Excessive activation of the nod-like receptor family pyrin domain containing 3(NLRP3) inflammasome plays a significant role in the progression of cardiac injury. In China, it has been well recognized that Chinese herbal medicine is markedly effective in treating cardiovascular diseases (CVDs). LuQi Formula (LQF) has been used clinically for more than 10 years and confirmed to be effective in improving cardiac function and inhibiting apoptosis. However, the specific mechanisms underlying its efficacy are mostly unknown. This study aimed to evaluate whether LQF could alleviate cardiac injury and apoptosis by regulating the NLRP3 inflammasome and the caspase-3/Bax pathway.  In this study, we investigated the effects of LQF on cardiac remodeling in a mouse model of myocardial infarction (MI) in vivo. n) of 10 mice in each group. Except the sham group, the other groups received left anterior descending (LAD) coronary artery ligation to induce MI and then treated with LQF, perindopril, or saline. Six weeks after MI, echocardiography was used to evaluate cardiac structure and function. Myocardial tissue morphology was observed by haematoxylin and eosin (H&E) staining, and heart samples were stained with Masson's trichrome to analyse myocardial fibrosis. Myocardial hypertrophy was observed by fluorescent wheat germ agglutinin (WGA) staining. The expressions of NLRP3, ASC, Cle-caspase-1, IL-1β, TXNIP, Cle-caspase-3, Bcl-2, and Bax in heart tissues were assessed by western blot analysis. mRNA expressions of ANP and BNP in heart tissues were measured by RT-PCR. The expression of reactive oxygen species in myocardial tissue was detected by using a DCFH-DA probe.  Forty male C57BL/6 mice were randomly divided into four groups: the sham group, the model group, the LQF group, and the perindopril group, with a sample size (P < 0.05), left ventricular internal diameter end diastole (LVIDd) and left ventricular internal diameter end-systole (LVIDs) were reduced (P < 0.05), and H&E and Masson's trichrome staining of cardiac tissues showed that LQF and perindopril could partially reverse ventricular remodeling and alleviate myocardial fibrosis (P < 0.05). WGA fluorescence results showed that compared with the model group, myocardial hypertrophy was significantly reduced in the LQF and perindopril group. We also found that LQF and perindopril reduce the oxidative stress response in the heart of MI mice. The protein expression of NLRP3, ASC, Cle-caspase-1, IL-1β, TXNIP, Cle-caspase-3, and Bax was downregulated in the LHF and perindopril treatment group, and Bcl-2 expression was upregulated.  Echocardiographic analysis showed that compared with the model group, the left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) in the LQF and perindopril group were increased (β cascade, decreased inflammatory infiltration, delayed ventricular remodeling, and downregulated caspase-3/Bax signaling, which can effectively reduce the apoptosis of cardiomyocytes. Perindopril showed the same mechanism. LQF and perindopril significantly attenuated cardiac injury and apoptosis in the MI model. In addition, we found that LQF effectively inhibited the activation of the NLRP3/ASC/caspase-1/IL-1 Chronic heart failure (CHF), characterized by high mortality and morbidity, is the final outcome of many CVDs . MyocardGrowing evidence suggests that an aseptic inflammatory response during acute myocardial ischemia (AMI) plays a crucial role in the formation of scar and remodeling of the damaged myocardium . An earlβ (IL-1β), thereby initiating the inflammatory response [β, cleaved caspase-1, and ASC was increased [Many studies have found that activation of the NLRP3 protein leads to an enhanced inflammatory response and cardiac dysfunction and remodeling, which aggravates acute myocardial injury in MI animals , 7. In cresponse . Recent response . Gao et response . Wang etncreased . TherefoAstragalus membranaceus, Codonopsis pilosula, Cassia twig, and, Semen lepidii, aiming at “warming kidney and Eliminating Dampness.” LQF can effectively relieve chest tightness, edema, and other uncomfortable symptoms of CHF patients. However, its effect on ventricular remodeling remains to be studied. As the cornerstone of clinical treatment of HF, angiotensin-converting enzyme inhibitors (ACEIs) improve ventricular remodeling [Traditional Chinese medicine (TCM) has been widely used in the prevention and treatment of diseases over the years and has been clinically validated as a good means to promote health. In China, it has been well recognized that TCM is markedly effective to treat CHF. LQF, an effective prescription to treat CHF, has been used widely in clinical practice; LQF  is an empirical compound prescription of several common medicinal herbs in TCM, composed of antler, safflower, modeling , 14. Hown = 10 for each group), Shanghai University of Chinese Medicine. All the animal experimentation was complied with the ARRIVE guidelines, and this study was conducted in accordance with safe animal testing specifications . All mice were maintained under a controlled temperature (20 ± 5°C) and humidity (55 ± 15%) with a 12 h light/12 h dark cycle. LQF dry powder, made by Shanghai Chinese Traditional Pharmaceutical Technology Co., Ltd. (China), was dissolved in distilled water to produce the equivalent of the crude drug concentration of 1.78 g/kg and was administered intragastrically. The gavage dose (1.65 g/ml) in the present study was determined according to the equivalent patient dose. Perindopril tablets (4 mg/tablet) were obtained from Servier Pharmaceutical Co., Ltd.  and used as a positive control. Perindopril tablets were crushed and dissolved in double-distilled water at a concentration of 0.06 mg/ml. The sham group mice received water. The starting dose for all mice was from the day after LAD artery ligation, and all mice were treated for 6 weeks.Male wild-type (WT) C57BL/6J mice (body weight 20–23 g), aged 8 weeks, were obtained from the Experimental Animal Center  in mice under anesthesia with isoflurane . Briefly, the mice were anesthetized using 1.0–2.5% isoflurane, and the heart rate was maintained at ∼450 to 550 beats/min. The heart was examined in the long-axis view at the papillary muscle level, and an M-mode echocardiogram of the mid ventricle was recorded. Cardiac function indices including LVEF, LVFS, LVIDd, and LVIDs were recorded .β , Bax, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) . Protein bands were visualized using the chemiluminescence system  for the required time. Quantitative analysis was performed using Image J software .Proteins were isolated from the frozen cardiac tissue, and their concentration was calculated using the bicinchoninic acid (BCA) method . Immunoblotting was performed as previously described . In thisμm-thick slices were cut for H&E staining to explore changes in heart size and for Masson's trichrome staining to visualize fibrosis [Heart tissues were isolated, rinsed with phosphate buffered saline (PBS), and fixed in 4% paraformaldehyde (PFA) over 24 h. Then, the tissues were dehydrated and paraffin-embedded. Next, 5 fibrosis . After sμm-thick slices were cut for IHC and IF analyses. Paraffin sections were dewaxed with water, and antigens were retrieved by heating in sodium citrate buffer. Endogenous peroxidase was removed by adding 30% H2O2, and an immunohistochemical pen was used to draw a circle around the tissue. Subsequently, 10% fetal bovine serum was added to block nonspecific binding to the tissue. The sections were incubated with NLRP3 and anti-IL-1β primary antibodies overnight at 4°C. Goat-anti-mouse secondary antibodies were then added and incubated at room temperature for 60 min, followed by washing with PBS three times for 5 min. Then, 30 μL of 4′, 6-diamidino-2-phenylindole (DAPI) solution was added and incubated for 10 min, followed by three PBS washes. For immunofluorescence staining, WGA was diluted to 0.1 mg/mL, added to the sections, and incubated at room temperature for 2 h. Sections were then washed three times with PBS for 5 min. Then, 30 μL of DAPI solution was added to the section and incubated for 10 min, followed by three PBS washes. A fluorescence microscope was used to observe and collect images in a darkroom. Semiquantitative analysis was performed in four randomly selected fields per section using Image J software.Heart tissues were isolated, rinsed with PBS, and fixed in 4% PFA over 24 h. The hearts were then dehydrated and paraffin-embedded. Next, 5 μm slices. After dewaxing to water, 20 μg/mL proteinase K was added to cover the tissue. Twenty minutes later, the sections were washed with PBS three times for 5 min, and TUNEL dye  was added and incubated at 37°C for 1 h. The slices were then washed with PBS three times for 5 min. DAPI was added to slices and incubated for 10 min. The slices were sealed with an antifluorescence quenching sealant. Fluorescence microscopy was performed in a darkroom for observation and image collection.Paraffin-embedded cardiac tissues were sectioned into 5 μm-thick sections were generated using a freezing microtome for ROS staining. Frozen sections were fixed at room temperature with 4% PFA for 5 minutes and washed three times with PBS for 1 minute each, and then, a tissue pen was used to draw a circle around the tissue. 5 μM 20,70-dichlorofluorescein diacetate (DCFH-DA) was added to the circled area and incubated at 37°C for 20 min, avoiding light, followed by PBS washes. DAPI staining was carried out for 10 min in the dark at room temperature. Slices were then washed with PBS three times for 5 min each. The slices were sealed with an antifluorescence quenching sealant. Fluorescence microscopy was performed in a darkroom for observation and image collection.Heart tissue was separated, washed with PBS, and rapidly frozen in liquid nitrogen. The tissue was then embedded with an optimal cutting temperature (OCT) embedding agent. After embedding, 6 The contents of MDA , SOD , and GSH  were determined by colorimetry. The assays were performed according to the manufacturer's recommendations.ΔΔCt values [Total RNA was isolated from individual hearts using commercial kits to evaluate mRNA expression of atrial natriuretic peptide (ANP) and brain type natriuretic peptide (BNP). Reverse-transcribed cDNA was obtained using the Total RNA rapid extraction kit . The primers used to amplify the genes are listed in t values .P < 0.05 was considered statistically significant.All results are expressed as the mean ± standard deviation (SD) and were analyzed using one-way analysis of variance (ANOVA) with the least significant difference- (LSD-) t's multiple comparisons, using SPSS version 20.0 software . ANP and BNP are sensitive markers of myocardial dysfunction, which correlate positively with the severity of HF. Thus, ANP and BNP are important indicators to determine the progression and prognosis of HF [Anp and Bnp mRNA expression were partly alleviated in LQF and perindopril mice compared with model mice  signaling molecules, leading to the increase of mitochondrial ROS and activation of NLRP3 inflammasome. To clarify whether the inhibition of the NLRP3 inflammasome by LQF and perindopril is related to the inhibition of ROS, we detected the ROS levels in the cardiac tissue Figures . The ROSTo identify whether LQF protects the postinfarct myocardium against apoptosis, we assessed apoptotic biomarker levels using western blotting and TUNEL staining. The results show that there was a significant increase in Bcl-2 and a decrease in Cle-caspase-3 and Bax levels on the border of the MI hearts after LQF and perindopril treatment Figures , suggestChronic heart disease is associated with a persistent low-grade inflammatory response that promotes adverse cardiac remodeling and is correlated with disease progression . CurrentAstragalus membranaceus, and atractylodesin III from Codonopsis pilosula, have shown anti-inflammatory effects in CVDs [β and downregulating the activity of caspase-1. The results suggested that LQF can be potentially developed as NLRP3-targeting medicine against MI-associated diseases.As a macromolecular complex, the NLRP3 inflammasome has been reported to be involved in many CVDs, including atherosclerosis, ischemia/reperfusion (I/R) injury, and HF . A growi in CVDs –35. In t+ outflow, lysosomal breakdown, and production of ROS. Mitochondrial dysfunction leads to changes in the ROS concentration, and the thioredoxin interacting protein separates from the thioredoxin and binds to NLRP3, inducing activation of the NLRP3 inflammasome [The common upstream mechanisms of the NLRP3 inflammasome activation are believed to include intracellular Kammasome . The finammasome –39. In tammasome . TherefoIn summary, our study demonstrates that LQF prevents ventricular remodeling after MI by regulating NLRP3 inflammasome. The application of Chinese medicine reflects advantages of simplicity and efficiency. Therefore, LQF should be used as a carrier to explore the mechanism of improving ventricular remodeling after MI.In this study, we only verified the effect of LQF on inhibiting the NLRP3 inflammasome and reducing myocardial apoptosis in animal experiments; therefore, which active component in LQF exerts these specific effects needs to be further clarified in our subsequent studies.