Circulating bacterial peptides and linked metabolomic signatures are indicative of early mortality in pediatric cirrhosis.

BACKGROUND: Patients with pediatric cirrhosis-sepsis (PC-S) attain early mortality. Plasma bacterial composition, the cognate metabolites, and their contribution to the deterioration of patients with PC-S to early mortality are unknown. We aimed to delineate the plasma metaproteome-metabolome landscape and identify molecular indicators capable of segregating patients with PC-S predisposed to early mortality in plasma, and we further validated the selected metabolite panel in paired 1-drop blood samples using untargeted metaproteomics-metabolomics by UHPLC-HRMS followed by validation using machine-learning algorithms. METHODS: We enrolled 160 patients with liver diseases (cirrhosis-sepsis/nonsepsis [n=110] and noncirrhosis [n=50]) and performed untargeted metaproteomics-metabolomics on a training cohort of 110 patients (Cirrhosis-Sepsis/Nonsepsis, n=70 and noncirrhosis, n=40). The candidate predictors were validated on 2 test cohorts-T1 (plasma test cohort) and T2 (1-drop blood test cohort). Both T1 and T2 had 120 patients each, of which 70 were from the training cohort. RESULTS: Increased levels of tryptophan metabolites and Salmonella enterica and Escherichia coli-associated peptides segregated patients with cirrhosis. Increased levels of deoxyribose-1-phosphate, N5-citryl-d-ornithine, and Herbinix hemicellulolytic and Leifsonia xyli segregated patients with PC-S. MMCN-based integration analysis of WMCNA-WMpCNA identified key microbial-metabolic modules linked to PC-S nonsurvivors. Increased Indican, Staphylobillin, glucose-6-phosphate, 2-octenoylcarnitine, palmitic acid, and guanidoacetic acid along with L. xyli, Mycoplasma genitalium, and Hungateiclostridium thermocellum segregated PC-S nonsurvivors and superseded the liver disease severity indices with high accuracy, sensitivity, and specificity for mortality prediction using random forest machine-learning algorithm. CONCLUSIONS: Our study reveals a novel metabolite signature panel capable of segregating patients with PC-S predisposed to early mortality using as low as 1-drop blood.

Comparative evaluation of effectiveness of three versus four mini-implants for simultaneous intrusion and retraction of maxillary anterior teeth: A 3D FEM study.

OBJECTIVE: To evaluate and compare the displacement pattern of maxillary anterior teeth in the sagittal and vertical planes and evaluate the stress distribution in pdl, bone, teeth of the maxillary anterior region, and around the mini-implants during simultaneous en-masse retraction and intrusion using two, three, and four mini-implants combinations. METHODS: A three-dimensional FEM model of maxillary teeth and periodontal ligament housed in the alveolar bone with extracted first premolarswasgenerated. The models were broadly divided into three groups according to the number of mini-implants. Mini-implants were placed bilaterally between the second premolar and molar in group I, and along with bilateral implants, an additional mid-implant was placed between the central incisors as in group II, whereas in group III, anterior mini-implants were placed in between lateral incisors and canine bilaterally. RESULTS: The two mini-implant model showed the maximum amount of retraction in the sagittal plane followed by three and four mini-implant models. In the vertical plane, all six anterior teeth showed intrusion only in the four mini-implant model. The stress in cortical bone, cancellous bone, PDL, around the mini-implants, and in lateral incisor was maximum in the three mini-implant model, followed by four mini-implants with the least stress in the two mini-implant model. CONCLUSION: The four mini-implant model is better than the three and two mini-implants model as there is a more even distribution of force in the four mini-implants model as compared to the three mini-implants model.

Comparative metabolome analysis reveals higher potential of haemoperfusion adsorption in providing favourable outcome in ACLF patients.

