Risperidone is a commonly prescribed antipsychotic drug with a potential side effect of weight gain. However, the pathophysiological mechanism is still poorly understood. Here, we sought to identify potential biomarkers of risperidone-induced weight gain by using a targeted metabolomics approach.We enrolled 30 subjects who received risperidone monotherapy for 8  weeks from a prospective longitudinal cohort study for drug-naïve schizophrenia patients. Plasma metabolites were measured by targeted metabolomics Biocrates MxP® Quant 500 Kit at baseline and 8-week follow-up.After 8  weeks of risperidone treatment, the levels of 48 differential metabolites were upregulated, including lysophosphatidylcholines (2), phosphatidylcholines (PC) (8), cholesteryl esters (CE) (3), and triglycerides (35), while 6 differential metabolites namely PC aa C38:6, methionine (Met), α-aminobutyric acid (AABA), TrpBetaine, CE (22:6), and Taurocholic acid (TCA) were downregulated. Interestingly, the reduction of PC aa C38:6, AABA and CE (22:6) was linearly related with increased BMI. Further multiple regression analysis showed that the changes of PC aa C38:6 and AABA were independent contributors of increased BMI. In addition, baseline levels of PC aa C36:5, CE (20:5) and AABA had positive relationships with the change of BMI.Our findings indicate phosphatidylcholines and amino acids may serve as biomarkers for risperidone-induced weight gain. Exclusion criteria included: (1) alcoholism or other substance (other than tobacco) abuse or dependence; (2) severe physical diseases; and (3) pregnant or lactating women. As a natural observational study, patients received antipsychotic treatment at the discretion of their clinicians. In the present study, 30 of the 200 patients (15.0%) were treated for 8 weeks with risperidone monotherapy. During the 8-week follow-up, the daily dose of risperidone was based on the doctor’s judgment. The combinations of benzodiazepines and drugs for the extrapyramidal side effects were acceptable. But subjects treated with other antipsychotics, valproic acid, lithium carbonate, carbamazepine, or antidepressants were excluded. All the participants were hospitalized and on a uniform diet. The main exercises were doing gymnastics and walking organized by nurses for 30 min a day.The study was approved by the Medical Ethics Committee of Tianjin Anding Hospital (Number 2017–03). All participants signed informed consent forms prior to participation.Clinical assessment and anthropometric data were obtained at baseline and 8 weeks. The severity of psychotic symptoms was assessed with the PANSS  by raterSubjects were fasted overnight before blood samples were obtained. The antecubital vein was collected as a baseline sample on the morning of day 0 of the study. The sample was also taken approximately 12 h after the last dose of risperidone at the end of 8 weeks. The plasma was frozen at-80°C until further analysis after centrifuging all blood samples at 1000 rpm for 15 min at 4°C.https://patents.google.com/patent/ EP1875401B1 and https://patents.google.com/patent/ EP1897014B1.The targeted metabolomic analysis of plasma samples was carried out by tandem mass spectrometry with the Biocrates MxP® Quant 500 Kit . Six hundred and thirty endogenous metabolites belonging to 26 biochemical classes, including amino acids, fatty acids, lysophosphatidylcholines, and phosphatidylcholines, have been quantified in the kit. A detailed description of experimental metabolomics measurement techniques can be found at via a 6,500 QTRAP® instrument .In brief, sample preparation was conducted using a 96-well device to quantify metabolite profiles. In this device, inserts have been impregnated with internal standards. And, a predefined sample amount has been added. Next, some analytes were derivatized with a phenyl isothiocyanate (PITC) solution, and after derivatization, the analytes were extracted by organic solvents and then diluted. Finally, the extracts were examined by Liquid chromatography–mass spectrometry (LC–MS/MS) and Flow Injection Analysis Tandem Mass Spectrometry (FIA-MS/MS) methods p < 0.05. The heat map and pathway enrichment analysis of differential metabolites were performed based on MetaboAnalyst 5.0 platforms.Data were imported into Biocrates MetIDQ™ software for calculation of analyte concentrations, data evaluation, etc. All metabolite concentrations were calculated in ng/ml and normalized. Variables missed in 50% or greater of all samples were removed, and the rest missing measurements were substituted with the arithmetic mean of all valid measurements for further statistical analysis. SIMCA software  was used for orthogonal partial least squares discriminant analysis (OPLS-DA) and variable importance in the projection (VIP) values were obtained. To evaluate the fitting of the OPLS-DA model, 100 permutation validations were carried out. According to the grouping of samples, T-tests were utilized to assess the significance of the variables. Differential metabolites were selected with VIP > 1 and T-test was for the comparison of clinical variables before and after treatment. We used Pearson correlation to evaluate the relationship between the changes of anthropometric parameters  and differential metabolites. A multiple linear regression analysis was used to assess the effects of several Independent variables, including age, sex, baseline BMI and the changes of differential metabolites on the change of BMI. Partial correlation analysis adjusting for age, sex, and baseline values to assess the effects of baseline levels of metabolites on the changes of anthropometric parameters . A two-tailed p < 0.05 was considered statistically significant.SPSS 23.0 was utilized to analyze the clinical variables. Paired The present study was composed by 30 patients with schizophrenia  who received risperidone monotherapy for 8 weeks. The average age was (40.47 ± 11.50) years and the average onset age was (36.40 ± 12.19) years. The shortest duration of illness was 1 month and the longest was 240 months, with an average of (51.77 ± 63.96) months. There were 7 patients who smoked and 7 patients with a family history of mental illness.p < 0.05). Similar results were found in BMI, WC, and HC. But there were no significant differences in TG, TC, HDL-C, LDL-C, or FPG (p > 0.05). It was noteworthy that 3 of 30 patients (10.00%) developed metabolic syndrome (MetS) which diagnosed according to the National Cholesterol Education Program Adult Treatment Panel (NCEP-ATP) III guidelines  experienced weight gain, of them 4 patients (13.33%) with weight gain ≥7% and 11 patients (36.67%) with weight loss/unchanged. The mean change in body weight from baseline was 1.35 kg. The difference between baseline and 8 weeks follow-up was statistically significant , AABA  and PC aa C38:6 . Only the change of PC aa C38:6 had a negative relationship with the change of HC . However, no correlation was found between the changes of WC and metabolites. Based on multiple linear regression analysis, the changes of PC aa C38:6  and AABA  had significant effects on BMI changes.Pearson correlation analysis indicated that the change of BMI was negatively correlated with the changes of CE (22:6) , but no correlation was found in WC and metabolites.Specifically, partial correlation analysis adjusting for age, sex, and baseline values showed that baseline levels of PC aa C36:5, CE(20:5), and AABA were positively correlated with the change of BMI , phosphatidylcholines (8), cholesteryl esters (3), and triglycerides (35), while 6 differential metabolites, namely PC aa C38:6, Met, AABA, TrpBetaine, CE (22:6), and TCA, were downregulated. Further analysis showed that these compounds were mainly divided into 3 clusters, which were TG and amino acid-related and phosphatidylcholines-related metabolites. Numerous studies have shown that phospholipids played a key role in the function and structure of membranes and phospholipids metabolism abnormalities occurred in patients with schizophrenia treated with antipsychotics, including the upregulated phosphatidylcholines classes . A metabPrevious metabonomics studies imply that phosphatidylcholines and lysophosphatidylcholines were closely related to overweight/obesity , 33. PhoStudies have reported that plasma amino acids were closely correlated with obesity and metabolic dysfunction , 41. DetIn addition, we also examined the correlations between the baseline metabolite levels and the change of BMI, the results indicated that the higher baseline levels of PC aa C36:5, CE (20:5) and AABA in drug-naive patients with schizophrenia, the higher degree of weight gain. This finding suggests that these metabolites can be used as biomarkers to predict weight gain after risperidone treatment.The present study has some limitations. First, although the patient’s diet was served by the nutrition canteen of our hospital, the amount of food intake and daily activity was not recorded. This might affect body weight and metabolites. Second, the relatively small sample size which reduced the statistical power. Third, as previous reported, clinically significant weight gain is defined as an increasing ≥7% of their baseline body weight . In our In summary, our study identifies that PC aa C38:6 and AABA might be particularly involved in the biological mechanism of weight gain after risperidone treatment. In addition, baseline levels of PC aa C36:5, CE (20:5), and AABA may be predictors of weight gain caused by risperidone. Our findings might provide preliminary insight into the underlying mechanism of weight gain after taking risperidone in drug-naïve patients with schizophrenia. Further exploration and verification of these biomarkers in a larger cohort would be of great significance.The datasets presented in this article are not readily available because it involves the personal privacy of the subjects and copyright for further use of data. Requests to access the datasets should be directed to corresponding author.The studies involving human participants were reviewed and approved by the Ethical Committee of Tianjin Anding Hospital. The patients/participants provided their written informed consent to participate in this study.JL designed the study and wrote the study protocol, and manuscript preparation. YQ, YD, and WS recruited the patients, performed the statistical analyzes, and drafted the initial manuscript. GL, ML,YZ, and CJ performed the clinical rating and collecting the clinical data. All authors contributed to the final drafting and critically revised the manuscript.This work was reported by Tianjin Key Medical Discipline  Construction Project, Award Number:TJYXZDXK-033A; Tianjin Key Medical Discipline  Construction Project of Tianjin Health Commission, Award Number:TJWJ2022XK039; and Tianjin Health Commission Project, Award Number: KJ20067.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.