β pathway activation, thereby alleviating cardiac inflammation, fibrosis, and apoptosis. We found that LQF demonstrated excellent performance in protecting cardiac function and ameliorating cardiac inflammation and apoptosis. Interestingly, we also found that perindopril reduced cardiomyocyte apoptosis by inhibiting NLRP3 inflammasome. The present study delineated the therapeutic properties of LQF and its value for subsequent research and clinical applications.In summary, we verified that, by inhibiting the production of ROS in cardiac tissue, LQF inhibits NLRP3/ASC/caspase-1/IL-1


Nocardia species are ubiquitous in natural environments and can cause nocardiosis. In the present study, the use of Resazurin salt and Spectrophotometry were proposed as alternative methods to reduce subjectivity in the interpretation of susceptibility results to antimicrobials by the broth microdilution method for Nocardia spp.Nocardia spp. isolates to Amikacin, Ciprofloxacin, Minocycline and Trimethoprim-Sulfamethoxazole was evaluated by Minimum Inhibitory Concentration (MIC) determinations by the broth microdilution method. To verify cellular growth, the colour-changing dye Resazurin was applied, the Optical Densities were measured on a spectrophotometer, and both were compared to Clinical and Laboratory Standards Institute (CLSI) Gold Standard method . Percentages of essential and categorical agreements and interpretative categorical errors were calculated within each method (intra-reading) and between them (inter-reading). The Gold Standard visual reading demonstrated 100% of essential and categorical intra-reading agreements for Amikacin, and there was no error when compared with the alternative methods. For Ciprofloxacin, the comparison between the Gold Standard and the Spectrophotometric reading showed 91.5% of essential agreement. In the categorical intra-reading analysis for Minocycline, there were 88.1 and 91.7% in the Gold Standard and in the Spectrophotometric readings, respectively, and 86.4% of concordance between them. High rates of categorical agreement were also observed on the Trimethoprim-Sulfamethoxazole analyses, with 93.7% for the Gold Standard, 84.9% for the Resazurin readings, and 80.5% between them.The susceptibility of The alternative methods with Resazurin and Spectrophotometric readings showed high agreement rates with the Gold Standard. Nocardia sp. is a large group of gram-positive bacteria, aerobically living in soil, organic matter, and fresh or salt water. Nocardia belong to the family Nocardiaceae, from the suborder of aerobic actinomycetes that also includes Mycobacterium, Corynebacterium, Gordona and Tsukamurella [The genus amurella , 2.Nocardia can cause disease in humans, with a wide spectrum of clinical manifestations. In immunocompetent individuals, nocardiosis commonly occurs after traumatic inoculation of the microorganism, while the respiratory tract is often the initial site of infection in the immunocompromised [Nocardia is challenging. These bacteria grow in specific media for mycobacteria and form partially acid-fast beaded branching filaments, similarly to mycobacteria, which frequently results in Nocardia being mistaken for Mycobacterium [Several species of promised . Despiteacterium .Nocardia spp. Since nocardiosis can be fatal in the absence of treatment and there were reports of Nocardia resistance to sulfonamides, the susceptibility testing becomes even more important. In addition, there are new antibiotic agents that could be even more effective than the usual sulfonamide treatment [The identification of the species is necessary for an adequate treatment, but its effectiveness may depend on the antimicrobial susceptibility profile, widely diverse among reatment , 6.Nocardia spp. isolates [The Clinical and Laboratory Standards Institute (CLSI) currently recommends the Broth Microdilution (BMD) method to determine the Minimum Inhibitory Concentrations (MIC) of antimicrobial agents to establish the susceptibility of isolates . Howeverisolates  already isolates –11. Likeisolates –14.Nocardia spp. by broth microdilution assay.In the present study, the application of Resazurin and OD readings were investigated as alternative methods that could reduce subjectivity in the interpretation of susceptibility to antimicrobials testing on Nocardia spp. isolates were evaluated in this study by the BMD method, and the use of Resazurin salt  and the OD reading (spectrophotometric determined MIC by ODs) were evaluated as alternative methods of result reading in comparison with the Gold Standard visual reading .The Nocardia isolates, with respective modal MIC and ranges for Amikacin (AMK), Ciprofloxacin (CIP), Minocycline (MIN), and Trimethoprim-Sulfamethoxazole (TMP-SMX) by BMD method are presented on Table The susceptibility profile of the evaluated P-values of < 0.05 indicate a significant correlation  and Categorical (susceptibility classification) Agreements were calculated for intra-  and inter-reading  comparisons, considering ±1 two-fold dilution for concordant results. The Pearson Correlation Coefficients (Pearson’s r) and respective 95% Confidence Intervals (95% CI) were determined on the inter-reading methods comparisons of MIC values. The Pearson’s r-values range from - 1 to + 1: values from 0 to + 1 indicate a positive correlation, from 0 to - 1 indicate a negative correlation, and 0.0 indicates no correlation at all. on Table .Table 2AP < 0.0001 for all comparisons). The EA percentages for CIP assays between the Gold Standard and the Resazurin readings, and between the Gold Standard and the OD readings were 90.7 and 89.4%, respectively. For the AMK assays, all three reading methods showed 100% of Categorical Agreement (CA) in both intra- and inter-reading comparisons  for the three drugs on both intra- and inter-reading comparisons were higher than 80.0%, and there were positive and significant correlations between the Gold Standard and both alternative methods, Resazurin and OD readings ; and Very Major error, a resistant result in the Gold Standard method, but susceptible in the alternative assay . Acceptable error levels were ≤ 10% for Minor, ≤ 3.0% for Major, and ≤ 1.5% for Very Major errors .There were no categorical errors on the results of AMK. For CIP, on the other hand, 0.85% of Very Major errors were observed between the Gold Standard and the Resazurin readings, and 1.52% of Major errors between the Gold Standard and the OD readings. For the MIN assays, there were no Very Major and Major errors in the inter-reading comparisons, and the percentage of Minor errors ranged from 12.3 to 14.4% . The inter-reading comparison between the Gold Standard and the OD readings showed 91.7% of CA with ±1 two-fold and 93.9% with ±2 two-fold dilutions. Despite of CA being greater than 90%, the percentage of Very Major errors for ±1 two-fold and ± 2 two-fold dilutions were 8.33 and 6.06%, respectively  and Categorical (93–99%) Agreements with the visual MIC determination [The comparisons between the Gold Standard and the OD readings showed high percentages of Essential (75.0–99.2%) and Categorical (75.0–100%) Agreements. Similarly, Meletiadis et al. demonstrated that the European Committee on Antimicrobial Susceptibility Testing (EUCAST) spectrophotometric MIC determination for antifungal susceptibility testing with mination .Nocardia spp., but our results demonstrate its applicability for these bacteria, as it is currently used for fungal agents.To the best of our knowledge, there are no studies using Spectrophotometry (OD readings) for Nocardia sp., however, their documents are free of charge and easily accessible to clinical laboratories for other microorganisms. CLSI documents, on the other hand, are only available through a costly payment, which can restrict their access and hinder their use by emerging countries. We showed that the use of Resazurin and Spectrophotometry could help defining a novel document and breakpoints for Nocardia spp. isolates.EUCAST, unlike CLSI, does not have a specific recommendation or defined breakpoints for Nocardia spp. isolates and evaluated drugs were the main limitations of this study. Nevertheless, the results herein disclosed the promising use of Resazurin and OD readings in future investigations on antimicrobial susceptibility of Nocardia spp. isolates.The small number of Nocardia spp.Our data suggest that Resazurin and Spectrophotometry are efficient, reproducible and easy-to-use alternative methods for determining the susceptibility profile of Nocardia spp. isolates from the Collection of Cultures of the Laboratory of Medical Research in Mycology (LIM53) were included in this study -1180 (MW348983), N. beijingensis-1181 (MW348984), Nocardia brasiliensis(N. brasiliensis)-95 (MW348931), N. brasiliensis-129 (MW348933), N. brasiliensis-519 (MW348935), Nocardia farcinica(N. farcinica)-788 (MW348936), and N. farcinica-2901 (MW348955) were identified by Gram and Ziehl Neelsen staining methods, morphological characteristics and molecular sequencing. A 606 bp fragment of the 16S rRNA gene was amplified with the primers Noc1 (5′-GCTTAACACATGCAAGTCG-3′) and Noc2 (5′-GAATTCCAGTCTCCCCTG-3′), and sequenced as previously described [http://www.ncbi.nlm.nih.gov/BLAST). Similarity values of ≥98.0% for 16S rRNA were deemed to indicate the same species.Staphylococcus aureus (S. aureus ATCC 29213) [Nocardia spp. isolates were cultured on Tryptone Soy Agar  at 35 °C for 7 days and the ATCC strain was also cultured on TSA at 35 °C for 24 h.CLSI-recommended quality control ranges for Amikacin (AMK), Ciprofloxacin (CIP), Minocycline (MIN) and Trimethoprim-Sulfamethoxazole (TMP-SMX) were performed by using a strain of C 29213) . NocardiThe 0.01% Resazurin solution was prepared by dissolving the powder dye  in 1× PBS, pH 7.2 . After complete dissolution, the solution was sterilized by a 0.22 μm membrane filtration, stored at 4 °C, protected from light, and used within 3 days.The antimicrobial solutions were prepared as recommended by the CLSI document M100-S24 . BrieflyNocardia [Nocardia isolate were prepared by dissolving the bacterial content with saline and glass microbeads , vortexing vigorously and then allowing it to precipitate for 10 to 15 min. The concentration was adjusted by dilution on Cation-Adjusted Mueller-Hinton Broth .The MICs