criteria: target false discover rate  0.01, validation based on q-value. Proteins that passed this “high” confidence cutoff were examined further. Differential abundance (DA) of proteins was determined using PEAKS label-free quantitation based on the MS1 peak integration approach with the following parameters: algorithm: PEAKS-Q, retention time tolerance: 1 min, and mass tolerance: 40 ppm. Data were then filtered for 1% protein FDR with ≥1 unique peptide and ≥2-fold change. Spectral counting was used to determine differential protein abundance. Data were filtered for 1% protein FDR with ≥1 unique peptide and then >4 spectral counts per protein (mean of replicates). Protein abundance was described using the normalized spectral abundance factor (NSAF). Biological function was determined for the 200 most abundant proteins per group  as determined by NSAF. Biomarker candidates were evaluated according to their abundance in GF, tissue specificity, low or absent concentration in normal dog OP, and increased concentration in vomiting/regurgitating compared to normal dog OP. Candidate proteins were then assessed in coughing dogs to look for an evidence of EERD.The LTQ Orbitrap XL raw files were submitted to the Proteome Discoverer search engine for protein identification . The protein database for p ≤ 0.05 significance level. Principal component analysis was performed for all proteins identified across groups. Differential abundance of proteins between groups based on pairwise comparisons was evaluated by Fisher's exact test, with p < 0.0004 considered significant after correction for multiple comparisons. Coefficients of variation were calculated for each protein demonstrating a differential abundance between groups. A one-way analysis of similarity (ANOSIM) using the Bray–Curtis similarity index was used to test for similarity between groups based on the biological function of the detected proteins with p ≤ 0.001 significance level. Normality was evaluated by Shapiro–Wilk test. Post-hoc analysis (Dunn's method or Bonferroni correction for multiple comparisons) was applied where appropriate.Statistical analysis was performed using Past (version 3.21) and SigmaPlot (version 14.0) data analysis software. Descriptive statistics were applied where appropriate as median . Differences in patient demographics between groups  were evaluated by Wilcoxon signed rank test with n = 5), Labrador retriever (n = 3), Yorkshire terrier (n = 2), standard poodle (n = 2), and one each for Australian shepherd, Welsh corgi, basset hound, Jack Russel terrier, beagle dog, golden retriever, German Shepherd dog, Boston terrier, bull terrier, miniature poodle, and Catahoula leopard dog. Twelve dogs were spayed females, seven were castrated males, two were intact males, and two were intact females. No significant differences were detected between groups  for age, weight, or body condition score (BCS) (Twenty-three companion dogs were prospectively enrolled. The breeds represented included mixed breed (re (BCS) . These rre (BCS) .From all samples, a total of 504 individual proteins were identified . Within-p <0.0004). Seventy proteins were significantly greater in abundance in GF; 60 were significantly greater in OP swabs from normal dogs. The coefficients of variation between dogs ranged from 16.9 to 244.9 for the DA proteins.One hundred thirty proteins showed a significant DA (p < 0.0004). Thirteen proteins were significantly greater in abundance in OP swabs from vomiting/regurgitating dogs; seven were significantly greater in OP swabs from normal dogs. The coefficients of variation between dogs ranged from 45.5 to 264.6 for the DA proteins.Twenty proteins showed a significant DA (p < 0.0004). Twelve proteins were significantly greater in abundance in OP swabs from coughing dogs; 10 were significantly greater in OP swabs from normal dogs. The coefficients of variation between dogs ranged from 41.7 to 244.9 for DA proteins.Twenty-two proteins showed a significant differential abundance . Seventy-three percent of gastric metabolic proteins were pancreatic in origin, which is over-represented compared to salivary (14%), gastric (10%), and intestinal (3%) proteins (p ≤ 0.05). Pancreatic metabolic proteins were also increased in overall abundance compared to salivary, gastric, and intestinal metabolic proteins by NSAF. Pepsin A was identified in GF from all dogs but was less abundant compared to other non-gastric metabolic proteins (p < 0.05) as were proteins with an immunologic function (p < 0.05). The coughing dogs had increased numbers of proteins involved in cellular differentiation and stimulus response compared to normal and regurgitating/vomiting dogs (p < 0.05).For the biological function of proteins, all groups were found to be dissimilar on ANOSIM, with p ≤ 0.05 . The prip ≤ 0.05 . The metproteins . MetabolIn this pilot study, untargeted LC–MS was used to evaluate the canine GF proteome and identify several candidate biomarker proteins demonstrating DA in health and disease in the dog. Proteins specific to the GI tract were identified in the OP of vomiting/regurgitating and coughing dogs but not of healthy dogs. This suggests a potential role for reflux in the pathogenesis of respiratory disease in dogs and the possible utility of protein biomarkers as a screening tool for dogs with EERD and aspiration. Reflux and aspiration contribute significantly to the pathogenesis and progression of respiratory disease in people –9, 24. WUnderstanding the link between reflux and aspiration in clinical patients is critically important as people with a reflux-associated respiratory disease have demonstrated reductions in both disease exacerbation and declines in lung function following treatment targeting GERD and EERD . A similIdentifying dogs with naturally occurring GERD and EERD poses a diagnostic challenge as reliable, inexpensive, and minimally invasive diagnostic tests are lacking. In people, biomarkers have been effectively utilized as a part of a multimodal approach to diagnosing reflux and aspiration-associated respiratory syndromes (AARS) , 20, 34.Statistically