BACKGROUND AND AIMS: Acute-on-chronic liver failure (ACLF) is a serious illness associated with altered metabolome, organ failure and high mortality. Need for therapies to improve the metabolic milieu and support liver regeneration are urgently needed. METHODS: We investigated the ability of haemoperfusion adsorption (HA) and therapeutic plasma exchange (TPE) in improving the metabolic profile and survival in ACLF patients. Altogether, 45 ACLF patients were randomized into three groups: standard medical therapy (SMT), HA and TPE groups. Plasma metabolomics was performed at baseline, post-HA and TPE sessions on days 7 and 14 using high-resolution mass spectrometry. RESULTS: The baseline clinical/metabolic profiles of study groups were comparable. We identified 477 metabolites. Of these, 256 metabolites were significantly altered post 7 days of HA therapy (p < .05, FC > 1.5) and significantly reduced metabolites linked to purine (12 metabolites), tryptophan (7 metabolites), primary bile acid (6 metabolites) and arginine-proline metabolism (6 metabolites) and microbial metabolism respectively (p < .05). Metabolites linked to taurine-hypotaurine and histidine metabolism were reduced and temporal increase in metabolites linked to phenylalanine and tryptophan metabolism was observed post-TPE therapy (p < .05). Finally, weighted metabolite correlation network analysis (WMCNA) along with inter/intragroup analysis confirmed significant reduction in inflammatory (tryptophan, arachidonic acid and bile acid metabolism) and secondary energy metabolic pathways post-HA therapy compared to TPE and SMT (p < .05). Higher baseline plasma level of 11-deoxycorticosterone (C03205; AUROC > 0.90, HR > 3.2) correlated with severity (r2 > 0.5, p < .05) and mortality (log-rank-p < .05). Notably, 51 of the 64 metabolite signatures (ACLF non-survivor) were reversed post-HA treatment compared to TPE and SMT(p < .05). CONCLUSION: HA more potentially (~80%) improves plasma milieu compared to TPE and SMT. High baseline plasma 11-deoxycorticosterone level correlates with early mortality in ACLF patients.

Ocimum sanctum Alters the Lipid Landscape of the Brain Cortex and Plasma to Ameliorate the Effect of Photothrombotic Stroke in a Mouse Model.

Stroke-like injuries in the brain result in not only cell death at the site of the injury but also other detrimental structural and molecular changes in regions around the stroke. A stroke-induced alteration in the lipid profile interferes with neuronal functions such as neurotransmission. Preventing these unfavorable changes is important for recovery. Ocimum sanctum (Tulsi extract) is known to have anti-inflammatory and neuroprotective properties. It is possible that Tulsi imparts a neuroprotective effect through the lipophilic transfer of active ingredients into the brain. Hence, we examined alterations in the lipid profile in the cerebral cortex as well as the plasma of mice with a photothrombotic-ischemic-stroke-like injury following the administration of a Tulsi extract. It is also possible that the lipids present in the Tulsi extract could contribute to the lipophilic transfer of active ingredients into the brain. Therefore, to identify the major lipid species in the Tulsi extract, we performed metabolomic and untargeted lipidomic analyses on the Tulsi extract. The presence of 39 molecular lipid species was detected in the Tulsi extract. We then examined the effect of a treatment using the Tulsi extract on the untargeted lipidomic profile of the brain and plasma following photothrombotic ischemic stroke in a mouse model. Mice of the C57Bl/6j strain, aged 2-3 months, were randomly divided into four groups: (i) Sham, (ii) Lesion, (iii) Lesion plus Tulsi, and (iv) Lesion plus Ibuprofen. The cerebral cortex of the lesioned hemisphere of the brain and plasma samples were collected for untargeted lipidomic profiling using a Q-Exactive Mass Spectrometer. Our results documented significant alterations in major lipid groups, including PE, PC, neutral glycerolipids, PS, and P-glycerol, in the brain and plasma samples from the photothrombotic stroke mice following their treatment with Tulsi. Upon further comparison between the different study groups of mice, levels of MGDG (36:4), which may assist in recovery, were found to be increased in the brain cortexes of the mice treated with Tulsi when compared to the other groups (p < 0.05). Lipid species such as PS, PE, LPG, and PI were commonly altered in the Sham and Lesion plus Tulsi groups. The brain samples from the Sham group were specifically enriched in many species of glycerol lipids and had reduced PE species, while their plasma samples showed altered PE and PS species when compared to the Lesion group. LPC (16:1) was found in the Tulsi extract and was significantly increased in the brains of the PTL-plus-Tulsi-treated group. Our results suggest that the neuroprotective effect of Tulsi on cerebral ischemia may be partially associated with its ability to regulate brain and plasma lipids, and these results may help provide critical insights into therapeutic options for cerebral ischemia or brain lesions.