were determined by the BMD method, according to CLSI document M24 for Nocardia . BrieflyMedium control wells had 200 μL/well of drug-free CAMHB, and inoculum control wells had 100 μL/well of drug-free CAMHB and 100 μL/well of bacterial inoculum. All the other wells were inoculated with 100 μL/well of bacterial inoculum on microplates with antimicrobial solutions, previously prepared. Microplates were incubated in a moist chamber at 35 °C.Following incubation of 24 h, 20 μL/well of 0.01% sterile resazurin solution were added to all wells and microplates were re-incubated for 48 h for colour development. A change in colour from blue to pink indicated bacterial growth.Nocardia isolates, except for the incubation period. After incubation for 17 h, 20 μL/well of 0.01% sterile resazurin solution were added to all wells and the microplates were re-incubated for more 3 h for colour development.The ATCC strain inoculum suspension was prepared as described for Nocardia isolates (48 and 72 h), the microplates were evaluated by the Gold Standard visual reading without any dye, the visual reading with Resazurin, or by OD readings without any dye, at 560 nm in the Versa Max Microplate Reader spectrophotometer .Following the stablished incubation periods for the ATCC strain (17 and 20 h) and for Growth in medium control wells indicated contamination or absence of growth in inoculum control wells indicated unviability of the isolate, and the corresponding assay was discarded. At least two wells were matched among the triplicates for a valid result.Nocardia [Nocardia spp. isolates were based on the M24 CLSI document: a zone of ≥35 mm indicated susceptibility, and a zone of ≤15 mm indicated resistance.The Disk Diffusion test was performed with the isolates suspensions, prepared as described on M24 CLSI document for Nocardia , and plaFor the visual interpretation of the results, the MIC well was determined by its comparison with medium and inoculum drug-free control wells. The MIC well was the one with the lowest drug concentration that prevented the bacterial growth and the colour change within the incubation period, being similar to the medium control well (no visible growth and blue with Resazurin). The inoculum with drug and the inoculum control drug-free wells should show visible growth and should be pink with resorufin. For the spectrophotometric readings, the percentages of bacterial growth were calculated with the following eq. : bacteriThe Pearson Correlation Coefficients (Pearson’s r) and respective 95% Confidence Intervals (95% CI) were determined by the Prism 5.01 statistical software .


Adipocyte-derived adiponectin may play a role in the host inflammatory response to cancer. We examined the association of plasma adiponectin with the density of tumor-infiltrating lymphocytes (TILs) in colon cancers and with vitamin D, clinicopathological features, and patient survival.Plasma adiponectin and 25-hydroxyvitamin D [25(OH)D] were analyzed by radioimmunoassay in 600 patients with stage III colon cancer who received FOLFOX-based adjuvant chemotherapy (NCCTG N0147 [Alliance]). TIL densities were determined in histopathological sections. Associations with disease-free survival (DFS), time to recurrence, and overall survival were evaluated by multivariable Cox regression adjusting for potential confounders . All statistical tests were 2-sided. = 6845 vs 8984 ng/mL; P = .04). A statistically significant reduction in adiponectin was also observed in obese (body mass index >30 kg/m2) vs nonobese patients , in men vs women , in Blacks vs Whites or Asians , and in those with fewer lymph node metastases . Insufficiency of 25(OH)D (<30 ng/mL) was detected in 291 (48.5%) patients. In multivariable analyses, neither adiponectin nor 25(OH)D were associated with a statistically significant difference in DFS, overall survival , or time to recurrence in models adjusted for potential confounders. We found a statistically significant association of TILs with prognosis, yet no such interaction was observed for the association of adiponectin with TILs for DFS.We found a statistically significant reduction in adiponectin, but not 25(OH)D, levels in tumors with high vs low TIL densities D was independently prognostic. Colorectal cancer (CRC) is the third most common cancer and is second only to lung cancer as a cause of cancer-related mortality in the United States . ObesityPlasma adiponectin was found to be associated with vitamin D levels in non-CRC patients . VitaminTo date, adiponectin has not been analyzed in relationship to the tumor immune microenvironment. We sought to test the hypothesis that adiponectin may play a role in the host inflammatory response to colon cancer, indicated by tumor-infiltrating lymphocytes (TILs). In patients with CRC, studies indicate that TILs can independently predict recurrence and survival . HoweverAmong 2686 patients with resected stage III colon cancer who had been randomly assigned to adjuvant FOLFOX alone or combined with cetuximab, we randomly selected 600 patients (300 per arm) for analysis of adiponectin and 25(OH)D . Right-sided tumors were defined as located proximal to the splenic flexure. N1 tumors had 1-3 metastatic regional lymph nodes; N2 tumors had at least 4 nodes. All patients provided written informed consent at the time of clinical trial enrollment. All data analyses shown here were approved by Mayo Clinic Institution Review Board.P = .99), we report total adiponectin. 25(OH)D was extracted from serum or plasma with acetonitrile, and samples were then assayed using an equilibrium radioimmunoassay procedure using an antibody specific for 25(OH)D (Plasma adiponectin and 25(OH)D were analyzed in processed and stored blood obtained at adjuvant study registration. Biomarker concentrations were measured by radioimmunoassay  as previously described . Circula 25(OH)D . BlindedA representative hematoxylin and eosin-stained tumor section from each patient was scanned at low power to identify areas with the most intraepithelial TILs. Once identified, 5 consecutive 40X fields were counted, and mean TILs per high power field (HPF) were calculated by dividing the total number of TILs by 5. All cases were scored independently by 2 gastrointestinal pathologists blinded to clinical and molecular data . CutoffsBRAF (c.1799T>A V600E) mutation in exon 15 and KRAS mutations in codons 12 and 13 of exon 2 was previously described , time from randomization to death from all causes; and time to recurrence (TTR) as time from randomization to recurrence. Distributions of DFS, OS, and TTR were estimated by the Kaplan-Meier method. Biomarkers were analyzed in relationship to clinical outcome as continuous variables using a relative risk model with cubic splines and as binary variables in multivariable Cox regression. Multivariable models included variables that were considered to be potential confounders. Interaction tests were performed. A 2-sided P value of less than  .05 was considered statistically significant and was not adjusted for multiple comparisons. SAS software version 9.4 and R version 3.6.2 were used .Adiponectin and 25(OH)D were analyzed as continuous variables in the primary analysis. As binary variables, adiponectin was dichotomized at the median, and 25(OH)D was dichotomized at 30 ng/ml with lower levels regarded as insufficiency . The ass = 6845 vs 8984 ng/mL; P = .04) ( = 6608 ng/mL) than did those of normal weight (median = 10 693 ng/mL) or underweight . A statistically significant decrease in adiponectin was observed in men vs women , in those aged 65 years or younger , and in Blacks vs Whites or Asians  . Adiponectin was not associated with a statistically significant correlation with 25(OH)D  or with T stage, DNA MMR, or the mutational status of KRAS or BRAF genes.The frequency distribution and median levels of plasma adiponectin and 25(OH)D are shown in P = .04) . FurtherP < .03) . FindingP < .001) (P < .001) and sex (P < .03) D, as a continuous variable, was found in Blacks vs Whites or Asians ( < .001) . Differe = 0.98, 95% confidence interval [CI] = 0.74 to 1.29; P = .88) shown in a Kaplan Meier plot  or TTR  (data not shown). We then constructed a multivariable model that included adiponectin and potential confounding variables based on observed associations  or a dichotomous (data not shown) variable in a multivariable model adjusted for treatment arm, race, BMI, TILs, and N stage  and N2 vs N1 stage   . Adipone = 0.85 to 1.47; P = .44) , or TTR . Similarly, the continuous 25(OH)D was not statistically significantly associated with DFS  that achieved statistical significance with 3-year survival rates of 65.4% vs 80.5%, respectively. In a multivariable model that included potential confounders, 25(OH)D as a continuous variable was not associated with patient DFS   and N stage  were statistically significantly associated with DFS D was not statistically significantly associated with DFS  , OS (HR with DFS  or with  = 1.00)  or with with DFS . We alsoP = .50) (data not shown). Furthermore and given observed differences in adiponectin and 25(OH)D levels by patient sex (We constructed 3-way interaction models that examined the association of adiponectin and 25(OH)D (adjusting for race and BMI) by TILs and N stage subgroups. No statistically significant relationships were found indicating that the observed associations were not interdependent. Of note, TIL density did not differ by BMI category  D in postsurgical blood samples from patients with stage III colon cancer treated in a phase III trial of adjuvant chemotherapy. Based on preclinical data suggesting that adiponectin may influence the host inflammatory response to cancer, we determined the association of adiponectin with TIL density in the tumor immune microenvironment. Lower adiponectin levels were associated with a statistically significant increase in TIL density, indicating an inverse relationship between adiponectin and antitumor immunity. This result is consistent with data in a murine model where adiponectin deficiency was associated with tumors with an increased inflammatory infiltrate compared with tumors in nonadiponectin-deficient mice . Adipone T cells . We founADIPOQ) in obesity D was found to be prognostic.Research reported in this publication was supported by the National Cancer Institute (NCI) of the National Institutes of Health (NIH) under award numbers U10CA180821, U10CA180882; U10CA180863, CCSRI 021039 (CCTG); U10CA180868 (NRG); U10CA180888, and UG1CA233163 (SWOG). NCCTG N0147 received funds from Sanofi Aventis. The study was also supported by NCI R01CA210509 (to FAS).Role of the funders: The funders had no role in the design of the study or in the data collection, analysis, or interpretation. Furthermore, the funders did not participate in the writing of the manuscript or in the decision to submit the manuscript for publication.Disclosures: FAS is a co-inventor of intellectual property with Roche Ventana Medical Systems  and may receive royalties paid to Mayo Clinic. No other relevant conflicts related to the subject matter of this manuscript are reported by the study authors.Author contributions: Study conceptualization: FAS. Data curation: QS. Formal analysis: SBJ, NRF, QS. Funding acquisition: FAS, SRA. Investigation: TCS, RMG, SJC, SG, MSK, SN, AFS, BNJ, MNP, SRA. Methodology: MNP. Project administration/supervision: FAS. Writing: original draft: FAS. Writing-review and editing: all authors.concepts@allianceNCTN.org). Requests to utilize biomarker data published in this manuscript should also be directed to the corresponding author.Data from published Alliance or North Central Cancer Treatment Group (NCCTG) trials can be accessed by submission of an Alliance Data Sharing Request Form (pkab070_Supplementary_DataClick here for additional data file.