Nucleocytoviricota are ubiquitous in ocean waters and play important roles in shaping the dynamics of marine ecosystems. In this study, we leveraged the bioGEOTRACES metagenomic dataset collected across the Atlantic and Pacific Oceans to investigate the biogeography of these viruses in marine environments. We identified 330 viral genomes, including 212 in the order Imitervirales and 54 in the order Algavirales. We found that most viruses appeared to be prevalent in shallow waters (<150 m), and that viruses of the Mesomimiviridae  and Prasinoviridae  are by far the most abundant and diverse groups in our survey. Five mesomimiviruses and one prasinovirus are particularly widespread in oligotrophic waters; annotation of these genomes revealed common stress response systems, photosynthesis-associated genes, and oxidative stress modulation genes that may be key to their broad distribution in the pelagic ocean. We identified a latitudinal pattern in viral diversity in one cruise that traversed the North and South Atlantic Ocean, with viral diversity peaking at high latitudes of the northern hemisphere. Community analyses revealed three distinct Nucleocytoviricota communities across latitudes, categorized by latitudinal distance towards the equator. Our results contribute to the understanding of the biogeography of these viruses in marine systems.Viruses of the phylum  Nucleocytoviricota, also known as “giant viruses”, are a diverse group of eukaryotic viruses with particle sizes typically larger than 0.2 μm in diameter and genome sizes reaching up to 2.5 Mbp [Algalvirales, Imitevirales, Pimascovirales, Pandoravirales, Asfuvirales, and Chitovirales, and up to 32 potential families [Imitervirales, Algavirales, and Pandoravirales infect a wide range of algae and various heterotrophic protists, members of the Asfuvirales, Chitovirales, and Pimascovirales infect a wide range of protist and metazoan hosts [Large DNA viruses of the phylum  2.5 Mbp –4. Knownfamilies , 7, and families –15. Gianan hosts –19. The an hosts , 21.Nucleocytoviricota were widespread in a variety of marine environments [4–106 viruses per milliliter of seawater, with higher abundances typically recovered during algal blooms [Although giant viruses are ubiquitous in the biosphere, they appear to be particularly abundant and diverse in marine environments. Early studies focusing on amplification and sequencing of the viral Family B DNA polymerase from seawater found that algal viruses within the ronments , 23, an ronments , 25. A rronments . Field sl blooms –32. Gianl blooms , 33, 34,l blooms –37, indil blooms . There iHere, we undertook a genome-based global survey of giant virus assemblages across the oceans and compare the diversity of these viruses in different geographic regions by leveraging the large number of metagenome-assembled genomes (MAGs) of these viruses that have been generated , 39–41. Nucleocytoviricota viruses, we constructed a phylogenetic tree of the viruses and 1188 Nucleocytoviricota reference genomes  of the 11 viruses found exclusively in GP13 were Imitervirales  and in the waters closer to the coast (eastern samples in transect GA10) where we observed a high abundance throughout the water column  were ses Fig. . Virusesmn Figs. ,  5C. Vicromonas , which hcromonas . Althougse hosts , 54.Fig.Imitervirales and Algavirales. We observed six MAGs within the Imitervirales (5 genomes) and the Algavirales (1 genome) that were particularly widespread in oligotrophic waters  , Chrysochromulina ericina virus (CeV), and Chrysochromulina parva virus (CpV) [Algavirales virus belonged to the Prasinoviridae family . The most broadly distributed genome, ERX556088.18.dc, a Mesomimivirus , was recovered in 122 samples  at 34 sampling locations. All the five Mesomimiviruses were extensively distributed in the Pacific transect GP13, while the prasinovirus, TARA_IOS_NCLDV_00011, was more widespread in the Atlantic Ocean  Table . The Mesus (CpV) –57. Otheus (CpV) , suggestean Fig. .Pimascovirales, Pandoravirales, and Asfuvirales were present to a lesser degree in the four transects, with average contributions of 9.1%, 1.5%, 0.3%, and 0.03% of giant virus occurrence, respectively. Mirusviruses were prevalent across all four transects   Fig. . The PanP < 0.001) , while in waters close to the coast at deeper than 200 m, members of the Algavirales are also present with relatively high occurrence and abundance, together with Imitervirales viruses  Fig. . In tran01) Fig. . In deepWe analyzed the giant virus communities along the transect GA02 in more detail to assess possible latitudinal gradients in giant virus diversity. The GA02 transect sampling sites follows the Americas-Atlantic Ocean coastline, spanning across a long range of latitudes from the parallel 50° North to 50° South and tracing a clear latitudinal gradient from the North Atlantic in the summer of 2010 to the south Atlantic in the austral summer of 2011. This sampling scheme may facilitate the detection of subtle latitudinal gradients that may be more difficult to resolve through comparison of samples collected across different ocean basins.Imitervirales communities, while varying marginally in the Algavirales communities for both viral genome richness and alpha diversity  harbored 93 unique genomes, while the mid-latitude zone (20° to 40° latitude) had 68 and the low latitude zone  had 24 genomes.In terms of community composition at each sampling location, the les Fig. . A non-mtor Fig. . The giator Fig. . This cltor Fig. , all of Mesomimiviridae and the Prasinoviridae. Complete or near-complete set of 9 giant virus core genes, including major capsid protein (MCP), A32-like packaging ATPase (A32), superfamily II helicase (SFII), family B DNA Polymerase (PolB), virus late transcription factor 3 (VLTF3), large and small RNA polymerase subunits , TFIIB transcriptional factor (TFIIB), and Topoisomerase family II (TopoII) were found in all of the Mesomimiviridae genomes, indicating that these are high quality genome assemblies  damage, and oxidative stress. We found genes involved in oxidative stress regulation, including thioredoxin, glutaredoxin, and superoxide dismutase (SOD) to be common among all genomes. Thioredoxin and SOD have been found expressed in several members of the rvirales , 76, 77 rvirales . It has rvirales , 79, 80.rvirales  to be prrvirales , 82, 83 We also identified various genes predicted to encode enzymes for synthesizing glycans, which may be involved in the decoration of capsids with sugar moieties. These viral-encoded fibril structures are potentially useful for viruses to extend their host range and persist in the open waters. First, the oligosaccharides may enable the modification of virion surface to mimic the host’s normal food source, e.g. organic debris and bacteria , 85, proImitervirales and Algavirales are particularly widespread and abundant in epipelagic waters. Giant virus communities vary markedly by latitude, and in the GA02 transect in the Atlantic Ocean we detected a latitudinal pattern of diversity that peaks at high northern latitudes and plateaus towards the south. Lastly, we identified five genomes of the Mesomimiviridae family of the Imitervirales and one genome of the Prasinoviridae of the Algavirales that are particularly widespread in oligotrophic waters. Our comparative genomic analysis revealed that these genomes encoded diverse genes involved in central carbon metabolism, stress responses, and lectin-domain proteins potentially involved in host-virus interactions. We hypothesize that these genes may collectively expand the host range of these viruses, possibly explaining their particularly broad distribution. Overall, our study provides genomic insights into the distribution of giant viruses in the ocean and sheds light on the biogeography of these ecologically-important community members.In this study, we conducted a metagenomic survey of giant viruses in the Atlantic and Pacific Oceans using the bioGEOTRACES datasets. We show that giant viruses of the orders Nucleocytoviricota genomes from the Giant Virus Database [Nucleocytoviricota, except those of the recently-discovered Mirusviricota lineage, which has a herpesvirus-like capsid and likely belongs to the realm Duplodnaviria. Although Mirusviruses represent a lineage distinct from the Nucleocytoviricota, we included them here because they represent a widespread lineage of marine large DNA viruses, and their genomes appear to be a chimera of different viral lineages, including the Nucleocytoviricota. To remove possible contamination from cellular sources, we screened all viral genomes using ViralRecall [Nucleoviricota and 111 Mirusviricota) for metagenomic read mapping.We downloaded 1382 Database  and 696 Database . All of alRecall  and remoalRecall  using anWe examined the metagenomic data from the >0.2-μm size fraction microbial communities of 480 samples collected by the international GEOTRACES program from May 2010 to December 2011. The samples were collected in four major cruise transects  across the Atlantic and Pacific Oceans at 2-10 depths in each sampling location, ranging from 6 to 5601 m. The collection time of each of the transects are as follows: GA02 (May-June 2010 and March 2011), GA03 (October 2010 and November-December 2011), GA10 (October-November 2010), GP13 (May–June 2011). Accession number, time and location of collection for each sample are listed in Table Nucleocytoviricota nucleotide sequences using coverM v0.6.1 (https://github.com/wwood/CoverM) in mode ‘genome’, with the parameter --min-read-percent-identity 0.95. We calculated relative abundance in reads mapped per kilobase of genome, per million mapped reads (RPKM). To avoid the false detection of viral genomes due to spurious read mapping, we only retained genomes with breadth coverage >20%  in subsequent analyses. This cutoff is based on recent work which suggested that a genome coverage of at least 20% is appropriate to indicate the presence of that genome in a sample [Nucleocytoviricota genomes for subsequent analysis.We downloaded and trimmed reads from each of the metagenome samples with Trim Galore v. 0.6.4 using parameters “–length 50 -e 0.1 -q 5 –stringency 1 --phred33”. We then mapped the trimmed reads onto the a sample . After tImitervirales order using a set of 7 marker genes: family B DNA Polymerase (PolB), A32-like packaging ATPase (A32), Poxvirus late transcription factor 3 (VLTF3), superfamily II helicase (SFII), alpha RNA polymerase subunits (RNAPL), TFIIB transcriptional factor (TFIIB), and Topoisomerase family II (TopoII). The concatenated alignment of these 7 markers was generated with the ncldv_markersearch.py script (github.com/faylward/ncldv_markersearch) and then trimmed with TrimAl v. 1.4.rev22 [Nucleocytoviricota as previously described [To provide phylogenetic context for the giant virus genomes that we identified, we constructed a multilocus phylogenetic tree of the .4.rev22  (paramet.4.rev22  with theescribed .https://cran.r-project.org/web/packages/vegan/). Variation among community composition was analyzed with NMDS ordination based on Bray–Curtis dissimilarity using the function ‘metaMDS’, parameters k = 2, trymax = 100. Statistical analyses of difference in community composition were performed using a PERMANOVA test with the ‘adonis’ function, 9999 permutations.Comparison of diversity among samples, especially alpha diversity, may be erroneous due to differing library sizes . To ensuWe performed interpolation of viral depth distribution using the program Ocean Data View v5.6.0  in DIVA Genes were predicted from genomes using Prodigal v 2.6.3 with default parameters, consistent with previous approaches , 96. We Supplementary FiguresTable S1Table S2Table S3