significant DA for multiple proteins was identified between all groups compared to normal dogs. This finding suggests that the canine proteome differs not only by the site of collection [GF vs. OP ] but also in health and disease . The principal component analysis demonstrated an overlap between GF, vomiting/regurgitating, and coughing proteomes. Furthermore, GI-specific proteins were identified in dogs with vomiting/regurgitation as well as dogs with cough. No such proteins were found in healthy dogs. Though further studies investigating larger sample sizes are needed, this supports the hypothesis that GF biomarkers may be identified in vomiting/regurgitating and coughing dogs.in vivo and ex vivo, may have reduced the abundance of this protein below our limit of detection. The low abundance of pepsin A in GF and the lack of detection of pepsin A in OP swabs in vomiting/regurgitating dogs suggest this to be a less-than-ideal protein biomarker regardless of cause and represent a significant departure from the human literature (Gastric pepsin (pepsin A) is a component of the human gastric refluxate which has been used as a biomarker to identify patients with EER and aspiration , 20, 21.terature , 34–38. terature . Salivarterature . Increasterature . A limitThe presence of gastric proteins demonstrating an increased abundance in OP swabs from vomiting/regurgitating and coughing dogs compared to normal dogs opens doors for future biomarker validation studies in dogs with EERD and AARS. Important to future biomarker studies was the finding that the protein concentrations between dogs were extremely variable, echoing similar findings in people, thus suggesting limited utility for individual protein biomarkers and emphasizing the need for biomarker panels –43.Biological protein function was determined for the 200 most abundant proteins per group, and a statistical evaluation by ANOSIM demonstrated that the OP swabs from normal, vomiting/regurgitating, and coughing dogs were statistically dissimilar, representing discrete functional proteomic profiles. This suggests that the differences in composition between groups may be enough to alter the functional resident proteome in health and disease. In vomiting/regurgitating dogs, proteins with metabolic  functions were found in increased abundance compared to those in normal dogs. This may reflect a contribution from the GF proteome, though the overall functional proteomic profiles were still considered dissimilar between these two groups.MSV000087178.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: massive.ucsd.edu (proteomxchange), identifier The animal study was reviewed and approved by University of Missouri IACUC #9154. Written informed consent was obtained from the owners for the participation of their animals in this study.MG, CR, and HR contributed to the writing of the manuscript. MG and HR were involved with sample preparation and data analysis. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


An inverse association with ALS risk was noted for vitamin D  and for vitamin E . The sensitivity analyses illustrated similar trends. No causal effect was observed between essential amino acids and minerals on ALS. Our study profiled the effects of diet-derived circulating nutrients on the risk of ALS and demonstrated that vitamin D and vitamin E are protective against the risk of ALS, and LA is a suggested risk factor for ALS. Previous studies have suggested a close but inconsistent relationship between essential nutrients and the risk of amyotrophic lateral sclerosis (ALS), and whether this association is causal remains unknown. We aimed to investigate the potential causal relation between essential nutrients  and the risk of ALS using Mendelian randomization (MR) analysis. Large-scale European-based genome-wide association studies’ (GWASs) summary data related to ALS  and essential nutrient concentrations were separately obtained. MR analysis was performed using the inverse variance–weighted (IVW) method, and sensitivity analysis was conducted by the weighted median method, simple median method, MR–Egger method and MR–PRESSO method. We found a causal association between genetically predicted linoleic acid (LA) and the risk of ALS (OR: 1.066; 95% CI: 1.011–1.125;  Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease associated with muscle weakness and atrophy, and patients usually die as a result of respiratory failure within 3–5 years of symptom onset ,2. No trEssential nutrients accessible from the diet include essential amino acids, essential fatty acids, vitamins, and minerals. Supplementing essential nutrients is considered a promising preventive intervention for many diseases due to its ability to maintain metabolic homeostasis and to mitigate oxidative stress ,8,9. WitMendelian randomization (MR) analysis is a genetic statistical approach to estimate the causal association between an exposure and an outcome using single nucleotide polymorphisms as instrumental variables (IVs), which mimic the random assignment to groups of RCTs by naturally allocating alleles and can lessen residual confounding .In this study, we performed a two-sample MR approach to systematically decipher the causal association between nutrition status and ALS.Essential amino acids, essential fatty acids, essential minerals, and essential vitamins were considered essential nutrients. We searched PubMed to extract identified exposure-related SNPs from the most recent and largest genome-wide association studies (GWASs) based on the European population.SNPs related to amino acids were extracted from seven datasets, namely, isoleucine, leucine, lysine, methionine, phenylalanine, tryptophan, and valine ,23. AracWe obtained large-scale European-based ALS GWAS summary data from a recently published study, which included 27,205 ALS patients and 110,881 controls . All pat2 = 0.01, KB = 5000) with genome-wide association levels (p < 5 × 10−8) for exposure events; for exposures without genome-wide association significant IVs, suggestive level genome-wide association significant (p < 1 × 10−5) IVs were employed as