Callithrix jacchus) is a New World monkey that is widely used to study brain function. Here, we present a natural marmoset model of generalized epilepsy. In this unique marmoset family, generalized epilepsy was successfully induced by handling operations in some individuals. We mapped the marmoset family with handling-sensitive epilepsy and found that the epileptic phenotype can be inherited. These marmosets were more sensitive to the epilepsy inducers pentylenetetrazol. Using electrocorticogram (ECoG) recordings, we detected epileptiform discharge in marmosets with a history of seizures. In summary, we report a family of marmosets with generalized seizures induced by handling operations. This epileptic marmoset family provides insights to better understand the mechanism of generalized epilepsy and helps to develop new therapeutic methods.Epilepsy has been extensively studied as a common neurological disease. Efforts have been made on rodent and other animal models to reveal the pathogenic mechanisms of epilepsy and develop new drugs for treatment. However, the features of current epilepsy models cannot fully mimic different types of epilepsy in humans, hence non-human primate models of epilepsy are required. The common marmoset (The online version contains supplementary material available at 10.1186/s13041-022-00901-2. Epilepsy is a common chronic brain disorder characterized by recurrent seizures that affects over 70 million people worldwide , 2. A thCallithrix jacchus) is a small New World monkey that has been frequently used because of its genetic constitution, body size, and unique reproductive characteristics (twice a year to either twins or triplets) [The common marmoset (riplets) –10. Genoriplets) , 12. Thiriplets) –15.Here, we showed for the first time a marmoset family with generalized epilepsy. In this marmoset family, some individuals showed significant seizure phenotypes in response to handling stimulation. The seizure symptoms include limb convulsions, movement disorders, vomiting, and salivation, which are typical phenotype of human epileptic seizures. Moreover, we found that this phenotype was stably inherited from generation to generation. We suggest that this natural epileptic marmoset is a potential non-human primate model for understanding the mechanism of epilepsy.Those epileptic marmosets were discovered as a result of routine handling for health checks, such as weight measurement. The first epileptic marmoset was born in 2002 by tracing the history of seizures in this family. The main branch of this epileptic marmoset family contains 51 marmosets across four generations. The incidence rate of epileptic seizures in the four generations are 2/2(G I), 15/22(G II), 8/16(G III), 3/11(G IV), respectively  and phase II (11–60 min). b Scratching, c Mouth cleaning, d Head shakes behaviors in asymptomatic and epileptic marmosets. Data are expressed as mean ± SEM. Two-way ANOVA followed by Fisher's LSD test was used to compare behaviors between groups. *P < 0.05, **P < 0.01, ***P < 0.001.


At the core-level, PRM-defined parenchymal disease, a potential readout of RAS, was found to correlate to neutrophil and collagen I levels (p < 0.05). We demonstrate the relationship of structural and biological markers to the CT-based distribution and arrangement of PRM-derived readouts of BOS and RAS.Chronic rejection of lung allografts has two major subtypes, bronchiolitis obliterans syndrome (BOS) and restrictive allograft syndrome (RAS), which present radiologically either as air trapping with small airways disease or with persistent pleuroparenchymal opacities. Parametric response mapping (PRM), a computed tomography (CT) methodology, has been demonstrated as an objective readout of BOS and RAS and bears prognostic importance, but has yet to be correlated to biological measures. Using a topological technique, we evaluate the distribution and arrangement of PRM-derived classifications of pulmonary abnormalities from lung transplant recipients undergoing redo-transplantation for end-stage BOS (N = 6) or RAS (N = 6). Topological metrics were determined from each PRM classification and compared to structural and biological markers determined from microCT and histopathology of lung core samples. Whole-lung measurements of PRM-defined functional small airways disease (fSAD), which serves as a readout of BOS, were significantly elevated in BOS versus RAS patients ( Transplanted lungs have one of the highest rejection rates among all solid organ transplantations, and approximately 50% of lung transplant recipients suffer from chronic lung allograft dysfunction (CLAD) within five years of transplantation ,2. The mWe previously demonstrated using a combination of CT and microCT that obstruction of pre-terminal airways is common in both BOS and RAS , while in RAS there is also parenchymal destruction leading to further distortion and remodeling of terminal bronchioles ,8. LungsParametric response mapping (PRM) is a quantitative CT-based algorithm that, when applied to paired inspiratory and expiratory high-resolution computed tomography (HRCT), has been shown to simultaneously detect parenchymal abnormalities associated with emphysema and small airways disease (SAD) in patients with chronic obstructive pulmonary disease (COPD) ,11. The In these studies, PRM classifications, inherently three-dimensional (3D) datasets of pulmonary abnormalities, have been reported simply as percentages of lung volume. We have expanded the clinical application of PRM by demonstrating how topological analysis of PRM (tPRM) provides information on the distribution and arrangement of PRM classifications that correlates to clinically meaningful measures of COPD severity . In thisAll patients who underwent double lung transplant reached a best postoperative forced expiratory volume at 1 s percent predicted (FEV1pp) > 80% and received azithromycin treatment for potential CLAD but were found to be nonresponsive. BOS and RAS were diagnosed according to the most recent International Society for Heart and Lung Transplantation guidelines. In brief, BOS patients showed a decline in FEV1pp > 20% compared to baseline but did not show evidence of concomitant restriction  < 90% or forced vital capacity pp (FVCpp) < 80% of baseline), without evidence of persistent opacities on CT. In contrast, RAS patients showed a simultaneous decrease in FEV1pp and TLCpp and persistent pleuroparenchymal opacities. Inspiratory and expiratory CT scans were acquired from all patients and the last CT scan prior to redo transplantation was used for further analysis. Clinical diagnosis was confirmed with pathological analysis of the contralateral lung with typical interstitial and (sub)pleural fibrosis in RAS and mostly inconspicuous parenchyma and airway obliteration in BOS. This study was approved by the local hospital’s ethical committee (S57752).CT data were obtained as whole lung volumetric CT scans at full inspiration (TLC) and at relaxed expiration ) on Somatom scanner  and reconstructed using a b60 or b70 reconstruction kernel. Slice thickness was 1 mm for all scans. All CT scans were checked for Hounsfield unit (HU) drift and if necessary corrected based on aortic blood (50 HU) and central air (1000 HU) as previously described .PRM was applied to all paired inspiratory and expiratory CT scans from both study groups. Briefly, lungs from paired scans were segmented from the thoracic cavity using an in-house algorithm written in MATLAB . The whole-lung inspiration CT scan was spatially registered and aligned to the CT scan obtained at expiration using a deformable image registration algorithm provided in Elastix, an open-source image registration software package ,21. Once3), Surface Area , Mean Breadth , and the Euler–Poincaré characteristic (χ) [3 voxels evaluated on a grid of every 5th voxel. V, S, and B were normalized by the Minkowski estimate of the mask within the same local window volume  and χ by the masked window voxel count. Minkowski measures were quantified per subject as the mean local normalized value over the entire lung volume for group comparisons and regression. Final displayed representations of spatially resolved indices have been linearly interpolated back to original dimensions. It is important to note that the volume density of a given PRM classification is proportional to relative volumes of PRM classification. All image processing was performed using in-house algorithms developed in a technical computing language .Topological properties of each PRM classification map were defined in this study through the Minkowski measures  associated with 3D distributions: Volume  . A detaistic (χ) . Maps of2O. They were then deflated to 10 cm H2O transpulmonary pressure, followed by freezing in the fumes of liquid nitrogen vapor. Lungs were scanned using HRCT . Following CT, the lungs were cut with a band saw, from apex to base, into slices of 2 cm and systematically sampled. Photographs were taken before and after sampling to ensure proper matching. Four cores per lung were selected for further analysis. These cores were chosen to present the different spectra of disease in the lungs. Specifically, for RAS patients, we included both fibrotic and non-fibrotic areas.Since 2009, explant lungs have been prospectively collected from all lung transplant patients undergoing lung redo transplantation or at autopsy . Permission for CT analysis was requested separately from the UZ/KU Leuven ethics committee via approval number S57752. Explant lungs were cannulated and inflated to total lung capacity by applying a transpulmonary pressure of 30 cm HTopological features of individual cores were determined through spatial alignment of the in vivo paired CT scans to a photograph of the explanted cored lung section. This method was previously reported . The proThe frozen cores were scanned in frozen condition with microCT  using a cooling stage . After scanning, the images were reconstructed with NRecon  and processed using CT for assessment of the parenchymal morphometry (tissue percentage of core and surface density) and by manually counting the number of terminal bronchioles (TB/mL) . A portip < 0.05).All data are presented as the mean ± the