Biomolecular map of albumin identifies signatures of severity and early mortality in acute liver failure.

BACKGROUND & AIMS: Acute liver failure (ALF) is associated with high mortality. Alterations in albumin structure and function have been shown to correlate with outcomes in cirrhosis. We undertook a biomolecular analysis of albumin to determine its correlation with hepatocellular injury and early mortality in ALF. METHODS: Altogether, 225 participants (200 patients with ALF and 25 healthy controls [HC]) were enrolled. Albumin was purified from the baseline plasma of the training cohort (ALF, n = 40; survivors, n = 8; non-survivors, n = 32; and HC, n = 5); analysed for modifications, functionality, and bound multi-omics signatures; and validated in a test cohort (ALF, n = 160; survivors, n = 53; non-survivors, n = 107; and HC, n = 20). RESULTS: In patients with ALF, albumin is more oxidised and glycosylated with a distinct multi-omics profile than that in HC, more so in non-survivors (p <0.05). In non-survivors, albumin was more often bound (p <0.05, false discovery rate <0.01) to proteins associated with inflammation, advanced glycation end product, metabolites linked to arginine, proline metabolism, bile acid, and mitochondrial breakdown products. Increased bacterial taxa (Listeria, Clostridium, etc.) correlated with lipids (triglycerides [4:0/12:0/12:0] and phosphatidylserine [39:0]) and metabolites (porphobilinogen and nicotinic acid) in non-survivors (r2 >0.7). Multi-omics signature-based probability of detection for non-survival was >90% and showed direct correlation with albumin functionality and clinical parameters (r2 >0.85). Probability-of-detection metabolites built on the top five metabolites, namely, nicotinic acid, l-acetyl carnitine, l-carnitine, pregnenolone sulfate, and N-(3-hydroxybutanoyl)-l-homoserine lactone, showed diagnostic accuracy of 98% (AUC 0.98, 95% CI 0.95-1.0) and distinguish patients with ALF predisposed to early mortality (log-rank <0.05). On validation using high-resolution mass spectrometry and five machine learning algorithms in test cohort 1 (plasma and paired one-drop blood), the metabolome panel showed >92% accuracy/sensitivity and specificity for prediction of mortality. CONCLUSIONS: In ALF, albumin is hyperoxidised and substantially dysfunctional. Our study outlines distinct 'albuminome' signatures capable of distinguishing patients with ALF predisposed to early mortality or requiring emergency liver transplantation. IMPACTS AND IMPLICATIONS: Here, we report that the biomolecular map of albumin is distinct and linked to severity and outcome in patients with acute liver failure (ALF). Detailed structural, functional, and albumin-omics analysis in patients with ALF led to the identification and classification of albumin-bound biomolecules, which could segregate patients with ALF predisposed to early mortality. More importantly, we found albumin-bound metabolites indicative of mitochondrial damage and hyperinflammation as a putative indicator of <30-day mortality in patients with ALF. This preclinical study validates the utility of albuminome analysis for understanding the pathophysiology and development of poor outcome indicators in patients with ALF.

Small molecule bio-signature in childhood intra-thoracic tuberculosis identified by metabolomics.

The diagnosis of pediatric tuberculosis (TB) remains a major challenge, hence the evaluation of new tools for improved diagnostics is urgently required. We investigated the serum metabolic profile of children with culture-confirmed intra-thoracic TB (ITTB) (n = 23) and compared it with those of non-TB controls (NTCs) (n = 13) using proton NMR spectroscopy-based targeted and untargeted metabolomics approaches. In targeted metabolic profiling, five metabolites (histidine, glycerophosphocholine, creatine/phosphocreatine, acetate, and choline) differentiated TB children from NTCs. Additionally, seven discriminatory metabolites (N-α-acetyl-lysine, polyunsaturated fatty acids, phenylalanine, lysine, lipids, glutamate + glutamine, and dimethylglycine) were identified in untargeted metabolic profiling. The pathway analysis revealed alterations in six metabolic pathways. The altered metabolites were associated with impaired protein synthesis, hindered anti-inflammatory and cytoprotective mechanisms, abnormalities in energy generation processes and membrane metabolism, and deregulated fatty acid and lipid metabolisms in children with ITTB. The diagnostic significance of the classification models obtained from significantly distinguishing metabolites showed sensitivity, specificity, and area under the curve of 78.2%, 84.6%, and 0.86, respectively, in the targeted profiling and 92.3%, 100%, and 0.99, respectively, in the untargeted profiling. Our findings highlight detectable metabolic changes in childhood ITTB; however, further validation is warranted in a large cohort of the pediatric population.