proxies; (2) nonrare SNPs (MAF ≥ 0.01); and (3) proxies in strong linkage disequilibrium (r2 > 0.8) were employed to replace SNPs that failed to provide corresponding information for ALS. Proxy loci not included in the ALS dataset were excluded. The LDlink online tool [We set strict selection criteria for eligible IVs: (1) independent SNPs (rine tool  was empl2 = 2 × beta2 × (1 − EAF) × EAF/SD2, where EAF = effect allele frequency and beta = effect of each SNP on exposures [2 × (N − 2)/((1 − R2))) [To measure the phenotypic variation explained by each SNP, Rxposures . We simi − R2))) . Detailep > 0.05) was indicated by analogical estimates for each IV. For the MR–PRESSO method [Aiming to ensure the validity of the IVs, heterogeneity and directional pleiotropy were tested using Cochran’s Q test and the MR–PRESSO method. For Cochran’s Q test, nonsignificant heterogeneity  theIn this MR framework, the multiplicative random effect inverse variance–weighted method (IVW) was utilized as the main approach to analyze causality, which regresses the effect of SNPs on the outcome by the effect of the SNP on the exposure . To ensup values were adjusted according to the strict Bonferroni correction. p < 0.05/k was considered a significant level (where k is the number of exposures), and a p value between 0.05/k and 0.05 was suggestive of a positive association. All analyses were performed in R software version 4.1.0 by the “TwoSampleMR” package (version 0.5.6) [n 0.5.6)  and the n 0.5.6) .The 29 dietary-derived potential risk factors for ALS that we evaluated involved five traits related to amino acids, seven to fatty acids, eight to minerals, and nine to vitamins, including 2–168 SNPs as genetic proxies for each of them. All associations analyzed by IVW random-effects models are shown in p value = 0.037; tryptophan: Cochran’s Q test p value = 0). Due to the limited number of IVs in isoleucine, we cannot conduct further analysis. After removing the tryptophan outliers, we obtained analyzable results .In our restricted analysis of amino acid profiles and ALS risk, no association was noted between them . Howeverp value = 0.019). The robustness of this result was supported by the MR–Egger approach , and estimates using the weighted median method  and simple median method  also showed similar trends. No heterogeneity was detected in the individual SNPs associated with LA and the risk of ALS . No indication of horizontal pleiotropy was found based on the MR–Egger intercept test , and the MR–PRESSO analysis showed no outlying SNPs  without the influence of heterogeneity. Due to only two proxy SNPs being available for DGLA, sensitivity analyses could not be performed. The estimated effect of DGLA on ALS by each SNP is displayed in With regard to the effect of fatty acids on ALS, the results are summarized in ing SNPs . Scattering SNPs A. In addp value = 0.034). However, because of the removal of SNPs with a null association with the overall iron status, this calculation was only based on three SNPs.We observed no causal relationship between circulating essential minerals and the risk of ALS . The MR–p value = 0.025; and relative alpha-tocopherol, OR: 0.461; 95% CI: 0.340–0.626; p value = 6.25 × 10−7)  and weighted median method  underpinned the causal effect of 25OHD on ALS, while the estimates of the simple median method  and weighted median method  were aligned with the significant relationship between relative alpha-tocopherol. No heterogeneity was detected using Cochran’s Q test separately . The MR–Egger intercept test showed no evidence of directional pleiotropy , and no outlying SNP was found. The scatter plots indicated the SNP-based causal estimates of absolute 25OHD on ALS  . Both asD on ALS C and relD on ALS D. The LOD on ALS . No effeD on ALS . A null D on ALS .In the present MR study, we classified and profiled the potential causal role of essential dietary nutritiona6l modifiers on ALS risk using GWAS summary statistics. Higher genetically predicted levels of 25OHD and relative alpha-tocopherol were calculated to be associated with a reduced risk of ALS, and increased LA levels showed a suggestive positive association with ALS risk. Our findings implied that the abnormal levels of these nutrients were not only a symptom of ALS but also independent risk/protective factors for ALS. Among the essential amino acids and minerals, no evidence supports that additional supplementation has an influence on the risk of ALS.Essential fatty acids are important components of cholesterol and phospholipids and are involved in the maintenance of membrane fluidity and beta-oxidation . Our resAmong the vitamins, we found an inversely causal association between two fat-soluble vitamins, namely, vitamin D and vitamin E, and ALS, while water-soluble vitamins showed negative results. Vitamin D is a steroid molecule that is produced in the skin by exposure to the ultraviolet spectrum of sunlight or is obtained from food and biological dietary supplements . It has Vitamin E is another common essential vitamin , which mThis pioneering study provided evidence for the primary prevention of ALS from a dietary perspective. We found that ALS-associated essential nutrients were concentrated in polyunsaturated fatty acids and fat-soluble vitamins, providing supportive evidence of the importance of lipid metabolism in ALS. No evidence has shown that diet-derived circulating essential amino acids and minerals influence the risk of ALS in the general population, even though some of them (e.g., branched-chain amino acids , iron, zEvidence from our study demonstrated that vitamin D and vitamin E are protective against the risk of ALS, and LA is suggested to be positively associated with ALS risk. It is unlikely that a clinical benefit for the primary prevention of ALS will be achieved through supplementing essential amino acids and minerals. Our study classified and profiled the effects of specific diet-derived circulating nutrients on the risk of ALS, providing evidence for medical interventions for ALS in the general population from the perspective of gene–environmental interactions.