standard deviation unless stated otherwise. Differences in metrics between CLAD subtypes were assessed by Mann–Whitney U Test for all continuous variables and Fisher Exact test for categorical variables. Correlation analysis was determined using Spearman rho. All statistical computations were performed using a statistical software package . Results were considered statistically significant at the two-sided 5% comparison-wise significance level (Patient characteristics are presented in Provided in fSAD), was on average more than double in BOS patients (0.48 ± 0.14) than in RAS patients (0.17 ± 0.13). The surface area, a readout of disease distribution (SfSAD), was also found to be elevated in the BOS group (0.66 ± 0.08 mm−1) compared to RAS (0.37 ± 0.25 mm−1), suggesting most patients have a diffuse spread of BOS throughout the lungs. Euler–Poincaré characteristic (χfSAD), a readout of disease pockets or holes, was negative for the BOS group (−0.007 ± 0.004). Negative values in χfSAD occur when PRM-derived fSAD is the dominant classification within the lungs with holes that may comprise of other PRM classes, or structures such as airways and blood vessels. In contrast, the observed mean value of χfSAD for RAS patients was positive (0.003 ± 0.001), which is associated with small pockets of PRM-derived fSAD. Although the mean curvature of PRM-derived fSAD, a readout of tunneling within this classification, was higher in RAS (0.04 ± 0.01 mm−2) than in BOS patients (0.02 ± 0.02 mm−2), this was not found to be significant.Volume density, surface area, and Euler–Poincaré characteristic metrics associated with PRM-derived fSAD were found to differ significantly between BOS and RAS . The volPD and SPD, patients with RAS  had higher values compared to patients with BOS (0.20 ± 0.03 (p = 0.145) and 0.36 ± 0.12 mm−1 (p = 0.091), respectively). Near identical values in mean curvature were observed between groups (BOS at 0.04 ± 0.01 mm−2 and RAS at 0.04 ± 0.03 mm−2). Although the mean value of χPD was lower in RAS (0.0003 ± 0.006) than in BOS (0.003 ± 0.001) patients, no significant difference (p = 0.132) was observed due to the large spread in values for RAS patients.In contrast to PRM-derived fSAD, the topology of PD did not result in significant differences between groups for any of the metrics. Notwithstanding, the volume density and surface area of PRM-derived PD generated expected trends. For VPRM-derived normal and emphysema provided mixed results. Only mean curvature for PRM-derived normal parenchyma showed a significant difference between groups . PRM-derfSAD . Here, patients with BOS had higher FVC and negative χfSAD, whereas RAS patients, except for one, had low FVC and positive χfSAD. VfSAD and SfSAD demonstrated significant correlations for FVC and FEV1/FVC . Nevertheless, VPD and χPD demonstrated mild correlations to FVC with p-values equal to 0.07 and 0.08, respectively. This trend was not observed when correlated to FEV1/FVC .Based on whole-lung analysis of individual tPRM metrics, the volume density of PRM-derived fSAD and PD were elevated for BOS and RAS, respectively. Using the total study population (N = 12), correlations to pulmonary function measurements were assessed for all tPRM metrics of these two PRM classifications . FVC andFEV1/FVC . For PRMNorm where normal lung parenchyma regionally coexists with local parenchyma classified as fSAD and PD (Norm). The low values in SNorm were a consequence of that entire lung region being classified as fSAD. The Euler–Poincaré characteristic provides an indication of the dominance of any PRM class, as well as its transition to or from another PRM class. As seen for PRM-derived fSAD, χfSAD generated negative values where VfSAD was highest. Within that same lung region, χNorm had positive values, suggesting an inferior contribution to this space (red regions). These trends are consistent with the whole lung analysis for χNorm and χfSAD in the BOS group . Only one case (231) was found to have similar contributions of PRM-derived fSAD (median VfSAD = 0.24) and PD (median VPD = 0.34) within the core sample. The surface area (S) and Euler-Poincaré characteristic (χ) were also consistent among BOS cases, except for 231. Unlike the BOS patients, no consistent topology of fSAD and PD were identified from the core samples obtained from lung sections of RAS patients. Two of the six RAS patients had core samples with elevated levels of VPD with median values above 0.6. Three of the six  had similar volume densities for fSAD and PD. The sixth case (143) had higher values of VfSAD than VPD, like trends observed in BOS patients.Topological values averaged over each core are presented for all cores sampled from each patient . As PRM-p = 0.06), neutrophil levels were more elevated in BOS than RAS subjects.p-value < 0.05) of tissue percentage of cores (T%) and surface density (SD) to nearly all the topology measures for PRM-derived fSAD. The strongest correlation was observed between VfSAD and T% . PRM-derived PD was also found to demonstrate significant correlations with the volume density (VPD) to T%  and the number of terminal bronchioles per mL (TB/mL) . MPO levels were found to correlate significantly to the volume density and surface area (S) of both PRM classes, as well as to the Euler–Poincaré characteristic of PD (χPD). CD117 was found to correlate with V and S of fSAD and COL I with χPD.For PRM classes fSAD and PD, correlations were determined for topology metrics and structural and biological markers . This anPD and TB/mL . Cores obtained from RAS patients showed significant correlations between tPRM metrics and structural and biological markers. Like the pooled data, TB/mL, MPOs, and COL I were found to significantly correlate with various topology metrics from both PRM classes. The strongest correlation, irrespective of pooling, was observed between VPD and MPO levels, with a R of −0.684 and p-value < 0.001.Correlations for individual groups generated different results from the pooled data. No significant correlations were observed within the BOS group. Nevertheless, non-significant correlations for BOS subjects were seen for VIn this study, we confirmed the role of PRM-derived fSAD and PD as CT-based readouts of BOS and RAS, respectively, in lung transplant recipients. We also demonstrated the relationship between the extent, distribution, and arrangement of our PRM-derived classifications of pulmonary abnormalities, and the structural and biological microenvironment of lung parenchyma in these same CLAD subtypes. We provide further support for our findings by demonstrating elevated levels of fSAD and PD in BOS and RAS patients, respectively, and a correlation between topological measures of fSAD and PD and microCT-measured surface density and MPO levels in cored lung samples.fSAD was found to be on average around 0.48 ± 0.18 of the total lung volume. This is equivalent to saying 48% of the lungs are PRM-derived fSAD. This value was higher than the ~30% reported previously, which has been demonstrated as a link to prognosis [fSAD over the lungs is equal to the percentage of PRM-derived fSAD normalized to 100. Taking this into account, we attribute the differences in the extent of fSAD reported here and in earlier studies to the severity of the disease and not to differences in calculation. Unlike the previous studies where PRM was determined at the time of diagnosis, patients in our cohort were scheduled for second lung transplants due to end-stage CLAD. With respect to the extent of PRM-derived PD, elevated values found in RAS patients  that was highly diffuse (elevated SfSAD) and was the dominant pathology within the cores  (negative χfSAD) , surface area (S) and Euler–Poincaré characteristic (χ) for PRM-derived fSAD and PD. Out of the 6 BOS cases, 5 were found to have large differences in the median values over all cores per case in fSAD and PD. The cores from these cases had extensive small airways disease (Ve χfSAD) . This ise χfSAD) , where sPD and SPD and negative χPD, which is like the topology of PRM-derived fSAD in BOS patients. Further, case 143, who had radiographically-identified scarring, presented several similarities consistent with the BOS group. Among these were trends in V, S and χ of the core samples, and volume densities of 0.37 for fSAD and 0.11 for PD when averaged over the lungs. This patient also had FVC (L) and FEV1/FVC of 1.95 L and 0.68, respectively, again consistent with BOS. It is important to note that PRM is defined within a range of −1000 HU to −250 HU on both the inspiration and expiration CT scans. Unlike air trapping due to BOS, pulmonary fibrosis, a hallmark of RAS, may have densities well outside this range. As such, PRM only detects and quantifies parenchyma with mild opacities that may be attributed to pneumonitis [A unique trend in topological features could not be identified in lung transplant recipients diagnosed with RAS. Only cases 184 and 233, as seen in umonitis  and fibrPD and MPOs  but also the positive correlation between VPD and COL I levels .Topology measures for PRM-derived fSAD and PD demonstrated strong correlations to microCT and biological measures when CLAD subtypes were pooled (N = 45). The most pronounced correlations for microCT and biology to topologies were identified in percentage of core tissue and MPOs . These fOur study was subject to some limitations worth addressing. First, the number of cases N = 12) accrued as part of this study limited our statistical power. Nevertheless, whole-lung analysis of our cohort generated volume densities of PRM-derived fSAD and PD  consiste accrued We have offered a view of the PRM landscape in a group of lung transplant recipients who developed specific CLAD subtypes. We identified a connection between the structural and biological makeup of the lung microenvironment and the topology of a clinically relevant CT-based readout of BOS and RAS. Our investigative group is working on developing additional techniques to elucidate the nature of small airway and parenchymal abnormality identified by PRM topology. These steps could further confirm the potential use and benefit of PRM for monitoring CLAD progression in lung transplant recipients.