Periodontal disease is a ubiquitous disease in small animal veterinary medicine. Currently regular professional dental cleaning and daily tooth brushing are considered gold standards in the prevention of periodontal disease. Efforts to find a noninvasive, cost effective and easy to use preventative for periodontal disease are ongoing. The primary objective of this double-blind randomized clinical trial was to determine if a single application of silver diamine fluoride (SDF) 38% on the buccal surface of all teeth would reduce gingivitis within 3 months in dogs with stage two periodontal disease.Twenty-nine client-owned dogs 3–12 years old, 6–35 pounds were randomized 1:1 into active and placebo-control groups. Both groups underwent a baseline treatment and a three-month follow-up under general anesthesia. Gingival Index (GI), Plaque Index (PI), and Calculus Index (CI) were assessed and recorded at each event.A single application of SDF 38% did not significantly lower GI relative to the control group. However, the GI score dropped significantly in both groups relative to baseline, with a 53% reduction in the average GI score for dogs that received SDF 38% treatment and a 44% reduction for dogs that received placebo treatment. There were no differences in PI or CI scores compared to control groups.Further research is needed to determine if a more frequent application or a longer study duration would yield a different outcome. Periodontal disease is a persistent concern in small animal veterinary medicine. As many as 80 percent of dogs will have gingivitis or periodontitis by 2 years of age . Small bThe pathophysiology of early gingivitis and advanced stages of periodontal disease is a result of the complex interplay between bacterial pathogens and the host immune response  often exRegular professional dental cleanings combined with daily tooth brushing is the gold standard for maintaining oral and dental health . ProfessSilver diamine fluoride (SDF) 38% was cleared by the Food and Drug Administration in 2014 as a topical treatment in human dentistry . Its usePorphyromonas, a genus of gram-negative bacteria, is the most abundant species in both healthy and diseased dogs with a 2.7-fold increase in dogs with periodontal disease . Owners provided written informed consent. The study was reviewed and approved by the University of Washington Animal Care and Use Committee  after consideration of the risks and benefits.Enrollment and treatment were carried out at two facilities: Location site one: A dentistry/oral surgery referral practice, Inland Northwest Veterinary Dentistry and Oral Surgery, Coeur D’Alene, Idaho. Location site two: A general practice clinic, Lower Columbia Veterinary Clinic in Longview, Washington. Recruitment began on September 14, 2021, and the last follow-up data were collected on November 01, 2022.Enrollment in the study was based on visual oral examinations done on awake dogs at the recruitment examination. The included dogs were required to have no worse than stage two periodontal disease, as defined by the American Veterinary Dental College  Table 1Table 1 aTo be included the dog needed to have an American Society of Anesthesia score no greater than two . A minimA dog was excluded if systemic immunosuppressants, antibiotics or localized periodontal medications, including Doxirobe gel® (doxycycline hyclate 8.5%) had been administered within the past 3 months of beginning the study. Additional exclusion criteria included receiving over the counter 1-TetraDecanol Complex supplements, other VOHC approved supplements, treats, additives, or oral rinses that might impact the condition of the periodontium within the past 3 months. Dogs that received regular teeth brushing or those that were fed prescription dental diets within the past 3 months were also excluded. No dental therapies, other than those described in the study were permitted during the duration of the dog’s participation in the study.Prior to each session, a general physical examination including heart and lung auscultation, abdominal palpation, body temperature, oral health and body weight was performed and recorded. A brief history was collected from the owner and recorded to confirm each dog was clinically healthy and the owner had no new concerns following their recruitment visit/examination. Physical examination parameters and history were collected and recorded by the same operator (AK).2 and systolic/diastolic and mean arterial pressure. A Doppler was used to monitor systolic blood pressure in dogs under 10 kg for improved accuracy. Thermoregulation support was provided. Few variations of this anesthesia protocol were employed, and no emergency drugs were needed. All parameters and remarks were recorded on an anesthesia chart at each treatment session.The anesthesia protocol and patient monitoring were performed identically at each facility, as well as at each therapy session. Each dog received gabapentin 50 mg/mL; 5–7.5 mg/kg by mouth upon admission to the clinic the morning of their procedure to reduce anxiety. Premedication was a selection/combination of; Methadone 10 mg/mL: 0.2–0.4 mg/kg intramuscularly, or Butorphanol 10 mg/mL: 0.2–0.4 mg/kg intramuscularly and Dexmedetomidine 500mcg/ml: 2-4mcg/kg intramuscularly. Following appropriate time to onset of action for the selected premedication, a 20- or 22-gauge intravenous catheter was placed in the right or left cephalic vein. Maropitant 10 mg/mL: 1 mg/kg was given slowly over 5 min intravenously. The patient was preoxygenated with flow-by oxygen for 5 min prior to induction with Propofol 10 mg/mL: 4 mg/kg intravenously to effect. After induction, endotracheal intubation was performed, and general anesthesia was maintained with 2% Sevoflurane. Local anesthesia was utilized when teeth were extracted or when periodontal therapies were provided. Bupivacaine (5 mg/mL): 1-2 mg/kg total volume. Each patient’s vitals were monitored and recorded during general anesthesia. Parameters included electrocardiogram, respiratory rate, oxygen percent saturation, temperature, end tidal COModified Löe and Silness Gingival Index (GI), modified Logan and Boyce Plaque Index (PI) and the Warrick and Gorrel Calculus Index (CI) were collected  Table 2Table 2. Separate randomization lists were generated for each site by the study biostatistician. Dogs were randomized 1:1 to treatment arms.The owners, study personnel, including treating clinician, examiner and recorder, and the biostatistician were blind to treatment allocation.R, Elevate Oral Care LLC, West Palm Beach, FL). The comparator treatment was identical except that it contained neither fluoride nor silver. Test and placebo solutions were prepared by Cascade Chemistry, Eugene, OR and a certificate of analysis was obtained from the laboratory certifying solution contents.The active treatment was 38% aqueous silver diamine fluoride solution . The amount of Solutions A and B applied followed manufacturer recommendations. The buccal surface of each tooth was gently dried using the dental unit air/water syringe. To apply the solution, a manufacturer supplied microbrush was dipped into the solution and applied to the buccal surface of the crown of every tooth, with focus on the free gingival margin. The solution was left on the tooth surface for 1 min and then the surface was rinsed with distilled water for 1 min.Following data collection, a comprehensive oral examination including periodontal probing, notation of oral pathology, and review of intraoral dental radiographs taken prior to data collection was completed. Then, necessary therapies, limited to extractions, closed root planing and post-operative intraoral dental radiographs (if indicated) were performed by the same operator (AK). A professional dental cleaning including ultrasonic scaling, hand scaling of each tooth, followed by polish and a final oral rinse with dental unit air/water syringe was completed. Following completion of oral therapies, either Solution A or B was applied topically to the buccal surface of all teeth.Owners were instructed to not perform at home dental therapies, including tooth brushing until after the three-month follow-up. If extractions were done, owners were instructed to monitor for complications. A recheck examination was advised if signs of inflammation, infection, oral malodor, or discomfort developed.Three months, mean (SD) = 3.2 months (0.4), following the initial treatment each dog returned for the second session. Study parameters were collected under general anesthesia in the same manner as at baseline. Following data collection a professional dental cleaning including ultrasonic scaling, hand scaling of each tooth, followed by polish and a final oral rinse with dental unit air/water syringe was completed.If extractions were done at baseline treatment, confirmation of oral surgery healing was completed at the three-month follow-up. The owner was encouraged to initiate at-home dental care, with daily tooth brushing being the primary recommendation.The primary outcome measure was the GI score at 3 months post-treatment. A recent study by Mateo et al.  showed aThe GI, PI and CI scores for each dog were calculated as the mean score of all scored teeth. Descriptive statistics, including mean, standard deviation, minimum and maximum values, median and 25th and 75th percentiles, are reported by intervention for gingivitis, plaque, and calculus scores at baseline, and 3 months post-treatment. The distribution of the outcomes was assessed by the skewness, normal quantile-quantile plots, and the mean and median. The distributions were symmetrical, and outcomes were approximately normally distributed.Age and weight of the dogs at baseline were compared between treatments using Welch’s t-test and the number of teeth was compared using Fisher’s exact test. The outcomes were compared between treatments at baseline and 3 months using Welch’s t-test and within each treatment the change between baseline and 3 months was compared using paired t-tests. Linear regression with heteroskedasticity robust standard errors was used to compare outcomes at 3 months adjusting for baseline values of the outcomes, age, and weight and the follow-up time between baseline and 3 months . Additiohttps://www.R-project.org/.Similar analyzes were performed for plaque and calculus scores. A two-sided 0.05 significance level was used to determine statistical significance and 95% confidence intervals (C.I.) were reported for all treatment effects to describe the precision of the treatment effect estimates. All statistical analyzes were performed using R statistical software (Version 4.2.0). R Core Team (2022). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. URL n = 20), male (n = 10), weighing 6.25 to 34.6 pounds were enrolled. One dog was eliminated at the end of the study due to advanced periodontal disease found under general anesthesia, leaving 29 dogs in the final analysis. The number of teeth each dog had was similar between both treatment groups. Treatment solution A (n = 15) had five dogs with one or more teeth extracted (four dogs with one tooth extracted and one dog with three teeth extracted). Treatment solution B (n = 14) had five dogs with one or more teeth extracted (three dogs with one tooth extracted and two dogs with two teeth extracted). When groups were combined, the number of study teeth present ranged from 18/18 teeth in 83% of dogs enrolled (24/29) to 14/18 teeth in 3.4% of dogs enrolled (1/29) (n = 5), chihuahua/mix (n = 6), other (n = 18) including 19 females (18 spayed) and 10 males (7 castrated). Neither age, weight, sex, or breed was associated with GI, PI, or CI.Thirty dogs, 3 to 12 years old, female (d (1/29) . FifteenNine dogs were treated at Inland Northwest Veterinary Dentistry and Oral Surgery: 33% (5/15) of dogs were allocated to the SDF 38% treatment group and 29% (4/14) of dogs were allocated to the placebo treatment group at this location. Twenty dogs were recruited and treated at Lower Columbia Veterinary Clinic, 67% (10/15) of dogs were allocated to the SDF 38% treatment group and 71% (10/14) of dogs were allocated to the placebo treatment group at this location.p value = 0.82).The GI was similar between both groups of dogs at baseline following treatment group allocation and prior to application of either SDF 38% (Solution A) or placebo (Solution B). At baseline the GI for dogs who received SDF 38% was, mean (SD) = 1.7 (0.5), versus GI for dogs who received placebo treatment, mean (SD) = 1.8 . There was no difference between treatment groups when adjusted for baseline GI, age, weight, sex or breed and follow-up time .The GI remained similar between SDF 38% treated dogs and placebo treated dogs at the three-month follow-up examination. GI for dogs who received SDF 38% treatment, mean (SD) = 0.8 (0.4), and GI for dogs who received placebo treatment, mean (SD) = 1.0  with a 53% reduction in the average GI score for dogs that received SDF 38% treatment and a 44% reduction for dogs that received placebo treatment .The PI was also similar between both groups of dogs at baseline following treatment group allocation and prior to application of either SDF 38% or placebo solution. Dogs that received SDF 38% treatment had a PI of mean (SD) = 8.9 (2.0), and dogs that received placebo treatment had a PI of mean (SD) = 9.2  but the difference was no longer significant when adjusted for baseline PI, age, weight, and follow-up time .At the three-month follow-up examination the PI was lower in placebo treated dogs, mean (SD) = 7.7 (1.8), than in SDF 38% treated dogs, mean (SD) = 9.2  dogs and non-significant reduction of 16% for the placebo condition  (At the three-month follow-up examination there was non-significant reduction of 3% for SDF 38% treated (= 0.079) .p value = 0.16).At the baseline examination following treatment group allocation and prior to application of