The effects of dual antiplatelet therapy (DAPT) adherence on survival in patients undergoing revascularization and the determinants of DAPT adherence.

BACKGROUND: The prevalence and burden of coronary heart disease (CHD) has increased substantially in India, accompanied with increasing need for percutaneous coronary interventions (PCI). Although a large government-funded insurance scheme in Maharashtra, India covered the cost of PCI for low-income patients, the high cost of post-PCI treatment, especially Dual Antiplatelet Therapy (DAPT), still caused many patients to prematurely discontinue the secondary prevention. Our study aimed to investigate the effectiveness of DAPT adherence on all-cause mortality among post-PCI patients and explore the potential determinants of DAPT adherence in India. METHOD: We collected clinical data of 4,595 patients undergoing PCI in 110 participating medical centers in Maharashtra, India from 2012 to 2015 by electronic medical records. We surveyed 2527 adult patients who were under the insurance scheme by telephone interview, usually between 6 to 12 months after their revascularization. Patients reporting DAPT continuation in the telephone survey were categorized as DAPT adherence. The outcome of the interest was all-cause mortality within 1 year after the index procedure. Multivariate Cox proportional hazard (PH) model with adjustment of potential confounders and standardization were used to explore the effects of DAPT adherence on all-cause mortality. We further used a multivariate logistic model to investigate the potential determinants of DAPT adherence. RESULTS: Out of the 2527 patients interviewed, 2064 patients were included in the analysis, of whom 470 (22.8%) discontinued DAPT prematurely within a year. After adjustment for baseline confounders, DAPT adherence was associated with lower one-year all-cause mortality compared to premature discontinuation (less than 6-month), with an adjusted hazard ratio (HR) of 0.52 (95% Confidence Interval (CI) = (0.36, 0.67)). We also found younger patients (OR per year was 0.99 (0.97, 1.00)) and male (vs. female, OR of 1.30 (0.99, 1.70)) had higher adherence to DAPT at one year as did patients taking antihypertensive medications (vs. non medication, OR of 1.57 (1.25, 1.95)). CONCLUSION: These findings suggest the protective effects of DAPT adherence on 1-year mortality among post-PCI patients in a low-income setting and indicate younger age, male sex and use of other preventive treatments were predictors of higher DAPT adherence.

Clinical use of multiplex-PCR for the diagnosis of acute bacterial meningitis.

Background and Objectives: Prompt and accurate diagnosis of acute bacterial meningitis (ABM) is critical for patient management. We designed and evaluated two sets of multiplex-PCR assays for the simultaneous detection of six major etiologies of ABM i.e., Streptococcus pneumoniae, Haemophilus influenzae type b, and Neisseria meningitidis in one set and Listeria monocytogenes, Streptococcus agalactiae, and Escherichia coli in another set of multiplex-PCR in CSF of patients with suspected ABM. Methods: A total of 113 CSF specimens from patients of all ages having clinical features suggestive of meningitis were tested for bacteriological evidence by Gram's smear, culture, and our designed multiplex-PCR. Results: Multiplex-PCR assay performed excellently by increasing the overall detection rate by 6% when compared to culture as of total 113 samples tested, 17 (15%) were positive by multiplex-PCR whereas only 9% (10/113) were positive by culture. It detected the DNA in eight culture negative samples revealing the presence of S. pneumoniae in three and other possible bacterial pathogens in five of them. Our assay showed a DNA detection limit of 1 pg/µL. Compared to CSF culture, the sensitivity and specificity of the multiplex-PCR were 90% and 92.2%, respectively. Conclusion: This study accentuates the importance of multiplex-PCR assay that is efficiently fast and reliable for the diagnosis of acute bacterial meningitis that can substantially improve the diagnosis in culture negative cases, especially in patients who were previously started on antimicrobial therapy.