Cannabis sativa L.) is a crop bred and grown for the production of fiber, grain, and floral extracts that contribute to health and wellness. Hemp plants interact with a myriad of microbiota inhabiting the phyllosphere, endosphere, rhizoplane, and rhizosphere. These microbes offer many ecological services, particularly those of below ground biotopes which are involved in nutrient cycling, uptake, and alleviating biotic and abiotic stress. The microbiota communities of the hemp rhizosphere in the field are not well documented. To discover core microbiota associated with field grown hemp, we cultivated single C. sativa cultivar, “TJ’s CBD,” in six different fields in New York and sampled hemp roots and their rhizospheric soil. We used Illumina MiSeq amplicon sequencing targeting 16S ribosomal DNA of bacteria and ITS of fungi to study microbial community structure of hemp roots and rhizospheres. We found that Planctobacteria and Ascomycota dominated the taxonomic composition of hemp associated microbial community. We identified potential core microbiota in each community . We found 14 ASVs as hub taxa , and 10 BASV connected the root and rhizosphere soil microbiota to form an extended microbial communication in hemp. The only hub taxa detected in both the root and rhizosphere soil microbiota was ASV37 (Caulifigura coniformis), a bacterial taxon. The core microbiota and Network hub taxa can be studied further for biocontrol activities and functional investigations in the formulation of hemp bioinoculants. This study documented the microbial diversity and community structure of hemp grown in six fields, which could contribute toward the development of bioinoculants for hemp that could be used in organic farming.Hemp ( Plant microbiota are an important component that can influence essential plant functions positively or negatively. Better understanding of these host-microbe interactions has the potential to offer intervention tools to manipulate microbiota to enhance ecological services . Root-asCannabis sativa L. is an emerging crop for the production of fiber, grain, and floral extracts that contribute to health and wellness, but is also widely cultivated indoors with a variety of growing substrates, artificial light, and temperature control. Some initial studies have explored ways to maintain yield  was the prior crop. All rhizosphere soil and root samples were collected in three replicates per field. The root system of each plant was collected using a shovel (approximately 15 cm depth). Fine roots were then cut and put them in a 50 mL tube. Rhizosphere soils were also collected on site and put in a separate container. First, we separated shoots from roots. Roots were gently shacked to remove bulk soil. The remaining soil attached to the roots was collected in Ziploc bags and it was considered as rhizosphere soil  was used to quantify the extracted DNAs, which were then visualized using a 1 percent agarose gel and the GelDoc System . The bacterial 16S rDNA and fungal ITS regions were amplified by PCR. For the amplification of bacterial 16S rDNA, we used forward primer CS1_341 (5′-ACA CTGACGACATGGTTCTACACCTACGGGNGGCWGCAG-3′) and reverse primer CS2_806R (5′-TACGGTAGCAGAGACT TGGTCTGACTACHVGGGTATCTAATCC-3′), while fungal ITS region was amplified with CS1_ITS3_KYO2 (5′-ACACTG ACGACATGGTTCTACAGATGAAGAACGYAGYRAA-3′) and CS2_ITS4 , 0.25 μM of each primer, 1.5X PlatinumTM GC Enhancer, and 20 ng of template DNA in a 25 μL reaction volume. PCR reactions were run in an Eppendorf Mastercycler Pro S  with following cycling conditions: activation at 94°C for 2 min, followed by 35 cycles of denaturation at 94°C for 15 s, annealing at 60°C for 15 s, extension at 68°C for 20 s, a final extension at 68°C for 1 min with a hold at 10°C. Negative PCR controls without DNA were included in each PCR run. PCR products were visualized on 1% agarose gel stained by GelRed on a GelDoc system . Amplicons were sent to the Genome Quebec Innovation Centre for sequencing on an Illumina MiSeq sequencer  using 2 X 300 bp pair-end reads which were demultiplexed on the instrument.DNA extraction from roots and rhizosphere soils and PCR amplifications were carried out as described in J = H/In (S) where H is Shannon diversity index and S referred the total number of species in the dataset] using vegan package v 2.5-6 on R. Principle Coordination Analysis (PCoA) were calculated using Bray-Curtis distance matrix of Hellinger transformed counts using the R package vegan v 2.5.6. After the calculation of alpha diversity indices by ANOVA, we performed Tukey’s post-hoc tests to compare between hemp fields and sample types using package agricolae v1.3-3  in six fields in New York: Bluegrass Lane (BGL), Hudson Valley Lab (HVL), Long Island Horticultural Research and Extension Center (LIH), McCarthy CBD Trial (MC), McCarthy Stress Trial (MSC), and Wegman’s Organic Farm (WOF). Three plants per field, for a total of 18 plants, were sampled for root and rhizosphere soil. Illumina MiSeq produced a total of 11,617,362 pair-end raw reads . The number of reads per sample ranged from 41,561 to 98,764 for bacteria, and 34,891 to 106,235 for fungi. Using the DADA2 pipeline, 7160 bacterial and 3993 fungal ASVs were obtained. We also removed 26 ASVs from bacteria and 862 ASVs from fungi whose taxonomy belonged to mitochondria or chloroplast, leaving 7134 ASVs for bacteria and 3131 ASVs for fungi, respectively.We sampled root and rhizosphere soil of P = 1.81E-05) on bacteria across fields (P < 0.001), LIH (P = 0.02), MC (P = 0.008), MSC (P = 0.003), and WOF (P = 6.74E-06), and the LIH field was significant (P = 0.02) from WOF. The Pielou’s evenness index did not varied much but BGL field had the lowest mean evenness index and there is a significant difference (P = 0.04) of species evenness in the bacterial communities (P = 0.01). The Simpson diversity index is quite homogenous across fields  and fungal diversity . The Pielou’s evenness index also showed significant difference for sample type in the bacterial (Pielou P = 2.00E-05) and fungal communities  and fungal  community structure  found in root in WOF  and three of which were Unclassified Planctobacteria , BASV33 , BASV37 (Caulifigura coniformis), BASV57 (Unidentified Pirellulaceaes), BASV194 , BASV285 (C. coniformis), BASV322 (Unidentified Planctomycetes), and BASV425 (C. coniformis) were identified as hub taxa in the root (C. coniformis), BASV314 , BASV636 , BASV1686 (Pirellula sp.), and two fungal ASVs, FASV46 (Metarhizium marquandii) and FASV79 (Nectria ramulariae), were identified as hub microbes in the rhizosphere soil microbiome network (C. coniformis) was the only hub taxa found both in the root and rhizosphere soil  growing in six fields in New York’s Finger Lakes region  was unclassified at the genus level, indicating that the core hemp microbiota comprises bacteria that have yet to be isolated or documented. This resulted in a list of microbes that should now be prioritized for directed isolation or genome binning using sequencing to further understand their potentials in hemp. We observed very little evidence of core microbiota. We identified a limited list of microbes as core microbiota (four bacteria and three fungi), which is similar to recent field-grown hemp study, who identified five bacterial and fungal core taxa in the bulk soil . Fusariu. sativa . But theown hemp , not onl attacks . Beneficn canola , non-pater crops . HoweverChaetomium globosum as an indicator species in the rhizosphere soil of the WOF field  can be found below: all sequences are accessible in NCBI SRA database under the accession number BA: conceptualization, experimental design, processed data, and wrote the manuscript. LS: conceptualization, experimental design, sample collection, supervision, and manuscript editing. MH: concepts, design, supervision, and contribution to the manuscript writing. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Polyherbal formulations (PLFs) have been widely used for liver protection, treatment for hepatic dysfunction, and regeneration. They can also enhance appetite and protect the gastrointestinal tract from injury. In spite of the prevalent use, there is a need of scientific evidence on their effectiveness and safety. The objective of the present study was to assess the hepatoprotective effect of polyherbal formulations  in CCl4  on day 8. Twenty-four hours after CCl4 administration, the mice were monitored for the effects of PLFs on liver morphology, biochemical parameters including serum aspartate transaminase (AST), serum alanine transaminase (ALT), alkaline phosphatase (ALP), and total bilirubin. Phenobarbitone-induced sleeping time and histopathology changes in liver tissues were also monitored.  In this study, Swiss albino mice were treated for 7 days with distilled water or PLFs  followed by single subcutaneous injection of CCl4 administration caused significant hepatotoxicity as evidenced by marked elevation in AST, ALT, ALP, and total bilirubin. Phenobarbitone-induced sleeping time and infiltration of inflammatory cells and centrizonal necrosis on histological examination of liver demonstrated hepatic injury after CCl4 administration. However, the administration of Icturn and J-deenar polyherbal formulations at the higher dose significantly decreased the levels of AST, ALT, ALP, and total bilirubin. Moreover, pentobarbitone-induced sleeping time and histopathological analysis also revealed significant improvement as result of treatment with formulations Icturn and J-deenar.  CCl4-induced hepatotoxicity in mice, demonstrating their protective effect for liver. Our results confirmed that polyherbal formulations (Icturn and J-deenar) can significantly prevent CCl These free radicals are thought to interact with membrane lipids causing their peroxidation [γ-glutamyltransferase (γ-GT), the marker enzymes of hepatotoxicity, centrilobular necrosis, and steatosis, are the consequences of CCl4-induced lipid peroxidation [Liver injury can be caused by various undesirable chemical components like toxic chemicals , therapeutic agents , microbial agents  obesity, genetic defects, autoimmune disorders, chronic consumption of alcohol, certain herbs, hormones (such as birth control pills), and anabolic steroids –3. Hepatl models , 6. CCL4Almost all the allopathic medicines are being used to treat hepatotoxicity in which certain drugs like cholestyramine, ursodeoxycholic acid, spironolactone, loratadine, vasopressin, etc. have side effects including diarrhea, constipation, flatulence, abdominal discomfort, variable response and may cause encephalopathy in severe cases , 7. Due In the traditional medicine, plant extract formulation and combined extract of plants are used as therapeutics rather than single drug. There is a growing awareness in polyherbal formulation to treat various diseases including liver diseases worldwide and a good number of herbal formulations are well accepted for their hepatoprotective effect . About 64-induced hepatotoxic mice models. The objective of our present study is to evaluate the relative hepatoprotective effects of the above mentioned four polyherbal formulations based on the biochemical parameters and histopathology of the liver. Furthermore, phenobarbital-induced sleeping time (PST) was assessed to explore the physiological function of liver. It was evidenced that phenobarbital is generally metabolized through cytochrome p450 enzyme system in the liver. Drugs/chemicals that inhibit the cytochrome p450 enzyme will extend the duration of phenobarbital-induced sleeping time (PST) and vice versa [In the present study, four marketed hepatoprotective polyherbal formulations (PLFs), namely Heptaliv, Holyliv, Icturn, and J-Deenar, marketed in Bangladesh, were selected to evaluate their effect in CClce versa .ad libitum throughout the experimental period. All animals were subjected to euthanasia prior to surgery. The surgical procedure was carried out by anesthesia with 5% isoflurane in the presence of 100% oxygen. Animal handling and care for the present research was maintained according to the guidelines of National Institution of Health . The present protocol was approved by the Biomedical Research Center, University of Dhaka, Bangladesh (Ref. No. BMRC/EC/2016–17/97).Swiss albino mice  were used for the experiments. The animals were accommodated under standard environmental conditions (12 : 12 hr L : D cycle and 25 ± 2°C) with free access to standard food pellets and tap water 4 and phenobarbitone were procured from Merck, Germany, and Sigma Aldrich