N-acetyl-p-aminophenol; APAP) overdose is a common presentation to emergency departments around the world. While N-acetylcysteine (NAC) remains the cornerstone of treatment for APAP overdose, extracorporeal treatment, in the form of renal replacement therapy with intermittent hemodialysis (IHD) or continuous renal replacement therapy (CRRT) may provide benefit in cases associated with altered mental status and metabolic acidosis. One treatment with IHD is typically sufficient for resolution of acidosis and global improvement clinically. We describe a case of massive APAP overdose presenting with altered mental status and lactic acidosis, refractory to multiple treatments of IHD as well as CRRT and high-dose NAC along with fomepizole. Despite these interventions, fulminant liver failure progressed with cerebral edema, coagulopathy and death. This is the first description of a fatal acetaminophen ingestion refractory to both IHD and prolonged CRRT. This case highlights the need for further investigation in the management of massive APAP overdose, including optimal method and timing of renal replacement therapy.Massive Acetaminophen ( While numerous experimental. treatments are still being investigated to improve outcomes in APAP overdose [APAP . To the best of our knowledge, this is the first case of APAP toxicity and early lactic acidosis where repeated extracorporeal treatments were unsuccessful in elimination of the offending parent compound and prompt resolution of acidosis.2A 24-year-old woman was brought to a community hospital with decreased level of consciousness. Medical history was significant for endometriosis, chronic pelvic pain, depression with previous suicide attempts, and generalized anxiety disorder. Collateral history estimated an ingestion of 50 g of APAP, 25.5 g of gabapentin, and 60 mg of morphine sixteen hours before presenting. Initial vital signs were normal despite her reduced level of consciousness. Admission investigations were remarkable for: APAP level of 3661 μM, lactate of 3.8 mM, pH of 7.22 and bicarbonate of 9 mM. Creatinine was 56 μM. Alanine aminotransferase (ALT) and alkaline phosphatase (ALP) were 113 U/L and 64 U/L, respectively, with a total bilirubin of 10 μM and International Normalized Ratio (INR) of 1.6. NAC was initiated, with a bolus of 60 mg/kg over one hour followed by an infusion of 12 mg/kg/hr thereafter in addition to 600 mg IV fomepizole. Peak APAP level, two hours after presenting, was 3983 μM. Subsequently, the patient was intubated for airway protection and transferred to our tertiary Medical-Surgical Intensive Care Unit (ICU) for consideration of liver transplantation.2O and pressure support of 8 cm H2O. Without sedation, she had spontaneous eye opening and followed commands. Pupils were equal and reactive. Cardiovascular and respiratory physical examinations were unremarkable. Laboratory data showed APAP level of 2931 μM, and lactate of 4.5 mM. Arterial blood gas (ABG) analysis revealed pH 7.24, PaCO2 of 29 mm Hg, and PaO2 of 405 mm Hg. Electrolytes were within normal limits. Creatinine was unmeasurable due to presence of NAC. ALT rose to 206 U/L with ALP of 46 U/L. INR was 3.9, with total bilirubin that rose to 19.0 μM.The patient arrived to our ICU thirty-two hours after ingestion. She was afebrile with a blood pressure of 110/60 mm Hg without inotropic support, a heart rate of 92 beats per minute (bpm), and oxygen saturation 94 % on pressure support ventilation with PEEP 5 cm HNAC and fomepizole were continued at a rate of 12 mg/kg/hr and 600 mg IV every six hours, respectively. Laboratory investigations four hours later showed worsening acidosis despite bicarbonate infusion. Therefore, IHD was initiated fifty-eight hours after ingestion. The following prescription was used: Fx1000 filter, a target blood flow (Qb) rate of 400 mL/min, with a dialysate flow of 500 mL/min and a bath of 140 mM of Sodium, Potassium 4.0 mM, Calcium 1.25 mM, Bicarbonate 40 mM. Her APAP level prior to dialysis was 3071 μM with lactate level of 6.8 mM. She received one four-hour run of IHD with a volume of blood processed (VBP) of 100.2 L, followed by a second six-hour run of IHD with a VBP of 144.4 L within a twenty-four-hour period. Despite these two runs of IHD, the lactate level rose to 14.8 mM while her APAP level remained elevated at 437 μM . At thisDespite ongoing CVVHDF, her APAP levels increased again to 614 μM after twelve hours of CVVHDF. Transaminases exceeded 8000 U/L, twenty-two hours after initiating CVVHDF. After forty-eight hours of CVVHDF, lactate decreased, however APAP levels were refractory, remaining elevated at 465 μM. Whole bowel irrigation and colonic decompression were attempted but unsuccessful secondary to ileus development. Not until post-ingestion day eight, and after seven days of CRRT did the APAP level become undetectable. Throughout this time, the patient developed fulminant liver failure, with coagulopathy, hypoglycemia and encephalopathy.Computerized Tomography (CT) imaging of the head revealed widespread cerebral edema and petechial hemorrhage on post-ingestion day five. Measures to reduce cerebral edema including hypertonic saline and hypertonic dialysate targeting serum sodium of 150−155 mM were employed. Magnetic Resonance Imaging (MRI) of the brain post-ingestion day twelve showed diffuse axonal injury. Life-sustaining measures were withdrawn day sixteen after admission following discussions with family on the basis of her poor neurologic prognosis.3To our knowledge, this is the first case reported of a fatal, massive ingestion of APAP refractory to repeated treatments utilizing multiple modalities of renal replacement therapy in the context of NAC and fomepizole co-administration. Massive APAP overdose can present with altered level of consciousness and early metabolic acidosis due to mitochondrial toxicity from NAPQI production . Renal rAs with any antidote, the efficacy of NAC on preventing hepatotoxicity and its sequelae are dependent on both the timing from toxic ingestion and the dose of antidote. It is important to note that in this case, the patient presented sixteen hours after her ingestion. Ideally, NAC should be initiated as soon as possible following ingestion, as delayed NAC administration is associated with worse outcomes . The delN-Acetylcysteine above serum concentrations of 333 mg/L can interfere with the assay. In addition, NAPQI itself may also interfere with enzymatic measurement of creatinine using this enzymatic method. While some cases have highlighted NAC and/or NAPQI and APAP interference with serum creatinine, this is the first case report to suggest cystatin C as the preferred method of renal function assessment APAP overdose. Our laboratory uses the Cystatin C Cobas® enzymatic assay by Roche® as well, which does not demonstrate interference with NAC and/or NAPQI. In our case, measurement of serum cystatin C was detectable despite use of NAC. As cystatin C is a reliable biomarker of AKI [Lastly, AKI is commonly associated with APAP overdose, the severity of which correlates with mortality risk . Therefor of AKI , we sugg4APAP overdose is a common presentation to emergency departments around the world. NAC remains the cornerstone of management and most patients recover with NAC alone. However, in cases of massive APAP overdose, renal replacement therapy may be indicated in cases of acidosis and altered mental status. Our patient presented with early metabolic acidosis and an altered mental status that required airway protection. Her acidosis and APAP levels did not resolve despite multiple runs of intermittent hemodialysis within seventy-two hours of ingestion and several hours of CRRT thereafter alongside a continuous high-dose NAC infusion. She would eventually go on to develop fulminant liver failure with associated cerebral edema. This case prompts further investigation into the appropriate use of renal replacement therapy in cases of severe APAP overdose.This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.The authors declare no conflict of interest.


The severe acute respiratory coronavirus-2 (SARS-CoV-2) is the cause of the global outbreak of COVID-19. Evidence suggests that the virus is evolving to allow efficient spread through the human population, including vaccinated individuals. Here, we report a study of viral variants from surveillance of the Delaware Valley, including the city of Philadelphia, and variants infecting vaccinated subjects. We sequenced and analyzed complete viral genomes from 2621 surveillance samples from March 2020 to September 2021 and compared them to genome sequences from 159 vaccine breakthroughs. In the early spring of 2020, all detected variants were of the B.1 and closely related lineages. A mixture of lineages followed, notably including B.1.243 followed by B.1.1.7 , with other lineages present at lower levels. Later isolations were dominated by B.1.617.2 (delta) and other delta lineages; delta was the exclusive variant present by the last time sampled. To investigate whether any variants appeared preferentially in vaccine breakthroughs, we devised a model based on Bayesian autoregressive moving average logistic multinomial regression to allow rigorous comparison. This revealed that B.1.617.2 (delta) showed 3-fold enrichment in vaccine breakthrough cases . Viral point substitutions could also be associated with vaccine breakthroughs, notably the N501Y substitution found in the alpha, beta and gamma variants . This study thus overviews viral evolution and vaccine breakthroughs in the Delaware Valley and introduces a rigorous statistical approach to interrogating enrichment of breakthrough variants against a changing background. The global COVID-19 pandemic is caused by infection with the virus SARS-CoV-2 –5.–Widespread vaccination against SARS-CoV-2 was introduced in the United States in the winter of 2020–2021. First to be implemented were vaccines based on modified mRNAs, followed by adenovirus vector delivery. Vaccines are highly protective against infection, severe disease, and death. However, infection of vaccinated individuals has been widely detected, albeit typically with much milder disease course compared to that experienced by unvaccinated individuals –8. Thus,–7–Several criteria can be applied to assessing whether sequence changes in a new variant have likely evolved to promote infection and vaccine breakthrough. Substitutions found in viral spike proteins can be tested in laboratory experiments to determine whether they promote more efficient replication in human cells or reduce binding of human antibodies –18. Othe–21–One indication of selection for increased transmission in humans is that several new variants have spread globally and rapidly displaced preexisting strains. This was first documented for the D614G substitution, which spread around the world in the Spring of 2020 and displaced most strains lacking this substitution –28. More–We have investigated viral genomic evolution in the Delaware Valley, which encompasses the city of Philadelphia, in a sample that includes vaccine breakthrough cases. Our initial report on the first wave of infection in this area  revealedIn this study, samples were collected from 2621 surveillance samples from infected individuals and 159 vaccine breakthrough cases through September 2021, and the representation of different variants compared. We found that several waves of variants rose and fell in prevalence over the course of our sampling period, by the end of sampling all genomes were identified as VOC delta lineages, and the delta lineage B.1.617.2 was potentially 3-fold enriched among vaccine breakthrough samples compared to surveillance samples. The amino acid substitution N501Y, found in the alpha, beta, and gamma variants, was also notably enriched in vaccine breakthrough samples. We introduce a rigorous statistical approach, Bayesian autoregressive moving average logistic multinomial regression, that should be widely useful for assessing enrichment of viral variants while controlling for the changing background of circulating strains.Sampling was carried out from March 