Bile multi-omics analysis classifies lipid species and microbial peptides predictive of carcinoma of gallbladder.

BACKGROUND AND AIMS: Histopathological examination is the gold standard for detection of gallstone (GS) or gallbladder carcinoma (CAGB). Bile concentrated in the gallbladder (GB) is expected to recapitulate metagenomics and molecular changes associated with development of CAGB. APPROACH AND RESULTS: Bile samples were screened for lipidomics and metaproteome (metagenomics) signatures capable of early detection of cancer in GB anomalies. Analysis of the training cohort (n = 87) showed that metastability of bile was reduced in CAGB (p < 0.05). CAGB bile showed significant alteration of lipidome and microbiome as indicated by multivariate partial least squares regression analysis and alpha-diversity and beta-diversity indexes (p < 0.05). Significant reduction of lipid species and increase in bacterial taxa were found to be associated with patients with CAGB, CAGB with GS, and GS (p < 0.05, log fold change >1.5). A multimodular correlation network created using weighted lipid/metaproteomic correlation network analysis showed striking associations between lipid and metaproteomic modules and functionality. CAGB-linked metaproteomic modules/functionality directly correlated with lipid modules, species, clinical parameters, and bile acid profile (p < 0.05). Increased bacterial taxa (Leptospira, Salmonella enterica, Mycoplasma gallisepticum) and their functionality showed a direct correlation with lipid classes such as lysophosphatidylinositol, ceramide 1-phosphate, and lysophosphatidylethanolamine and development of CAGB (r2  > 0.85). Lipid/metaproteomic signature-based probability of detection for CAGB was > 90%, whereas that for GS was > 80% (p < 0.05). Validation of eight lipid species using four machine learning algorithms in two separate cohorts (n = 38; bile [test cohort 1] and paired plasma [test cohort 2]) showed accuracy (99%) and sensitivity/specificity (>98%) for CAGB detection. CONCLUSIONS: Bile samples of patients with CAGB showed significant reduction in lipid species and increase in bacterial taxa. Our study identifies a core set of bile lipidome and metaproteome signatures which may offer universal utility for early diagnosis of CAGB.

Yme2, a putative RNA recognition motif and AAA+ domain containing protein, genetically interacts with the mitochondrial protein export machinery.

The mitochondrial respiratory chain is composed of nuclear as well as mitochondrial-encoded subunits. A variety of factors mediate co-translational integration of mtDNA-encoded proteins into the inner membrane. In Saccharomyces cerevisiae, Mdm38 and Mba1 are ribosome acceptors that recruit the mitochondrial ribosome to the inner membrane, where the insertase Oxa1, facilitates membrane integration of client proteins. The protein Yme2 has previously been shown to be localized in the inner mitochondrial membrane and has been implicated in mitochondrial protein biogenesis, but its mode of action remains unclear. Here, we show that multiple copies of Yme2 assemble into a high molecular weight complex. Using a combination of bioinformatics and mutational analyses, we find that Yme2 possesses an RNA recognition motif (RRM), which faces the mitochondrial matrix and a AAA+ domain that is located in the intermembrane space. We further show that YME2 genetically interacts with MDM38, MBA1 and OXA1, which links the function of Yme2 to the mitochondrial protein biogenesis machinery.

Protocol for global proteome, virome, and metaproteome profiling of respiratory specimen (VTM) in COVID-19 patient by LC-MS/MS-based analysis.

In this protocol, we describe global proteome profiling for the respiratory specimen of COVID-19 patients, patients suspected with COVID-19, and H1N1 patients. In this protocol, details for identifying host, viral, or bacterial proteome (Meta-proteome) are provided. Major steps of the protocol include virus inactivation, protein quantification and digestion, desalting of peptides, high-resolution mass spectrometry (HRMS)-based analysis, and downstream bioinformatics analysis. For complete details on the use and execution of this profile, please refer to Maras et al. (2021).

Global metabolome profiling of COVID-19 respiratory specimen using high-resolution mass spectrometry (HRMS).