Chemicals, India, respectively. Rest of the chemical reagents and enzyme assay kits were obtained from Merck .In the present study, four polyherbal hepatoprotective formulations including Heptaliv, Holyliv, Icturn, and J-deenar were randomly selected from the local markets . These f4 challenge.The doses of polyherbal formulations (PLFs) for mice were calculated based on clinical doses as mentioned by the guideline of manufacturing company. Dose conversion  was done by utilizing a standard conversion table . It was n = 6 mice) were subjected for the evaluation of biochemical and histopathological parameters (n = 3 mice) were used for measuring phenobarbitone-induced sleeping time (4 subcutaneously (s. c.) in the lower abdomen in a suspension of olive oil in the ratio 1 : 1 v/v at the dose of 1 ml CCl4/kg BW of each animal.Animals were divided into 20 groups. Among all the groups, 10 groups , alanine transaminase (ALT), alkaline phosphatase (ALP), and bilirubin were tested. Serum bilirubin was determined by using a standard method using alkaline methanolysis of bilirubin followed by chloroform extraction of bilirubin methyl esters . The sepAfter administration of phenobarbitone, the sleeping time (in minutes) of animals was recorded between sleep and rev up time. The present protocol for investigation of sleeping time was slightly altered by administering phenobarbitone in substitution of hexabarbitone . In the p < 0.05). All statistics have been done using GraphPad Instat version 3.10.All data were expressed as Mean ± S.E.M. and represented in tables. The results were statistically analyzed using one way analysis of variance (ANOVA) followed by Dunnett's test. The differences between the control and treatment groups were tested in 95% significance level , ALT (168.10 ± 2.9 IU/L), and ALP (34.40 ± 3.1 IU/L) was significantly higher (p < 0.01) in comparison to normal control . The serum bilirubin level was significantly increased (p < 0.01) in the CCl4-treated group compared to the normal level (3.1 ± 0.10 vs. 0.3 ± 0.01). At the end of the study, the three polyherbal formulations namely Holyliv, Heptaliv, and J-Deenar significantly reduced AST at the higher dose (5.2 ml/kg), whereas Ictrum demonstrated significant reduction in serum AST level at both doses  when compared with the CCl4 treated group (p < 0.01) in the activity of ALT compared to the CCl4-treated group. On the other hand, there was a significant increase in ALT level (p < 0.01) in group treated with Holyliv as compared to the CCl4-treated control group (p < 0.05) as compared to the CCl4-treated control group. Nevertheless, group treated with Holyliv at the both doses showed no significant reduction in serum ALP level as compared to the CCl4-treated control group . However, treatment with the formulations namely Holyliv, Heptaliv, and J-Deenar at dose of 5.2 ml/kg/day for 7 days prior to CCl4 intoxication showed significant decrease (p < 0.05) in level of bilirubin as compared to the CCl4 control group, while Ictrum demonstrated significant reduction in serum bilirubin level at the both doses  when compared with the CCl4 treated group . A notable restoration in the phenobarbitone-induced sleeping time (p < 0.01) was observed with all the polyherbal drugs at the higher dose. However, the effect of Holyliv-treated groups insignificant at the dose of 2.6 mg/kg  had better hepatoprotective effects than other PLFs. They could significantly restore level of serum biochemical markers  in mice treated with CCl4 to the level of normal mice.Our present study has evaluated hepatoprotective effects of four commercially available PLFs from Bangladesh market in CCl4 causes hepatopathy along with the significant elevation in AST, ALT, ALP and bilirubin in serum as compared with normal mice [4 is metabolized by the liver into highly reactive metabolites which either directly or indirectly cause lipid peroxidation of the hepatocytes [Administration of CClmal mice . CCl4 isatocytes . Consistatocytes . Especiaatocytes .4-induced hepatoxic mice. This may result from the effect of Icturn on the stabilization of plasma membrane as well as the repair of hepatic tissue damage caused by CCl4 toxicity. A previous study has demonstrated that PLFs have hepatoprotective activity like an active compound silymarin, a flavonolignan from Silybum marianum [Icturn, a well-known commercial hepatoprotective PLF in Bangladesh, has been used as an effective treatment in liver diseases. Pretreatment with Icturn prevented the rise in liver enzyme parameters and kept them to near normal level when administered at 5.2 ml/kg BW/d dose in CClmarianum . The pro4 administration, it will cause longer mean duration of sleeping time as compared with mice with normal liver functions. Barbiturate will be poorly metabolized as a consequence of hepatic damage, which decreases in cytochrome P450s-mediated metabolic functional activity [4-treated mice. The results suggested that cytochrome P450s, which is the most critical xenobiotic metabolizing enzyme system in liver, are preserved by PLFs pretreatment in CCL4-challenged mice. It will be worthwhile to study the mechanisms how these PLFs protect cytochrome P450 in liver of CCl4-challenged mice.Phenobarbitone is a hypnotic drug that is metabolized generally in liver. When it is given in mice that have damaged liver caused by CClactivity , 26. Our4 treatment produced various changes in mouse hepatocytes, including centrizonal necrosis, cell inflammation, and infiltration of inflammatory cells. Pretreatment with Icturn and J-Deenar could prevent CCl4-induced changes in the hepatic structural design and protected the liver tissue from necrosis and degeneration (Our histological study revealed that CClneration . We specOur study demonstrated a comparative investigation on possible use of four commercial polyherbal formulations of Bangladesh for the prevention of liver injury. Finally, our findings revealed that Icturn and J-Deenar at the higher dose had better hepatoprotective effects than other PLFs. However, the mechanism in relation to hepatoprotective effects needs to be investigated further under various preclinical conditions.4-challenged mouse model, justifying their use as hepatoprotective agents in clinic. The efficacy of Heptaliv and Holyliv was low, and a dose adjustment may be necessary for their use in traditional medicine for human liver diseases.In conclusion, our study clearly demonstrated that PLFs Icturn and J-Deenar were the most effective in liver protection in CCL