2020 to September 2021. Over the course of the study, several waves of infection are evident as increased test positivity rates (n = 2621) were defined as those acquired from clinical diagnostic laboratories across the Delaware Valley (n = 2485), from hospitalized subjects (n = 116), and asymptomatic subjects testing positive in a university screening program (n = 20) (n = 159) were included only if detected at least 2 weeks after the final vaccine dose (second dose for Moderna and Pfizer-BioNTech mRNA vaccines or single dose for the Johnson & Johnson adenovirus vector platform) and the subject tested positive by clinical laboratory assay. Data were not available on subject immune responses following vaccination.Patient samples  used for SARS-CoV-2 whole-genome sequence analysis are listed in (n = 20) . Vaccine10.1128/mbio.03788-21.1TABLE S1Table S1, PDF file, 0.3 MB.Human subjects and SARS-CoV-2 genome sequences analyzed in this work, including genome quality metrics, viral variant designation, and accession numbers. Download Copyright © 2022 Marques et al.2022Marques et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the n = 172) were also compared. The TaqPath COVID-19 Combo kit by Thermo Fisher Scientific targets three regions of the SARS-CoV-2 genome for viral detection; the ORF1ab, nucleocapsid (N), and spike (S) genes. The region of the S gene interrogated by the assay overlies a characteristic deletion in the alpha variant (del 69–70), so samples containing alpha lineage virus are selectively negative for the spike amplicon, while the other two targets are detected. Samples with these characteristics were targeted for sequencing early during the wave of alpha infections to track the variant; here these spike target gene failures serve as positive controls for the statistical model.As a positive control, S gene target failure samples  variants emerged, with B.1.1.7 becoming predominant by spring 2021. Delta (B.1.617.2 and AY lineages) became detectable in late spring and predominant in early summer. By late summer, delta lineages were the only variants detected.n = 2621).Wide-spread vaccination was introduced in midwinter 2020–2021, but nevertheless breakthrough infections were detected in some vaccinated individuals, raising the question of whether specific viral features identifiable in sequence data might be associated with vaccine evasion. We sequenced 159 vaccine breakthrough samples collected between February 22 and September 3, 2021, and compared the distributions of lineages or genomic variations to those observed in surveillance samples from the same community  the distribution of variants in the surveillance samples is changing over time; (ii) sampling is uneven over time; and 3 sampling is subject to stochastic fluctuations. To address these issues, we developed a model based on Bayesian analysis that combines an autoregressive moving average model with multinomial logistic regression. The underlying variant proportions at each week of the study were estimated from the counts of SARS-CoV-2 variants assuming relatively smooth changes over time and accounting for stochastic fluctuations during sampling . These eExamples of the temporal profiles of the modeled lineage succession are shown in more detail in As a control, we assessed the variants associated with spike (S) gene target failure samples , where a10.1128/mbio.03788-21.3TABLE S3Table S3, PDF file, 0.03 MB.Estimated fold enrichment in odds of appearing in the spike gene target failure set for each SARS-CoV-2 lineage. Download Copyright © 2022 Marques et al.2022Marques et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the As expected, we found that the B.1.1.7 lineage was estimated to be highly enriched in the S gene target failure set over what would be expected from its frequency in the surveillance lineage counts  . Enrichment among S gene target failures was also seen for another 28 mutations , where aFor vaccine breakthroughs , the mos10.1128/mbio.03788-21.7TABLE S7Table S7, PDF file, 0.04 MB.Estimated fold enrichment in odds of appearing in the vaccine breakthrough samples for each substitution or deletion studied. Download Copyright © 2022 Marques et al.2022Marques et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Vaccination against SARS-CoV-2 has been highly effective at preventing infection, hospitalization and death. However, infections occasionally take place despite vaccination. It has been suggested that certain lineages of SARS-CoV-2 are more prone to evade vaccination, however, interpretation of counts of vaccine breakthrough is difficult due to variation over time in circulating lineages, the numbers of vaccinated individuals, times since vaccination and the application of nonpharmaceutical interventions. Here, we introduce modeling based on Bayesian autoregressive moving average multinomial logistic regression, which estimates the proportions of lineages in the surveillance data over time, allowing comparison of lineage counts in populations of interest to time-matched surveillance estimates. Multiple studies have investigated whether certain lineages are more likely to appear in vaccine breakthrough infections , 39–43. 39–Using this method, we found that there was evidence for enrichment in the odds of variant lineages appearing in the vaccine breakthrough population relative to the general surveillance population, with the delta variant B.1.617.2 showing the strongest signal with an estimated enrichment of 3-fold . Several previous studies have also noted delta variants to be enriched among vaccine breakthroughs , so it is possible that other variants predominate in subjects with lower viral loads. Studies have been initiated to address some of these concerns.In summary, similar to other regions in the US and around the world, VBM/VOC and other lineages waxed and waned in the Delaware Valley, with delta lineages ultimately comprising all recent samples in the fall of 2021. Widespread vaccination was introduced in the region in winter 2020–2021, and increasing numbers of breakthrough infections have since been detected. To compare the lineages found in breakthrough with those observed in general surveillance, we introduce analysis based on Bayesian autoregressive moving average multinomial logistic regression and show that delta variant B.1.617.2 showed 3-fold enrichment in vaccine breakthroughs. The N501Y substitution stood out among point substitutions for enrichment in vaccine breakthroughs. We expect that these modeling methods will be useful in monitoring the effectiveness of vaccination programs going forward as novel variants of SARS-CoV-2 continue to emerge.The University of Pennsylvania Institutional Review Board (IRB) reviewed the research protocol and deemed the limited data elements extracted with positive SARS-CoV-2 specimens to be exempt from human subject research per 45 CFR 46.104, category 4 (IRB #848605). For hospitalized subjects at the University of Pennsylvania, following informed consent (IRB protocol #823392), patients were sampled by collection of saliva, oropharyngeal and/or nasopharyngeal swabs, or endotracheal aspirates if intubated, as previously described . ClinicaSeveral sequencing methods were used to acquire viral whole-genome sequences.The POLAR protocol was used to acquire the majority of viral genome sequences . IlluminAnother sequencing method, used at CHOP, was Paragon. Paragon sequencing was carried out as in . BrieflyThe Thomas Jefferson University site carried out sequencing using the Illumina RPIP and CovidSeq methods essentially as per the manufacturer’s instructions. A nasopharyngeal swab specimen that was tested positive by PCR for SARS-CoV-2 was used for this study. Vaccine breakthrough specimens were identified by Jefferson Occupational Health Network for Employees and Students (JOHN) and were sequenced with a designation of VBT. Randomly selected residual positive specimens from the Molecular & Genomic Pathology Laboratory at Jefferson during the same period were sequenced for the purpose of epidemiology surveillance and labeled as SURV. RNA was extracted from 200 μl of the specimen and eluted in 110 μl using the bioMérieux EasyMag Extraction System, following EasyMag’s Generic protocol. Whole-genome sequencing for SARS-CoV-2 was subsequently performed at the Molecular & Genomic Pathology Laboratory at Thomas Jefferson University Hospital. With the exception of a few samples, samples were sequenced using Illumina COVIDSeq protocol (Illumina) following manufacturer’s guideline. Remaining specimens were sequenced using the Swift Normalase Amplicon Panels (SNAP) Core kit along with the SARS-CoV-2 Additional Genome Coverage Primer Panel following the manufacturer’s protocol. For data analysis, Illumina’s Local Run Manager’s GenerateFASTQ module was used to generate the fastq files for all specimens. The fastq files were transferred to UPenn’s secure data server for further processing to obtain QC metrics and lineage data.Samples with VSP numbers lower than VSP00256  were preTo process sequence data, sequence reads are trimmed to remove low quality base calls (< Q20) and aligned to the original Wuhan reference sequence (NC_045512.2) with the BWA aligner tool (v0.7.17) , after wVariants were assigned using the Pangolin lineage software (Pangolin version 3.1.11 with the PangoLEARN 2021-08-24 release). Note that these lineages are updated regularly and so are expected to change over time. Point mutations were assigned using a previously described bioinformatics pipeline .i, and assumes they are multinomially distributed where:i,w is the true proportion of variant or mutation i on week *,ww. p indicates the vector of all lineage probabilities for week w and n is the total number of variants or mutations observed. The underlying proportions for a given week are assumed to be centered around the proportions observed in the prior week (autoregressive) plus a change term that is itself correlated with the change observed in the prior week (moving average):changei,w is the change in log odds of variant or mutation i on week w. The initial starting proportions are given flat priors:Statistical analysis of variant enrichment and mutation enrichment in subsets of the data were separately assessed using a Bayesian model and Markov chain Monte Carlo sampling implemented in Stan . The modGamma prior distribution.The standard deviation for changes, σ, was given a w, were modeled as:iβ measures the log odds enrichment/depletion of variant or mutation i in the vaccine breakthrough population over the surveillance population, igroup indicates the WHO classification for Pango lineage i and groupiδ indicates the enrichment or depletion for a CDC VBM/VOC classification containing more than one Pango lineage, e.g., Delta containing B.1.617.2 and other variants with an AY prefix. In these data, Delta, Gamma and Non-VBM/VOC groupings contained more than one lineage above the abundance threshold. For WHO classifications containing only a single lineage, groupiδ was set to 0. The β and remaining δ were given DoubleExponential priors.To assess if there's enrichment of a particular variant or mutation in the vaccine breakthrough, counts of vaccine breakthrough samples for a given week, vaccineCountsw, modeled as:This was repeated equivalently in the samples collected as S gene target failures, with the counts, SFailCountsThe α were given DoubleExponential priors.δ terms.For the assessment of lineage enrichment, genomes from lineages that had more than 10 genomes assigned to them were included as their individual lineages, genomes from lineages with 10 or fewer assignments that were listed within VBM/VOC were included as miscellaneous classification for their particular WHO category, e.g., “Other delta” and all other genomes were grouped into an Other category. For the assessment of mutations, each mutation that was found in greater than 5% of genomes was independently modeled as above but replacing the Multinomial distributions with Binomial and removing the inapplicable lineage group https://doi.org/10.5281/zenodo.5888338. Sequence processing software and intermediate files used in this study are available at https://doi.org/10.5281/zenodo.5559699. A list of key reagents used in this study is in All viral genome sequences acquired in this study have been deposited in GISAID and at NCBI under accession numbers listed in 10.1128/mbio.03788-21.8TABLE S8Table S8, PDF file, 0.04 MB.Key reagents. Download Copyright © 2022 Marques et al.2022Marques et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the