either SDF 38% solution or placebo solution, there was an insignificant difference in the CI for dogs that received SDF 38% solution treatment, mean (SD) = 5.9 (2.5), compared to dogs that received placebo solution treatment, mean (SD) = 7.4 . When adjusted for baseline CI, age, weight and follow-up time, the CI was also not significantly lower in the placebo solution treated dogs than in the SDF 38% treated dogs .At the three-month follow-up examination, the CI was lower in placebo solution treated dogs, mean (SD) = 2.5 (1.3), than in the SDF 38% solution treated dogs, mean (SD) = 3.2 (1.6), but the difference was not statistically significant  and placebo solution treated dogs , with a 46% and 66% reduction in the average CI score for each, respectively .No owners reported clinical signs indicative of oral discomfort or concerns with staining of teeth at the follow-up call the day after the baseline treatment. Oral surgery healing was confirmed at the three-month follow-up. Owners were instructed to reach out with concerns prior to this visit as needed. No oral surgery complications were reported in the current study population.Overall, no harms were reported, and no animals were withdrawn by the owner or exited by the attending veterinarians for medical reasons or harms associated with the study drug.The ability of SDF 38% to reduce gingivitis in humans is a growing area of interest , 21. ItsA possible explanation for our failure to find a primary treatment effect may lie in the current study’s design. The protocols utilized in the papers looking at efficacy in reducing gingivitis in humans employed more frequent application, up to once weekly for multiple weeks . This stSilver ions have both antimicrobial and bactericidal capacity. The silver ions remain unchanged after application in dead bacterial cells which act as a reservoir for the silver particles. When these ions are released, they are bactericidal to organisms in proximity . DespiteIt is unclear whether the modified Gingival Index for humans is reliable and valid in dogs. One such variable impacting reliability and validity of measurement is gingival pigmentation. A human dentistry study by Eid et al. found that the degree of melanin induced pigmentation in the gingiva has a negative correlation with the degree of bleeding on probing . HoweverThe results of the current study suggest that regular professional dental cleanings are an effective therapy in reducing gingivitis. This is in accordance with the current standard of care for veterinary medicine when managing periodontal disease , 42.Additional areas of interest include whether SDF 38% could be a beneficial treatment for enamel hypomineralization in the dog. Enamel hypomineralization increases the risk of dentin sensitivity, periodontal disease, pulpitis, pulp necrosis, and dental trauma . In humaIn the current study dogs were recruited based on awake visual oral examinations. The limitations of diagnosing the stage of periodontal disease accurately with an awake visual oral examination are known. Wallis et al. highlighted this by reporting a disparity between awake visual oral examinations versus oral examinations under anesthesia and the prevalence of periodontitis. The prevalence of periodontitis based on an awake visual oral examination reported levels of 9.3–18.2% whereas a higher prevalence of periodontitis ranging from 44 to 100% was found when the dogs were examined under anesthesia or post-mortem . The studies were conducted in accordance with the local legislation and institutional requirements. Written informed consent was obtained from the owners for the participation of their animals in this study.AK: Investigation, Resources, Writing – original draft, Writing – review & editing. TR: Project administration, Resources, Writing – review & editing. MLR: Data curation, Investigation, Resources, Supervision, Writing – review & editing. LM: Data curation, Formal analysis, Resources, Software, Validation, Visualization, Writing – review & editing. PM: Conceptualization, Funding acquisition, Project administration, Supervision, Visualization, Writing – review & editing.The study was sponsored by Elevate Oral Care LLC., West Palm Beach, FL. through an unrestricted donation to the University of Washington and direct payment of travel expenses for the investigators to the two sites. Elevate Oral Care provided the test and placebo solutions at no-cost to the study. No Elevate Oral Care personnel had any contact with the participants or had any role in the analysis or interpretation of the study data.PM is a member of Advantage Silver Dental Arrest, LLC.The remaining 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.


Delayed return to gut function and prolonged postoperative ileus (PPOI) delay recovery after colorectal surgery. Prucalopride is a selective serotonin-4-receptor agonist that may improve gut motility.versus placebo in patients undergoing elective colorectal resection. Patients with inflammatory bowel disease and planned ileostomy formation were excluded, but colostomy formation was allowed. The study medication was given 2 h before surgery and daily for up to 6 days after operation. The aim was to determine whether prucalopride improved return of gut function and reduced the incidence of PPOI. The primary endpoint was time to passage of stool and tolerance of diet (GI-2). Participants were allocated in a 1 : 1 ratio, in blocks of 10. Randomization was computer-generated. All study personnel, medical staff, and patients were blinded.This was a multicentre, double-blind, parallel, placebo-controlled randomized trial of 2 mg prucalopride versus 4 (3–5) days respectively (P = 0.124). Prucalopride improved the median time to passage of stool  but not time to tolerance of diet  or median duration of hospital stay . In patients who underwent laparoscopic surgery , prucalopride improved median time to GI-2: 3 (2–4) days versus 4 (3–5) days for placebo (P = 0.012). The rate of PPOI, complications, and adverse events was similar in the two groups.This study was completed between October 2017 and May 2020 at two tertiary hospitals in New Zealand. A total of 148 patients were randomized, 74 per arm. Demographic data were similar in the two groups. There was no difference in median time to GI-2 between prucalopride and placebo groups: 3.5 (i.q.r. 2–5) http://www.clinicaltrials.gov).Prucalopride did not improve time to overall recovery of gut function after elective colorectal surgery. Registration number: NCT02947269 ( This multicentre double-blind placebo-controlled RCT used prucalopride in patients undergoing elective colorectal surgery; 148 patients were recruited. Prucalopride did not improve overall time to return of gut function, but significantly improved time to postoperative gut recovery in patients who underwent laparoscopic surgery. Prucalopride was safe in this cohort. When delayed gut motility persists for 4 or more days after operation, this is called prolonged postoperative ileus (PPOI)2. PPOI is one of the most common complications after major colorectal surgery. PPOI occurs in around 10–25 per cent of patients who undergo elective colonic or rectal surgery, and leads to significant patient morbidity and increased healthcare costs. More than 50 per cent of patients who develop PPOI develop a further complication3, and patients with PPOI spend twice as long in hospital4. The inpatient economic burden of PPOI is estimated to be an additional €7880 per patient affected with ileus5, or €1.4 billion per year in the USA alone6.Delayed return of gut motility after surgery, or postoperative ileus, is a predominant cause of delayed discharge after elective colorectal surgery7. Postoperative ileus affects the entire gastrointestinal tract9, meaning that prokinetics which only selectively target the colon or stomach, for example, are unlikely to be effective. Recent evidence suggests that the pathophysiology of postoperative ileus is mediated by an initial neurologically mediated phase and then a delayed inflammatory response to surgery in the gut, and that this inflammatory response can be diminished by drugs that potentiate vagal nerve activity8–10.The inflammatory phase is thought to contribute to the development and duration of PPOI11.One of the key problems with delayed return to normal gut motility, and PPOI, is that there are few effective treatments, or preventative measures to reduce its burden12–15. A recent trial by Gong and colleagues16 showed that administration of prucalopride after elective colorectal surgery improved time to passage of flatus and stool. Recent studies have shown that prucalopride, when given before surgery and continued in the postoperative phase, reduces the inflammatory response associated with surgery17, and improves time to tolerance of diet18.Prucalopride is a serotonin-4-receptor agonist that increases presynaptic acetylcholine release from vagal neurones, improves colonic motility, and is a gastric prokineticThe primary aim of this double-blind placebo-controlled randomized trial was to investigate whether prucalopride administered before operation and continued afterwards improves time to return of postoperative gut motility in patients undergoing elective colorectal resection. The secondary aim was to determine whether prucalopride reduces the incidence of PPOI after elective colorectal surgery.ClinicalTrials.gov (NCT02947269).The study was approved by the New Zealand Health and Disability Ethics Committee, as well as by the Auckland District Health Board and Southern District Health Board Research Review Committees. The trial was registered prospectively with This multicentre, double-blind, parallel, placebo-controlled randomized trial was undertaken to investigate whether prucalopride improves time to recovery of postoperative gut function compared with placebo. Eligible patients were aged 18 years or older, who had elective colorectal surgery at one of two tertiary New Zealand hospitals  between October 2017 and May 2020. Patients and the public were not involved in the design or conduct of this study. There was no change to the trial methods after commencement.Patients who underwent right hemicolectomy, sigmoid colectomy/anterior resection, subtotal colectomy, Hartmann’s procedure, or abdominoperineal resection (APR) for colorectal cancer, diverticular disease, or volvulus, with or without colostomy formation, were included. All patients had to be able to give informed consent, and understand the risks and benefits of the study. All patients were seen before surgery in clinic, and given verbal and written information about the design, purpose, risks, and benefits of the study. Written consent was obtained from all study participants, and patients could withdraw themselves from the study at any stage.Patients were excluded if they had an ASA fitness grade of IV or higher, any allergy to serotonin-based medication, active inflammatory bowel disease, moderate-to-severe renal impairment (estimated glomerular filtration rate below 50 ml/min) or severe hepatic impairment, were pregnant, or were receiving preoperative intravenous nutrition (IVN). Patients in whom ileostomy formation was planned were excluded, as were those who had pre-existing gut dysmotility with endocrine, metabolic, or neurological causes. Patients who were unable to consent owing to dementia, cognitive impairment, language difficulties or delirium were not included.19, and then daily thereafter. Information on primary and secondary outcomes, blood tests, and complications were recorded on pre-prepared data collection sheets. Patients were telephoned to determine time to passage of stool, if this had not been achieved at the time of discharge. The primary outcome of this study was time to GI-219. Tolerance of oral diet was defined as the ability to eat a solid or semisolid diet of 25 per cent or more of preoperative meal intake without significant nausea or vomiting over two or more consecutive meals. Secondary outcomes recorded included: time until passage of flatus and stool, and tolerance of diet, preoperative and postoperative blood test results from days 1–3, including haemoglobin level, C-reactive protein (CRP) concentration, white cell count (WCC) and differential, renal function, and electrolytes. Duration of hospital stay, need for reoperation, and 30-day readmission rates were also recorded. Volumes or quantities of perioperative and postoperative intravenous fluid, analgesia, and antiemetic and anti-inflammatory medications were recorded. Adverse events were graded by system and severity using the Common Terminology Criteria for Adverse Events version 4 guidelines, and complications using the Clavien–Dindo classification system20. As a secondary endpoint, patients were also assessed for PPOI using the