Ananas comosus fruit peels using ecofriendly method steps. The characterization of the particles obtained was determined by using a UV-visible spectrophotometer (UV-Vis.), Fourier transform infrared spectroscopy (FTIR), X-ray diffraction diffractometer (XRD), Fourier scanning electron microscope (FESEM), and transmission electron microscopy (TEM). The nanoparticles showed maximum absorbance at 463 nm, measuring 11.61 in crystal nanosize, and presented spherical in appearance. An antimicrobial activity test was determined with the minimum inhibition concentration (MIC) method. The nanoparticles showed promising inhibitory activity on the Gram-positive and Gram-negative pathogen microorganisms  at low concentrations. The cytotoxic and growth inhibitory effects of silver nanoparticles on different cancer cell lines were examined via the MTT assay.Metallic nanoparticles are valuable materials and have a range of uses. Nanoparticles synthesized from plant wastes by environment-friendly methods have attracted the attention of researchers in recent years. Also, the advantages of biological resources and synthesis methods are attracting attention. In this study, silver nanoparticles were synthesized from  Nanomaterials synthesized by different methods, produced with metallic nanoparticles, are very valuable products that can be used in many areas. Nanoparticles show biological, optical, magnetic, and catalysis properties depending on their shape and size . Some fePhysical, chemical, and biological methods are used to synthesize nanoparticles . PhysicaFor nearly 5000 years, ancient civilizations used silver materials while consuming foods and beverages or storing them for a long time . EspeciaCancer, with its different types and difficult treatment process that is often insufficient, is a tough disease to cope with, in today's world . A lot oAnanas comosus fruit peels economically and easily via an ecofriendly method that does not involve toxic chemicals, characterize them, and examine their anticancer and antibacterial activities.Antibiotic resistance of microorganisms poses a serious problem. Thousands of people succumb to these microorganisms and die due to hospital pathogens being resistant to the antibiotics used . StudiesThe AgNPs were characterized by using the UV-visible spectrophotometer , Fourier transform infrared spectroscopy , X-ray diffractometer , Fourier scanning electron microscope , transmission electron microscope , and energy dispersive X-ray diffraction . A high-speed OHAUS FC 5706 model centrifuge  was used to precipitate the nanoparticles. Silver nitrate solution (2.5% (w/v) AgNO3 in H2O) and standard antibiotics  were commercially purchased .Ananas comosus fruits were cut and removed. After washing with tap water several times followed by distilled water, the peels were dried at room temperature. These dried peels were cut into smaller pieces and prepared to weigh. 200 grams of the dried fruit peels was weighed and left to boil in 500 mL of distilled water, half covered. From the moment it started boiling, we waited for about 15 minutes and left it to cool fully covered. First, coarse filter paper and, subsequently, a filtering process with Whatman 0.1 mm filter paper were used. The extract was stored at + 4°C for use in the synthesis phase. A solution of 10 mM (millimolar) concentration was prepared from the solid AgNO3.The peels of The prepared 500 mL plant extract and 10 mM solution were mixed in a 1000 ml glass flask at room temperature by using a magnetic stirrer. The solution was observed for color changes.Related to color changes, samples were periodically taken  and measurements were made with a UV-Vis spectrophotometer to determine the formation and presence of AgNPs. FTIR device frequencies were used for determining the functional groups responsible for the reduction. After the reaction, the dark liquid sample was centrifuged at 10.000 rpm and the precipitate was dried for other characterization processes. Crystal nanosize and structure were evaluated with XRD data. The FESEM, TEM, and EDX data were used to determine morphological structure and element composition.Staphylococcus aureus American Type Culture Collection (ATCC) 29213 and Escherichia coli ATCC25922 strains and Candida albicans yeast) and the Microbiology Research Laboratory of Artuklu University (Bacillus subtilis ATCC 11774 and Pseudomonas aeruginosa ATCC27833).Microorganisms were provided by the Microbiology Laboratory of İnönü University's Medical Faculty Hospital  concentration for each of the microorganisms grown from the plates there in solid form.C. Albicans yeast) antibiotics to compare the inhibitory effects of AgNPs.Muller Hinton broth for bacteria, Roswell Park Memorial Institute (RPMI) broth for yeast, and solutions containing different concentrations of AgNPs were added to 96-well microplates. Firstly, wells were designed and then a series of dilutions were performed. After the microorganism suspension prepared for each strain was added to wells, the same procedures were repeated for vancomycin (used for Gram-positive strains), colistin (used for Gram-negative strains), and fluconazole .Glioblastoma (U118), human colorectal adenocarcinoma (Caco-2), and ovarian sarcoma (SKOV-3) cell lines were commercially purchased from the American Type Culture Collection (ATCC). Cytotoxic activity applications on these cells were carried out at the Cell Culture Laboratory of Dicle University's Scientific Research Centre.2, 95% air, and humidity conditions. After the cells reached approximately 80% confluency, the cell was counted by using a hemocytometer and 104 cells were seeded per well of 96-well plates and subjected to overnight incubation. After the incubation period, the cells were treated with nanoparticles in concentrations of 200 μg/mL, 100 μg/mL, 50 μg/mL, and 25 μg/mL and incubated for 48 hours. After this period of waiting, the MTT solution was added to the plate wells, and 3 hours after incubation with MTT reagent, the medium was aspirated gently and 100 μl of DMSO was added to each well and incubated for 15 min at RT with gentle shaking. The absorbance of the microplates at 540 nm wavelength was measured using the Multi ScanGo, Thermo device.U118 and Caco-2 cells used were cultured in 75 t-flasks with Dulbecco's Modified Eagle's Medium (DMEM). Skov-3 cells were cultured in 75 t-flasks with the Roswell Park Memorial Institute (RPMI) 1640 medium. The cultured flasks were incubated at 37°C, under 5% COUtilizing these absorbance values, the concentration in which the percentage of the viability of AgNPs is inhibited on cells was calculated.U/C ∗ 100 [U is the absorbance of cells treated with AgNPs and C defines the absorbance values of control cells.