N-oxide (TMAO) is linked to an increased risk for cardiovascular diseases. Trimethylamine (TMA), which is subsequently oxidized to TMAO in the liver, is formed by intestinal bacteria via distinct biochemical routes from dietary precursors that are enriched in animal product-based foods. To get a full picture of the entire process of the diet > gut microbiota > TMAO axis, we quantified potential TMA-forming gut bacteria and plasma metabolites using gene-targeted assays and targeted metabolomics on a subsample (n = 425) of a German population-based cohort study. We specifically compared persons reporting daily meat intake with those that rarely or never consume meat. While meat intake did not predict TMAO plasma levels in our study, two major bacterial TMA-forming pathways were linked to the metabolite’s concentration. Furthermore, advancing age was strongly associated with TMAO. Construction of a structural equation model allowed us to disentangle the different routes that promote higher TMAO levels with increasing age, demonstrating, for the first time, a functional role of gut microbiota in the process, where specific food items augmented abundances of TMA-forming bacteria that were associated with higher TMAO plasma concentrations. Analyses stratified by age showed an association between carotid intima-media thickness and TMAO only in individuals >65 of age, indicating that this group is particularly affected by the metabolite.The gut microbiota-dependent metabolite trimethylamine-IMPORTANCE Many cohort studies have investigated the link between diet and plasma TMAO levels, reporting incongruent results, while gut microbiota were only recently included into analyses. In these studies, taxonomic data were recorded that are not a good proxy for TMA formation, as specific members of various taxa exhibit genes catalyzing this reaction, demanding function-based technologies for accurate quantification of TMA-synthesizing bacteria. Using this approach, we demonstrated that abundances of the main components leading to TMAO formation, i.e., TMA precursors and TMA-forming bacteria, are uncoupled and not governed by the same (dietary) factors. Results emphasize that all levels leading to TMA(O) formation should be considered for accurate risk assessment, rejecting the simple view that diets rich in TMA precursors directly lead to increased plasma levels of this hazardous compound. The results can assist in developing strategies to reduce TMAO levels, specifically in the elderly, who are prone to TMAO-associated diseases. N-oxide (TMAO) has been linked to an increased risk for developing cardiovascular and renal diseases independently of traditional risk factors in several studies , carnitine oxygenase (CntA), and betaine reductase (GrdH) as the key enzymes of respective pathways , 8. WhilTo get a comprehensive picture of the entire diet > gut microbiota > TMAO axis, we quantified potential TMA-forming bacteria and plasma metabolites using gene-targeted assays and targeted metabolomics on samples from a German population-based cohort study. We specifically compared subjects who reported frequent meat consumption with those that rarely or never consumed meat in the general population.n = 425) were drawn from participants of two distinct dietary groups, i.e., persons who reported daily meat consumption versus those primarily fostering a vegetarian diet. Their main characteristics, along with plasma concentrations of the TMA precursors betaine, carnitine, and choline as well as TMAO and abundances of genes encoding betaine reductase (grdH), carnitine oxygenase (cntA), and choline TMA lyase (cutC) from main TMA-forming pathways of gut microbiota, are summarized in We investigated the associations of all major components of the diet > gut microbiota > TMA(O) axis, including data on general dietary habits and quantification of plasma metabolites as well as potential TMA-forming bacteria from 425 individuals of the SHIP cohort in northeast Germany. Samples  in individuals that reported daily meat consumption, whereas gene abundances of cutC and cntA displayed opposite patterns and were lower in abundance (P < 0.05) in that group; grdH abundance was not associated with any dietary group (P = 0.75) in our study. Increasing age, however, was associated with all variables, namely, TMA-forming bacteria , precursor concentrations , and TMAO levels (P < 0.01); descriptive visualizations of these associations based on stratified age groups are shown in P = 0.60), concentrations of both TMA precursors  and TMA-forming bacteria  were different between the two groups, even if adjusted for diet (P = 0.89). An observed positive association between body mass index (BMI) and TMAO concentrations (P = 0.032) was not sustained when adjusted for age (P = 0.274)  of ±5.31 μM observed . Out of ry group . Higher for diet . Sex was= 0.274) . BMI wasas found .10.1128/mSystems.00945-21.2TABLE S1grdH, cntA, and cutC) encoding key enzymes of main TMA-forming pathways in stool samples of subjects by age groups. Significance (p.age) was assessed based on continuous age data. Download Table S1, PDF file, 0.1 MB.Differences in plasma metabolite concentrations and abundances of genes  are given as well. Download Table S2, PDF file, 0.1 MB.Effect of sex on TMAO, TMA precursors, and TMA-producing bacteria. Diet and age were determined as important explanatory variables, and Copyright © 2021 Rath et al.2021Rath et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSystems.00945-21.4TABLE S3P values adjusted for this parameter (p.adj.age) are given as well. Download Table S3, PDF file, 0.1 MB.Effect of BMI on TMAO, TMA precursors, and TMA-producing bacteria. Age was determined as an important explanatory variable, and Copyright © 2021 Rath et al.2021Rath et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the P < 0.05) with gene abundances of cntA and cutC (P > 0.05), the consumption of specific foods, namely, salad (cntA), fruits (cutC), and vegetables (cutC), had a positive effect on gene abundances  are given as well. Download Table S4, PDF file, 0.1 MB.Effect of precursors and dietary items on TMA-producing bacteria. Age and sex were determined as important explanatory variables, and Copyright © 2021 Rath et al.2021Rath et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSystems.00945-21.6TABLE S5Table S5, PDF file, 0.1 MB.Effects of age on dietary habits, i.e., consumption of individual dietary items and contribution of overall dietary groups (rare meat consumption and daily meat consumption). Download Copyright © 2021 Rath et al.2021Rath et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 2 test, P > 0.05; comparative fit index [CFI] = 0.975; root mean square error of approximation [RMSEA] = 0.033; standardized root mean square residuals [SRMR] = 0.037). The model allowed us to disentangle different routes explaining the higher TMAO levels observed with increasing age while taking into account all other routes simultaneously. The results suggest that increased abundances of potential TMA-forming bacteria (route 1a) with progressing age play a role in the process and that higher abundances of those bacteria are governed by intake of certain foods, namely, salad, fruits, and vegetables (route 1b) that are associated with age (route 1c). This effect is present on top of the direct effect of age on TMAO, which is controlled for in this model (route 2). In other words, a part of the effect of age on TMAO levels is mediated through the abundance of TMA-forming bacteria. As univariable analyses indicated effects of sex on the variables included in the SEM, we estimated an additional multiple group model and compared it to the original (restricted) model. A likelihood ratio test indicated no significant increase in model fit  when sex was considered in the SEM.Based on results of pairwise comparisons between individual components across the TMAO formation process shown in P < 0.01) that was, however, not sustained when adjusted for age (P = 0.192). Interestingly, stratified analyses within different age groups revealed a significant association between TMAO and IMT in only the elderly (P = 0.018), defined as >65 years of age, indicating that the effects of TMAO are particularly pronounced in that group  as a marker of atherosclerosis. As expected, we found a significant association between the two parameters  formation process based on samples of a population-based cohort study in northeast Germany. For the first time, we were able to reveal a functional role of gut microbiota in this process. Furthermore, age was most strongly associated with plasma TMAO levels, as observed by many others in both animal experiments , 25 and Although we uncovered important aspects of increased TMAO formation with advancing age, SEM analysis indicated that other factors not considered here might also play a role. For instance, additional pathways for TMA formation, specifically from carnitine, were recently reported . Howeverin vitro investigations on human-derived material have highlighted the contribution of TMAO to endothelial senescence and vascular aging  and those never consuming meat (n = 33). The latter group was expanded by samples from participants that rarely eat meat (n = 181), defined as “once a month or less” (n = 80) and “a few times per month (but less than once a week)” (n = 101), in order to increase sample size. Subjects suffering from chronic kidney disease and those being treated with proton pump inhibitors were excluded, as they are known to have higher TMAO levels  and altered gut community profiles, respectively. Genes involved in TMA formation were analyzed in all stool samples, while plasma levels of choline, betaine, carnitine, and TMAO were measured only in 383, 390, 372, and 416 samples, respectively, due to sample volume limitations in plasma samples.Matched plasma and stool samples as well as associated metadata from 425 individuals were retrieved from the Study of Health in Pomerania (SHIP) , 33. Sam−1 in an artificial plasma matrix were prepared as well. All samples were analyzed using an Agilent 1290 Infinity II HPLC system coupled to an AB Sciex QTRAP 6500+ mass spectrometer. Peak areas of each sample and of the calibration curves were analyzed using MultiQuant 3.0 software (AB Sciex), and metabolites were identified based on respective ion pairs.Analytics were done using a label-free procedure as described previously . In briecutC, cntA, and grdH genes were targeted by degenerate primers and amplified in triplicate reactions in a LightCycler 480 (Roche Diagnostics) using the HOT FIREPol EvaGreen qPCR Mix Plus  and 10 ng of template DNA -derived values that were below the limit of detection were replaced by the lowest observed value divided by 2 for nguage R .The effects of microbiota on TMAO levels and of food intake on microbiota composition were assessed using linear regressions. In addition, all analyses were performed with age added as a covariate to adjust for confounding effects.cntA and cutC levels (bacteria), respectively. For both latent constructs, the unstandardized coefficient of the first predictor was fixed to one to assign a scale to the latent variable. Estimation was performed using weighted least squares with robust standard errors for robustness against deviations from normality. Model fit indices in terms of the model χ2, comparative fit index (CFI), root mean square error of approximation (RMSEA), and standardized root mean square residuals (SRMR) were computed to indicate model fit as recommended  to disentangle the complex interplay of effects. Analysis was performed using the package lavaan  in R. Diommended . However