definition provided by Vather and colleagues2, as well as need for a nasogastric tube (NGT) or IVN. Specifically, patients were diagnosed with PPOI if they met two or more of the following criteria on or after postoperative day 4: nausea or vomiting, inability to tolerate an oral diet over the past 24 h, absence of flatus over the past 24 h, abdominal distension, or radiological evidence of ileus2. All patients also completed a Gastroparesis Cardinal Symptom Index (GCSI) questionnaire before surgery and daily until discharge21. The GCSI comprises nine patient-reported components, each rated in severity from 0 to 5, with a total score of 45. The questions involve three subscales . Higher scores indicate worse symptoms of gastroparesis.Demographic data were collected on patient factors including: age, sex, procedure, indication, co-morbidities, and ASA grade. Patients were assessed twice daily (0.800 and 20.00 hours) by a blinded study investigator until they achieved GI-2, defined as passage of stool and tolerance of oral diet22, and continued daily in the morning for up to 6 days, or until the patient had achieved the primary endpoint or was discharged from hospital. All patients undergoing elective colorectal surgery at Auckland City and Dunedin hospitals follow a structured enhanced recovery after surgery (ERAS) protocol. The ERAS protocol included preoperative oral carbohydrate drinks, early introduction of postoperative diet, stepwise analgesia progression, minimization of intravenous fluids, bowel preparation and opiate use, early patient mobilization, subcutaneous thromboprophylaxis, and omission or early removal of drains, lines, NGTs, and catheters.Patients were randomized by study investigators on the morning of operation to receive either 2 mg prucalopride or 2 mg identical placebo capsule, using a computer-generated randomization list prepared by an external pharmacy. The study medication was prepared by an external pharmacy, block randomized into groups of 10 in a 1 : 1 allocation and numbered sequentially. Participants, study investigators, and clinical staff were all blinded to the allocation. Only the external pharmacy had access to the unblinded study medication allocation data. The study medication was given 2 h before operation, so that prucalopride reached its peak plasma concentration at time of surgerya priori power calculation was performed using retrospective data from a previous prospective trial4 on postoperative ileus at Auckland City Hospital. Pilot data were extracted from 76 patients undergoing elective bowel resection without ileostomy formation, which showed a mean(s.d.) time to GI-2 of 4.9(2.6) days. An α value  of 0.05 was used and a β value of 0.2 , with a study power of 80 per cent. The hypothesis was that prucalopride would reduce the time to GI-2 by a clinically meaningful 25 per cent. Using the computer-simulated bootstrapping technique for power calculation23, and a two-tailed test in a 1 : 1 allocation ratio, it was calculated that 65 patients per study arm would be required. An expected 15 per cent drop-out rate was used, for a total recruitment of 150 patients across both study sites.An t test, and data that had an asymmetrical distribution using the Mann–Whitney U test. The χ2 test was used for categorical univariate analyses. All tests were two-sided and P < 0.050 was deemed significant. SPSS® for Mac® version 24  was used for statistical analysis.Statistical analyses were conducted on an intention-to-treat basis and no interim analysis was performed. Data were assessed for normal distribution using a histogram plot and the Shapiro–Wilk test. Normally distributed data were analysed using an independent-samples Student’s Fig. 1). One patient in the prucalopride group was withdrawn before operation by the surgeon owing to co-morbidity, and one in the placebo group was withdrawn as their operation was cancelled on the day. Therefore, 148 patients were analysed, 74 per study arm. No patients were lost to follow-up. Eight patients in the prucalopride group and nine in the placebo group discontinued treatment early (11 and 12 per cent respectively), mainly because they developed postoperative ileus. None of the patients who discontinued medication early were excluded from the analysis. Patients received preoperative medication a mean(s.d.) of 138(60) min before surgery, and two patients (1 in each group) did not receive the preoperative dose of study medication.Some 330 patients were assessed for eligibility, of whom 180 were excluded; 150 patients were randomized, 75 per arm, and one patient in each group was excluded after randomization  and Auckland Hospital 57 patients (38 per cent). Demographic data, including age, sex, ethnicity, BMI, ASA grade, and indication for surgery, were not statistically different between groups. The surgical approach  was also similar in the two groups. All APRs occurred in the prucalopride group (5 patients); the groups were otherwise similar in terms of type of colonic resection. Perioperative management was similar in the prucalopride and placebo groups. There was no difference in the use of spinal or epidural block: 46 per cent of patients in the prucalopride group had a spinal and 8 per cent an epidural block, compared with 43 and 7 per cent respectively in the placebo group (P = 0.870). Of patients who underwent laparoscopic surgery, two had an epidural block (1 in each group). There was no difference in the volume of perioperative crystalloid (P = 0.716), units of non-steroidal anti-inflammatory drugs (P = 0.882) or units of opiates (P = 0.598) between groups.The demographics the cohort are described in versus 4 (3–5) days respectively (P = 0.124). The time to tolerance of oral diet was similar between groups: 2 (0.5–3.5) versus 2 (0.5–3.5) days (P = 0.669). Patients who received prucalopride had a shorter time to passage of stool than those in the placebo group: 3 (2–4) versus 4 (2.7–5.3) days (P = 0.027). Median time to flatus was 2 days in both groups, but there was a statistical difference (P = 0.029), although this was not considered clinically significant. Table 2 shows a full statistical analysis of primary and secondary outcomes. The rates of PPOI, NGT insertion, and requirement for IVN were similar in the two groups. Duration of hospital stay was also similar at median 4 (2–6) days in both groups (P = 0.929). There was no difference between groups in terms of symptoms reported by patient using the GCSI questionnaire on postoperative days 1 to 4 .There was no clinically, or statistically, meaningful difference in time to GI-2 (passage of stool and tolerance of diet) between the prucalopride and placebo groups: median 3.5 (i.q.r. 2–5) Table 3. The rates of postoperative complications and adverse events were similar. There was no difference between the prucalopride and placebo groups in rates of anastomotic leak  and reoperation , or in readmission rates within 30 days (P = 0.597). The rate of cardiac complications was similar . Patients in the prucalopride group had fewer renal complications .Complications and adverse events in the prucalopride and placebo groups are summarized in P = 0.465), 2 (P = 0.529), and 3 (P = 0.860). Postoperative levels of CRP on postoperative days 1 (P = 0.544), 2 (P = 0.860), and 3 (P = 0.725) were also no different.There was no difference in WCC between groups on postoperative days 1 (P = 0.041) and stool (P = 0.006) faster than those who received placebo, but did not have a shorter time to tolerance of diet (P = 0.446) or GI-2 (P = 0.177), or a shorter hospital stay (P = 0.850). Because all the APRs occurred in the prucalopride group, a further analysis was undertaken to determine whether this had skewed the results. There was no difference in GI-2 (P = 0.244), time to tolerance of diet (P = 0.419), time to flatus (P = 0.062), or duration of hospital stay (P = 0.691). However, patients in the prucalopride group had a shorter time to passage of stool (P = 0.014).A subgroup, per-protocol, analysis was performed that excluded patients who did not receive preoperative medication (2) and those who had unplanned ileostomy formation (10). The results were consistent with those of the intention-to-treat analysis. Patients in the prucalopride group passed flatus . Patients in the prucalopride group achieved GI-2 a median of 1 day faster than those in the placebo group: 3 (2–4) versus 4 (3–5) days (P = 0.012). However, there was no difference in median duration of hospital stay: 4 (3–5) versus 4 (2.5–5.5) days respectively (P = 0.469). The incidence of PPOI (P = 0.478), need for NGT insertion (P = 0.375), and IVN (P = 0.833) were similar in the two groups. There was no difference between groups in postoperative inflammatory markers (WCC or CRP) on days 1–3, or in rates of complications and adverse events, among patients who underwent laparoscopic surgery.A P = 0.177) or stool (P = 0.687), but there was a trend towards delay to tolerance of diet in the prucalopride group compared with the placebo group: 6.3 (1.5–8.7) versus 3 (1.5–4.5) days respectively (P = 0.054). Median time to GI-2 was 6.3 (5.1–7.5) days in the prucalopride group versus 5 (3–7) days in the placebo group (P = 0.204). There was no difference between prucalopride and placebo groups in duration of hospital stay (P = 0.147), or in complications or adverse events, among those who had open surgery.An analysis of patients who had open surgery or conversion to an open procedure  showed no difference in time to passage of flatus , in adequate numbers to suggest that these results may be meaningful. Inflammation plays a key role in the development and prolongation of postoperative ileus11. The inflammatory response after laparoscopic surgery is significantly less than that after open surgery25. It is, therefore, possible that prucalopride was ineffective in patients who underwent open surgery owing to the marked increase in inflammatory response to laparotomy. Further studies assessing the differences in postoperative inflammatory markers are planned.A possible advantage of prucalopride was noted in the laparoscopic surgery cohort. Prucalopride significantly improved time to GI-2 by 1 day among patients who underwent laparoscopic or laparoscopically assisted surgery. This is a clinically meaningful reduction in time to recovery of gut function, but it did not translate to a difference in duration of hospital stay. It is important to note that this was a 11.The strength of this study is that it is a large RCT comparing prucalopride with placebo in patients undergoing elective colorectal surgery. Giving prucalopride before and after surgery is a novel feature of this work. Preoperative administration is important so that prucalopride is active at time of surgery, which is when the neurally and inflammatory-mediated mechanisms of ileus commenceThere are some limitations to the present study. Although there were differences in ileostomy and APR rates between groups, the authors do not believe these would have affected the primary outcomes, as they were accounted for in subgroup analysis. All APRs occurred in the prucalopride group, but there were few such procedures in this cohort. The number of patients who had an unplanned ileostomy was also low in both groups, and fell below the study’s drop-out rate of 15 per cent. The results of the per-protocol analysis (with ileostomy excluded) were consistent with those of the primary analysis.12 reported a reduction in postoperative interleukin (IL) 6, IL-8 and tumour necrosis factor α levels in intestinal samples from patients who received 4 mg preoperative prucalopride, but no difference in serum samples. However, increasing the prucalopride dose from 2 to 4 mg did not significantly improve outcomes for patients with chronic constipation27, and both 2 and 4 mg prucalopride were sufficient to improve gastric emptying times in patients with gastroparesis in recent series28. It is unclear whether patients would benefit from additional preoperative dosing to fully benefit from the anti-inflammatory properties of prucalopride, or to achieve a steadier state of the drug before operation.One consideration is whether an adequate dose of preoperative prucalopride was provided. Stakenborg and colleaguesPostoperative ileus remains a significant problem for patients and healthcare professionals after colorectal surgery. Although prucalopride was ineffective in improving time to GI-2 and reducing the rate of PPOI in the overall cohort, it had an apparent advantage in time to passage of stool for all patients, and significantly improved time to GI-2 in patients undergoing laparoscopic surgery. Prucalopride may therefore be effective in improving time to return of postoperative gut function in selected patients undergoing elective minimally invasive colorectal surgery.