% viability = /C ∗ 100 , 26, whet-test and ANOVA. P < 0.05 was regarded as significant.The experiments were performed in triplicate by means of the 3 solution [Holoptelea integrifolia plant extract, 460 nm maximum absorbance data were evaluated with the presence of AgNPs [Colour transformation from yellow to dark brown was observed one hour after mixing the plant extract and the 10 mM AgNOsolution . This cosolution . The maxsolution . In the of AgNPs . In anotof AgNPs .−1 and 1635.35 and 1634.97 cm−1 occurred, suggesting that the functional groups of–OH (hydroxyl) [FTIR data were evaluated to examine the functional groups involved in the formation of AgNPs. The frequency shifts that occurred between 3334.96 and 3338.80 cmydroxyl)  and C=O ydroxyl) , respectθ were found to be 38.14, 44.09, 64.44, and 77.40, respectively (D = Kλ/(β cosθ)) [D = particle size, K: constant value (0.90), X-ray wavelength λ value: 1.5418 Å, β: value of the peak at the maximum height (FWHM), and Bragg θ: angle of a high peak. In some studies, the crystal nanosize of AgNPs 14.58 nm [The X-ray diffraction values of the peaks (111), (200), (220), and 311) at 2ectively . Based o1 at 2ect14.58 nm , 12.63 n14.58 nm , and 2.114.58 nm .FESEM, TEM, and EDX analysis data were used to determine the morphological structures and element compositions of AgNPs. FESEM and TEM images showed that the AgNPs obtained were spherical, and the presence of substantially strong silver peaks  were detMetallic silver ions are inert in their dry state. They show highly reactive properties when they are ionized in water. Ionized silver contacts microorganisms via its electrostatic attraction force. This causes an increase in reactive oxygen species (ROS). The structure of the cell wall is disrupted by the increase in ROS. The structure of the cell membrane and nucleus membrane also deteriorates. Structures such as DNA and RNA have a high affinity for these species. By affecting the activities of these structures, they disrupt their functions and cause cell destruction and death , 36.Antimicrobial effects of AgNPs were tested on pathogens such as Gram-positive and Gram-negative bacteria and yeast. The inhibitive effects of silver nitrate, AgNPs, and standard antibiotics on tested microorganisms were compared. Based on these results, the AgNPs were found to be effective at least 2 more times than the antibiotics used in the treatment .Pistacia terebinthus extract were said to be effective on S. aureus, E. coli, and C. albicans microorganisms at concentrations of 0.04, 0.66, and 0.16 μg/mL, respectively [B. subtilis, S. aureus, and E. coli were reported in size 0.8, 6, and 6 μg/mL, respectively [μg/mL was effective for Pseudomonas aeruginosa ATCC 27853 [AgNPs obtained with ectively , 33. In ectively . In anotCC 27853 . In anotCC 27853 , 38.Ananas comosus peel extract on U118, CaCo-2, and Skov-3, cancer cells were examined via the MTT method (μg/mL, it showed an inhibitory effect of 81% depending on the % viability of 18.20 on the cell line. Increases in the percentage of viability in some cell lines during transitions to high concentrations were due to the proliferative features of cancer cells [The cytotoxic activities of AgNPs synthesized using T method . Accordier cells .+ release can induce cytotoxic and genotoxic formations in biological structures, and it is important to evaluate these effects [AgNPs show a strong oxidative structure. Ag effects .AgNPs interact with the cell surface and cause the cellular composition to deteriorate . AgNPs sμg/mL showed toxic effects on Caco-2 cells [μg/mL [In a study conducted to examine the toxic effects of AgNPs, a concentration of 3.75 -2 cells . In anots [μg/mL .Some features can have a significant effect on the toxicity of nanoparticles. Concentration, exposure time, burden, the chemistry of surface composition, degree of accumulation, shape, and size are some of these , 23.The different cytotoxic concentrations of AgNPs in the studies may be due to the synthesis of AgNPs from different sources or their different sizes and morphological structures.Antibiotic resistance is a serious problem. AgNPs will significantly contribute to the research for antimicrobial agents to solve this situation. Studies have demonstrated that AgNPs synthesized with biological sources show biocompatible properties.AgNPs are effective on pathogenic species in many studies. However, the toxic effects of their use should also be noted. Cytotoxic activity studies are significant data that will help eliminate this concern.Waste is among the ever-growing problems in our world. A wide variety of methods are being developed to use such waste in the fields and areas that will benefit people.μg/mL concentration showed 25–81% inhibition in different cancer cell lines. In addition, the anticancer effect of AgNPs obtained by green synthesis on the U118 cancer cell line was investigated for the first time in this study. The resulting AgNPs can be developed and used in the medical and pharmaceutical industries.In the study, AgNPs were synthesized in an easy, economical, and ecofriendly method by using the bioactive components in pineapple fruit peels to reevaluate waste. The obtained AgNPs were characterized via UV-Vis, FTIR, XRD, TEM, FESEM, and EDX data in which they exhibited at 463 nm maximum absorbance, 11.61 nm in crystal nanosize, and spherical appearance. The AgNPs showed antimicrobial effects at low concentrations. To examine the usability of these particles as an anticancer agent, their cytotoxic effects were examined, and it was determined that 25