Bce). Our results indicate the presence of high steppe ancestry in the Somogyvár–Vinkovci culture. They were then replaced by the Kisapostag group, who exhibit an outstandingly high (up to ∼47%) Mesolithic hunter–gatherer ancestry, despite this component being thought to be highly diluted by the time of the Early Bronze Age. The Kisapostag population contributed the genetic basis for the succeeding community of the Encrusted Pottery culture. We also found an elevated hunter–gatherer component in a local Baden culture–associated individual, but no connections were proven to the Bronze Age individuals. The hunter–gatherer ancestry in Kisapostag is likely derived from two main sources, one from a Funnelbeaker or Globular Amphora culture–related population and one from a previously unrecognized source in Eastern Europe. We show that this ancestry not only appeared in various groups in Bronze Age Central Europe but also made contributions to Baltic populations. The social structure of Kisapostag and Encrusted Pottery cultures is patrilocal, similarly to most contemporaneous groups. Furthermore, we developed new methods and method standards for computational analyses of ancient DNA, implemented to our newly developed and freely available bioinformatic package. By analyzing clinical traits, we found carriers of aneuploidy and inheritable genetic diseases. Finally, based on genetic and anthropological data, we present here the first female facial reconstruction from the Bronze Age Carpathian Basin.In this study, we report 21 ancient shotgun genomes from present-day Western Hungary, from previously understudied Late Copper Age Baden, and Bronze Age Somogyvár–Vinkovci, Kisapostag, and Encrusted Pottery archeological cultures (3,530–1,620 cal  Bce) and Bronze  Ages. A number of cultural transformations occurred in the Carpathian Basin, often attributed to population changes and genetic influxes. The observed shifts in ancestry compositions sparsely covered intensive or direct European HG introgressions into EEF or steppe ancestry groups  , their ay groups   or human leukocyte antigen (HLA) genes and pigmentation markers, are beginning to thrive , Kisapostag culture , and the Encrusted Pottery culture  that are referred to as Bk-I, II, and III phases in this study, respectively  and clinical variant analyses. Moreover, we present a series of bioinformatic tools for biological relatedness, ploidy, and variant analyses implemented in a new bioinformatic package for ancient DNA analysis. We analyzed archeological finds from the Balatonkeresztúr-Réti-dűlő site in Western Hungary (Transdanubia), where—among others—Bronze Age features and human remains were found during roadwork in 2003. Three Bronze Age archeological horizons were distinguished based on ectively  online. ectively . Howeverectively . AdditioBce), excavated ∼30 km away from Balatonkeresztúr, was added to the data set. Our data highlight not only detailed population events in a microregion but also reveal hidden processes that formed the genetic landscape of East-Central Europe at the beginning of the Bronze Age.In order to provide an additional data point to our analysis of population ancestry of the region, a Late Copper Age individual from the Baden culture  and whole mitochondrial DNAs (mtDNAs) of all individuals. The shotgun- and the capture-sequenced samples ultimately resulted in an average ∼144k SNPs/individual using the 1240k SNP panel for genotype calling  see Mat online. mobility .Bce). A majority of the samples came from the site of Balatonkeresztúr-Réti-dűlő. We sampled and sequenced 20 individuals, labeled S1–S45, skipping numbers that did not belong to the Bronze Age horizon or were not suitable for genetic testing. One male individual (S9) that was associated with Bk-I by 14C data has a very long (ultradolichocran) skull type, which differentiates him from most individuals in Bk-II and Bk-III, who have very short (brachycranic) skull types ; however, with Bayesian analysis using the OxCal software  were possibly slightly earlier (Bce) with skeletal remains of eight people of various ages. This is an unusual burial treatment as most bodies were cremated or buried as single inhumations in that culture. For detailed descriptions of the sites and burials, see We included only one Infans II (late childhood) individual (BAD002) from the site of Balatonlelle belonging to the early phase of the Baden culture  lineages provides rough estimates of genetic composition and is essential to assess biological relatedness and social structure for the studied population. Additionally, we performed phylogenetic analysis using MrBayes software  on mtDNAMPMR; fig. 1b) of Bk-II approximately follows the distribution of individuals in A–B grave groups (fig. 1a), which were likely established along family relationships and chronology. Individuals buried in the Bk-III mass grave only show a few blood relations (fig. 1c): a half-brother or uncle–nephew, a father–son, and a dizygotic twin; to our knowledge, the latter is the oldest detection of such relatedness. None of the distant inhumations  show biological relationship to any other individuals up to second degree. For further details, see Contrary to the diverse mtDNA makeup, most male individuals in Bk-II and Bk-III belong to the Y chromosome haplogroup I2a-L1229, except for two individuals that belong to haplogroup R1b-Z2103 . SimilarInvestigating genetic disorders in archeological data sets can be useful to improve our knowledge of the history of health and medicine and also highlights the overall genetic health of past populations. Genetic disorders, if accompanied with severe phenotypic anomalies, could also explain unusual burial practices, for example, as it was described in cases of dwarfism in the Byzantine era  or in thZ-score–Adjusted Coverage (ZAC) for ploidy estimates by using a set of reference genomes. This can estimate ploidy for samples as low as 0.01x average genomic coverage, enabling genetic sex determination and aneuploidy assessment for all new samples . After running the panel, we excluded low-coverage transitional variants from the final evaluation due to the possible presence of DNA damage. We only made exceptions when skeletal features supported the presence of the low-quality variant or when more than one sample possessed the same allele. Nevertheless, we listed all alternate variant hits in We also examined the nuclear genomes to find regions with clinical significance. Since a complete panel for determining disease susceptibility only exists in commercial DNA kits with nonavailable descriptions similar to the 1240k panel, we created a new SNP calling panel focused on various conditions including amyotrophic lateral sclerosis, Alzheimer disease, autism, Crohn's disease, diabetes, lactose intolerance, mental disorders, Parkinson disease, schizophrenia, and ulcerative colitis. For this study, we used a ∼5k set of clinically significant SNPs, which were marked as “pathogenic” or “likely pathogenic” in the ClinVar database , by ignoLig4 syndrome is a transitional mutation (rs104894421) induced disease with skeletal abnormalities , for whic), although some variants for lighter pigmentation exist within this group too. Members of Bk-III on the other hand show a wide range from dark to light pigmentation tones and even the presence of variants for red hair , Bk-I is clearly separated from Bk-II and Bk-III, where Bk-II has a strong shift toward European HGs overlapping with only a fraction of known ancient samples  for assessing genetic components revealed ∼17% HG, ∼40% EEF, and ∼43% steppe ancestry for Bk-I, similar to average Bronze Age Europeans –related population and Ukraine_EBA with almost equal contributions , whereas “dilution” of Bk-II to Bk-III is mostly driven by contact with various local populations, genetically best represented by later Transdanubian Hungary_LBA or Serbia_Mokrin_EBA_Maros (Maros culture) groups  with software  based onsoftware  and ADMIsoftware  analysessoftware . Accordisoftware a, Bk-I i samples  and Bk-Ianalyses b for assuropeans   groups  online.qpAdm analysis of the basic composition resulted in EEF ∼40 ± 2%, Eastern HG (EHG) ∼39 ± 3%, Western HG (WHG) ∼13 ± 2.7%, and Caucasus HG ∼8 ± 2%, P = 0.0917, in par with Admixture analysis, pointing toward a rather EHG characteristic composition. Next, we performed an f4 test in the form of f4 , but only marginal relations with the EHG (Lithuania_Mesolithic) populations , or Scandinavia, although we can see some weak connections to older (down to sixth millennium Bce) Lithuanian HGs. These results may reflect the population turnover in the sixth millennium Bce in the Baltics (qpAdm provides negative weights for this component when we model Bk-II as a combination of WHG (Loschbour_WHG), EHG (Lithuania_Mesolithic), EEF (Turkey_N), and Yamnaya (Russia_EBA_Samara_Yamnaya), suggesting that Lithuania_Mesolithic is not a good proxy for the actual EHG component.We aimed to investigate further the exact composition of HG ancestry in Bk-II. buti.DG) , to see buti.DG) . Contrar Baltics , suggestDATES  on all of the groups  moved northward from the region of today's Eastern Romania, Moldavia, or Western Ukraine, subsequently mixed with FBC- or Globular Amphora culture (GAC)–related populations and then split into two groups: one taking a route to Transdanubia and the other moving further North. These results are highly in par with 87Sr/86Sr isotopes to evaluate whether individuals were born in the area of their burial. The results  excluded the contribution of BAD002 to Bk-II  shows affinity to the Iberian Bell Beaker culture–associated individuals  online. to Bk-II  online.PAPline (Performing Archaeogenetic Pipeline), written in linux bash, R, and python v3.8.10 programming languages. One can use this package primarily to analyze next-generation sequencing data of archeogenomic samples, supplemented by tools, including ploidy test, MPMR relatedness analysis, and clinical variant test. The standalone tools and the core workflow of PAPline are available at https://github.com/ArchGenIn/papline. In the future, PAPline is aimed to be compared with the EAGER  and genetic continuity for hundreds of years at the studied site. Observable dilution of HG ancestry in Bk-III compared with Bk-II can be connected to continuous female-biased admixture with nearby communities according to our and previous genetic  and arch87Sr/86Sr isotope data show local values for both sexes, which along with similar genomic makeup of females and males suggest exogamy most probably between villages of the same population. The overlap between outlier parallels of Bk-II/III and archeological contact regions is also noteworthy, as it suggests smaller scale migrations of Kisapostag/Encrusted Pottery individuals or groups along trading networks, such as mobility possibly connected to wandering merchants.In both periods, the homogeneity of paternal lineages suggests a patrilocal residence system, similarly to previously described social organizations . HoweverBce Maros culture individuals from Mokrin, Serbia  of the uniparental lineages are the same at the haplogroup level with the individuals from the Croatian Encrusted Pottery culture Jagodnjak site, despite high similarities in cultural traits, social structure, and genomic composition of the communities . This su, Serbia , that shThe relatively limited presence of female and children burials in both Bk-II and Bk-III periods may suggest distinctive treatment or another (here undiscovered) burial group for women and children at the same site. However, in other cemeteries of the culture, for example, Ordacsehi and Bonyhád in Hungary, males, females, and children were buried close to each other, suggesting high variance in burial practices .Although low genomic coverages did not allow fine SNP recovery, we did find evidence for malignant variants within all of the tested groups and undoubtedly showed the presence of LHON and Jacob's syndrome within Bk-II. Whereas it only remains a possibility, the presence of autism risk factor in the CNTNAP2 gene, signs for severe bruxism, and the distinctive burial treatment of individual S45 suggest the actual onset of symptoms. Additionally, the disease panel we created and made freely available can be extended and used in future studies, providing insight into past population health qualities.87Sr/86Sr isotope analyses on the first and third molars of the individuals in the BK-III mass grave indicate that subadult males—including a severely disabled individual (S15) with hip dysplasia—left their community for a while and then returned to their birthplace prior to their death, raising further questions for future studies on prehistoric lifeways and social organization.Considering the unstructured age and biological relatedness distribution in the mass grave of Bk-III compared with Bk-II, the coetaneous death of eight people at least, the absence of traumatic or ritual events on bones, and the noncremated nature of the burial all signal a sudden tragic event in the Middle Bronze Age period (Encrusted Pottery population), most likely an epidemic, as first suggested based on the anthropological analyses . Careful87Sr/86Sr isotope measurements were performed in the ICER Centre, Institute for Nuclear Research Debrecen, Hungary, and at Quinnipiac and Yale University, Connecticut, USA  with AmpFLSTR Yfiler PCR Amplification Kit (Applied Biosystems), having one blank and one positive control in each reaction. The workflow followed the recommended protocol except the PCR cycles were increased from 30 to 34, and reactions were halved in volume. Two repeats were done where at least four markers yielded results. Data analyses were carried out in GeneMapper ID Software v3.2.1 (Applied Biosystems), and results are summarized in PAPline (https://github.com/ArchGenIn/papline). We used the GRCH37.p13 reference sequence to call the pseudohaploid genomes. For relatedness inferences, we applied the READ software (MPMR; see MrBayes v3.2.6 (BEAST v1.10.4 (Popgenome (R package (see Yleaf v1 (Network v10.1.0.0 and Network publisher v2.1.2.5 (ADMIXTURE v1.3.0 software (f statistics and qpAdm were performed using the admixr v0.9.1 (admixtools v2.0.0 (R packages. The timing of the admixture events was inferred by using the DATES software (Illumina sequencing paired-end reads were processed by the software  and a cus v3.2.6  and the  v1.10.4  softwareopgenome  R packagkage see  online. Yleaf v1  softwarev2.1.2.5  programsv2.1.2.5  online. v2.1.2.5  using thv2.1.2.5 ; for othv2.1.2.5  was usedv2.1.2.5  online. software . f statir v0.9.1  and the s v2.0.0  R packagsoftware .msad182_Supplementary_DataClick here for additional data file.


Fusobacterium and the reductions of some bacteria genera, including Faecalibacterium, Bifidobacterium, Ruminococcus, Subdoligranulum, Parabacteroides, Collinsella, and Olsenella, etc., were closely related to diarrhea associated with BRV and BCoV infection. Metabolites in the feces of BRV and BCoV-infected calves with diarrhea were significantly changed. Phosphatidylcholine , lysophosphatidylethanolamine , lysophosphatidylcholine  and LysoPE (0:0/18:0) were significantly higher in the feces of BRV-infected calves with diarrhea. In contrast, some others, such as desthiobiotin, were significantly lower. BRV infection affects glycerophospholipid metabolism and biotin metabolism in calves. Two differential metabolites were significantly increased, and 67 differential metabolites were significantly reduced in the feces of BCoV-infected calves with diarrhea. Seven significantly reduced metabolites, including deoxythymidylic acid (DTMP), dihydrobiopterin, dihydroneopterin triphosphate, cortexolone, cortisol, pantetheine, and pregnenolone sulfate, were enriched in the folate biosynthesis, pantothenate and CoA biosynthesis, pyrimidine metabolism, and steroid hormone biosynthesis pathway. The decrease in these metabolites was closely associated with increased harmful bacteria and reduced commensal bacteria. The content of short-chain fatty acids (SCFAs) such as acetic acid and propionic acid in the feces of BRV and BCoV-infected calves with diarrhea was lower than that of healthy calves, which was associated with the depletion of SCFAs-producing bacteria such as Parabacteroides, Fournierella, and Collinsella. The present study showed that BRV and BCoV infections changed the composition of the calf fecal microbiota and were associated with changes in fecal metabolites. This study lays the foundation for further revealing the roles of intestinal microbiota in neonatal calf diarrhea associated with BRV and BCoV infection.The changes in the composition of intestinal microbiota and metabolites have been linked to digestive disorders in calves, especially neonatal calf diarrhea. Bovine rotavirus (BRV) and bovine coronavirus (BCoV) are known to be the primary culprits behind neonatal calf diarrhea. In this study, we analyzed changes in the fecal microbiota and metabolites of calves with neonatal diarrhea associated with BRV and BCoV infection using high-throughput 16S rRNA sequencing and metabolomics technology. The microbial diversity in the feces of calves infected with BRV and BCoV with diarrhea decreased significantly, and the composition changed significantly. The significant increase of  The whole experiment process is in strict accordance with the National Standard Guidelines for the Ethical Review of Animal Welfare (GB/T 35892-2018).2.2.The calves involved in this study were from a large-scale dairy farm in Ningxia Hui Autonomous Region, China. The calves were artificially fed with 4 L colostrum within 2 h of birth and then transferred to the calf island for separate feeding. Calves are fed 2 L of pasteurized milk each morning and evening during individual feeding on the calf island. Calves are given free access to water and food during feeding. All calves were female, and no antibiotics and probiotics were used before sampling.Sample collection took place from June to September 2019. Calves with obvious diarrhea symptoms and healthy calves without any clinical symptoms within 30 days of age were selected as experimental subjects. The body temperature, respiration, and body condition of calves were examined before sampling. Fecal samples were collected from the rectum of calves using the rectum invasion method reported by 2.3.Escherichia coli K99, and Cryptosporidium spp. Firstly, antigen detection kits were used to detect BRV, BCoV, Escherichia coli K99, and BVDV in fecal samples . Subsequently, PCR was performed to detect BRV, BCoV, K99 Escherichia coli, BVDV, bovine norovirus (BNoV), and Cryptosporidium spp. in fecal samples (PCR primer information is shown in n = 16). Samples with a fecal score of 3 and only detected BRV were classified as group BRV (n = 8), while samples with a fecal score of 3 and only detected BCoV were classified as group BCoV (n = 8). A total of 32 samples were used for 16S rRNA gene sequencing. Afterward, eight samples were selected from the CK group, six samples were selected from the BRV group, and six samples were selected from the BCoV group for metabolome sequencing analysis , analysis .2.4.n = 32) using the E.Z.N.A. Stool DNA Kit . The quality and quantity of the DNA were assessed using NanoDrop 2000 ultraviolet–visible spectrophotometer . DNA was diluted to 1 ng/μL using sterile double distilled water. The V3–V4 hypervariable region of the bacterial 16S rRNA genes was amplified by PCR using primers . The Silva Database was used based on the Mothur algorithm to annotate taxonomic information for each representative read. Multiple sequence alignments were performed using the MUSCLE software (Version 3.8.31) to analyze the phylogenetic relationship of different OTUs and the difference between the dominant species in different groups. The OTUs abundance information was normalized using the sequence numbering standard corresponding to the samples with the least sequences. Alpha diversity and beta diversity analysis were conducted based on normalized data using QIIME software (Version 1.9.1). Chao1 index, Shannon index, and Simpson index were used to the identify alpha diversity of community. PCoA was performed to evaluate the differences between groups in species complexity based on the Bray-Curtis distance. Based on Spearman’s correlation coefficient to correlation analysis of differential microbiota between groups. The Phylogenetic Investigation of Communities by Reconstruction of Unobserved States  was used to predict the microbial metabolic function based on the 16S rRNA gene sequencing data.2.6.A total of 20 samples were used for untargeted metabolomic assays, including 8 CK group samples, 6 BRV group samples, and 6 BCoV group samples. The 50 mg sample was transferred to a centrifuge tube with 400 μL methanol: water solution  and then treated by a frozen tissue grinder  at 50 Hz for 6 min. Samples were ground and then extracted in a low-temperature ultrasonic at 40 Hz for 30 min and then incubated at −20°C for 30 min. After incubation, the samples were centrifuged at 4°C 13000 g for 15 min, and the supernatants were transferred to sample vials for LC–MS/MS analysis. Ten microliters of supernatant were injected into a UHPLC system  equipped with BEH C18  and coupled with a Thermo Q Exactive Mass Spectrometer equipped with an electrospray interface for analysis of fecal metabolites. The mobile phases consisted of 0.1% formic acid (v/v) and the acetonitrile: isopropanol  containing 0.1% formic acid (v/v). A gradient elution procedure was performed for sample analysis. The flow rate was 0.4 mL/min, and the column temperature was set at 40°C. The mass spectrometric data was collected using a Thermo UHPLC-Q Exactive Mass Spectrometer equipped with an electrospray ionization (ESI) source operating in either positive or negative ion mode. The raw data were imported into the metabolomics processing software Progenesis QI  for baseline filtering, peak identification, integration, retention time correction, and peak alignment. The relative abundance information of each metabolite ion in each sample was obtained after metabolite peak spectrum annotation, missing value filling, data normalization, QC sample RSD evaluation, and data conversion. Unsupervised principal component analysis (PCA) was performed to observe the overall distribution among the samples and the degree of dispersion among the different groups. Supervised Orthogonal Partial Least Squares Discrimination Analysis (OPLS-DA) was conducted to visualize differential metabolites among the different groups. KEGG pathway enrichment analysis was performed on the screened differential metabolites by Python software package scipy.stats.2.7.The samples for SCFAs measurement were identical to samples for non-targeted metabolomics analysis. The 100 mg sample was weighed in a 2 mL grinding tube with a grinding bead and 450 μL methanol solution. The 2-ethyl butyric acid was also added as an internal standard to the grinding tube. Supernatants were obtained by grinding, extraction, and centrifugation of the sample in sequence according to the aforementioned methods. One microliter of supernatant was injected into an Agilent 8890B-5977B GC/MSD system  equipped with HP FFAP capillary column  and electron bombardment ion source (EI) for GC/MS analysis. The split injection method was performed, and the split ratio was 10 to 1. Nitrogen was used as carrier gas at a flow ratio of 1.0 mL/min. The injection port temperature was 260°C. The column temperature was programmed at 80°C to 120°C at 40°C/min and from 120°C to 200°C at 10°C/min, and then held at 230°C for 1 min. The ion source temperature was 230°C. The quadrupole temperature was 150°C, the transfer line temperature was 230°C, and electron energy was set at 70 eV. The scanning mode was selected ion scanning mode (SIM). SCFAs were identified according to mass spectra and retention time of corresponding standards . The linear regression standard curves were done according to the concentrations of SCFAs standard in aliquots and the ratio of SCFAs peak area and internal standard peak area. The SCFA concentration of each sample was calculated from the standard curve. Based on the relative abundance of fecal microbiota and Spearman’s correlation coefficient of SCFAs content in feces to evaluate the correlation between fecal microbiota and SCFAs.2.8.T-test combined with the multivariate analysis OPLS-DA method was used to screen out the differential metabolites between groups. A 200-time permutation test was used to examine the fitting effect of the model. The SCFAs content in the two groups of samples was compared using the Kruskal-Wallis test in Origin 2021 (Version 9.8.0.200) and plotted using GraphPad Prism 8.0.1 (Version 8.0.1.244). The relationships between microbiota and SCFAs were analyzed using Spearman’s correlation test. All values were shown as mean ± standard error of the mean, and p < 0.05 was considered a significant difference.An analysis of alpha diversity indices  was performed to compare the different groups based on the Wilcox rank sum test. Permutational multivariate analysis of variance (PERMANOVA) was used to analyze differences in fecal microbiota structure between groups. A Wilcoxon test was used to analyze the difference in bacterial microbiota between groups. 3.3.1.A total of 2,042,707 valid reads were obtained from 32 fecal samples, with an average of 63,834 reads per sample . The samp < 0.01; p < 0.01; p < 0.01; The results of alpha diversity analysis showed that the Chao 1 indices of the BRV group were not significantly different from those of the CK group . While tp < 0.001). In contrast, the abundance of Actinobacteria and Euryarchaeota extremely significantly decreased , while the abundance of Actinobacteria extremely significantly decreased (p < 0.01), and the abundance of Euryarchaeota significantly decreased in the BCoV group , and the relative abundance of Subdoligranulum, Ruminococcus, Rikenellaceae_RC9_ gut_group, etc., significantly decreased in the BRV group , and the relative abundances of Collinsella, Fournierella, and Parabacteroides, etc., were decreased in BCoV group .At the genus level, The dominant bacterial genera in the CK group were um, etc. ,F. CompaFusobacterium was negatively correlated with Bifidobacterium, Faecalibacterium, and Parabacteroides in the CK group and significantly negatively correlated with Collinsella, Subdoligarnulum. Bifidobacterium was significantly positively correlated with Olsenella and negatively correlated with Parabacteroides and Erysipelatoclostridium. Subdoligarnulum was significantly positively correlated with Faecalibacterium and Fournierella. Subdoligarnulum showed a significant positive correlation with Faecalibacterium and Fournierella /16:1(9 Z)] and dethiobiotin, were enriched in glycerophospholipid metabolism and biotin metabolism pathways, respectively. In the BCoV group, there were seven differential metabolites that, including deoxythymidylic acid (DTMP), dihydrobiopterin, dihydroneopterin triphosphate, cortexolone, cortisol, pantetheine, and pregnenolone sulfate were enriched, respectively, in the pathways of folate biosynthesis, pantothenate and CoA biosynthesis, pyrimidine metabolism, and steroid hormone biosynthesis.3.3.p < 0.05; p < 0.05; Rotavirus and coronavirus infections differentially affect SCFAs content in calf feces. Compared with the CK group, the BRV group had significantly lower contents of total SCFAs, acetic acid, propanoic acid, and isohexanoic acid /16:1(9 Z)], LysoPE (0:0/22:0), LysoPC (P-16:0), and LysoPE (0:0/18:0) were increased significantly in the BRV infection-associated diarrhea group. Among them, PC [16:1 (9 Z)/16:1(9 Z)] was enriched in the Glycerophospholipid metabolism pathways. PC [16:1(9 Z)/16:1(9 Z)] is phosphatidylcholine (PE), while LysoPE and LysoPC belong to the lysophospholipid class of compounds. These compounds are also considered to be important components of the lipid droplet monolayers in mammalian cells. It has been reported that rotavirus induces the formation of lipid droplets in infected cells by upregulating the synthesis of lipid molecules, which may promote viral replication in host cells . In addia genera .Bifidobacterium, Faecalibacterium, and Subdoligranulum, as well as the over presence of Fusobacterium in feces. Reduction of dihydrotreterin and dihydrotreterin triphosphate can cause a deficiency of tetrahydrobiopterin (BH4), further resulting in impaired amino acid metabolism, and increased susceptibility to intestinal inflammation /DiMe ] and (R)-1-O-[b-D-Apiofuranosyl-(1 → 2)-b-D-glucopyranoside]-1, 3-octanediol were upregulated in the present study. PS [DiMe/DiMe] belongs to the phosphatidylserine class of organic compounds PS; . In mammammation . Metagen pathway . Panthen pathway . Accordi barrier . Previou barrier . The dec barrier . Pregnen barrier . Cortiso barrier . AldosteParabacteroides and Ruminococcus, etc., was closely associated with the reduction of acetic acid, while the missing of Parabacteroides, Ruminococcus, Fournierella, and Rikenellaceae_RC9_gut_group, etc., were closely related to the decreased contents of isohexanoic acid. However, a significant reduction in propanoic acid in the feces of calves with BCoV-associated diarrhea was positively associated with the depletion of Collinsella. It is also reported that Parabacteroides, Ruminococcus, Collinsella, and other bacteria genera can produce SCFAs well in the anaerobic environment of the intestine /16:1(9 Z)], LysoPE (0:0/22:0), LysoPC (P-16:0), LysoPE (0:0/18:0) and a lower concentration of dethiobiotin, which is closely correlated with Glycerophospholipid metabolism and Biotin metabolic pathway, respectively. There were two upregulated metabolites and 67 downregulated metabolites in BCoV infection-associated diarrhea group, which were closely correlated with the increase of harmful bacteria and the depletion of commensal bacteria. The downregulated metabolites were enriched in the pathways of Folate biosynthesis, Pantothenate and CoA biosynthesis, Pyrimidine metabolism, and Steroid hormone biosynthesis. The concentrations of some SCFA in BRV and BCoV groups were significantly lower than in healthy calves, which was associated with the depletion of SCFAs-producing bacteria.The diversity and composition of fecal microbiota were significantly changed in calves with diarrhea associated with BRV and BCoV infection. The significant increase of https://www.ncbi.nlm.nih.gov/, PRJNA928849.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found at: The animal study was reviewed and approved by Animal Experimental Ethical Review Committee of Ningxia University .SC, SG, and YY carried out the experimental design of this study. SC, QZ, and YY contributed to the experimental implementation and contributed to the article writing. SC contributed to the data analysis. SC, SG, YL, YM, and YY were involved in the sample collection and testing of this study. All authors contributed to the article and approved the submitted version.This research was supported by grants from Ningxia Hui Autonomous Region Key R&D Projects  and Natural Science Foundation of Ningxia (nos. 2022AAC03231 and 2023AAC03051).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.