Here we describe a protocol for identifying metabolites in respiratory specimens of patients that are SARS-CoV-2 positive, SARS-CoV-2 negative, or H1N1 positive. This protocol provides step-by-step instructions on sample collection from patients, followed by metabolite extraction. We use ultra-high-pressure liquid chromatography (UHPLC) coupled with high-resolution mass spectrometry (HRMS) for data acquisition and describe the steps for data analysis. The protocol was standardized with specific customization for SARS-CoV-2-containing respiratory specimens. For complete details on the use and execution of this protocol, please refer to Maras et al. (2021).

Baseline Plasma Metabotype Correlates With Direct-Acting Antiviral Therapy Nonresponse for HCV in HIV-HCV Coinfected Patients.

Introduction: With the advent of direct-acting antiviral (DAA) therapy for HCV, the cure is achieved at similar rates among HIV-HCV coinfected patients as in HCV mono-infected patients. The present study evaluates host plasma metabolites as putative indicators in predicting the treatment response in baseline HIV-HCV patients. Methods: Non-cirrhotic HIV-HCV (N = 43) coinfected patients were treated with sofosbuvir and daclatasvir for 12 weeks. Plasma metabolite profiling of pre- and post-therapy was analyzed in 20/43 patients. Of the 20 selected, 10 (50%) attained the sustained viral response [(SVR) (responders)] as defined by the absence of HCV RNA at 12 weeks after the treatment, and 10 (50%) did not attain the cure for HCV (nonresponders). Results: A total of 563 features were annotated (metabolomic/spectral databases). Before therapy, 39 metabolites differentiated (FC ±1.5, p < 0.05) nonresponders from responders. Of these, 20 upregulated and 19 downregulated were associated with tryptophan metabolism, nicotinamide metabolism, and others. Post therapy, 62 plasma metabolites (12 upregulated and 50 downregulated, FC±1.5, p < 0.05) differentiated nonresponders from responders and highlighted a significant increase in the steroid and histidine metabolism and significant decrease in tryptophan metabolism and ascorbate and pyruvate metabolism in the nonresponders. Based on random forest and multivariate linear regression analysis, the baseline level of N-acetylspermidine (FC > 2, AUC = 0.940, Bfactor = -0.267) and 2-acetolactate (FC > 2, AUC = 0.880, Bfactor = -0.713) significantly differentiated between nonresponders from responders in HIV-HCV coinfected patients and was able to predict the failure of treatment response. Conclusion: Increased baseline levels of N-acetylspermidine and 2-acetolactate levels are associated with the likeliness of failure to attain the cure for HCV in HIV-HCV coinfected patients.

Attitude toward Public Health Dentistry as a career among dental students in Odisha: A Cross-sectional study.

BACKGROUND: Knowledge of dental students' expectations of their profession as well as their attitudes to study a particular specialty of dentistry is of great importance. These attitudes and expectations make studying dentistry meaningful to dental students and society and understanding these factors facilitate workforce planning in the dental sector The aim of the study was to assess the attitude of dental students towards considering Public Health Dentistry as their future career. MATERIALS AND METHODS: A questionnaire-based, cross-sectional survey was conducted, which included the 3rd year, 4th fourth year and dental interns studying in the State of Odisha. It consisted of 27 questions that were graded on 5-point Likert scale. The responses for the attitude questions toward selecting Public Health Dentistry for postgraduation were categorized into three factors, which are a negative attitude (includes score 0-21), neutral attitude (score 22-44), and positive attitude (score 45-64). Differences between groups were examined using Chi-square test for proportions. The level of statistical significance was set at P < 0.05. RESULTS: Among 886 respondents, 302 (34.08%) were males and 584 (65.91%) were females. One-third (33.52%) of them had a positive attitude toward selecting public health dentistry as their future career, and nearly two-third of them (58.23%) had neutral attitude, with very few students having a negative attitude (8.23%) toward the specialty for pursuing postgraduation. CONCLUSION: Respondents had a considerable amount of interest in pursuing postgraduation in this specialty. Efforts should be intensified, both by the dental council and by the dental colleges, to develop this specialty, keeping in mind the increasing attitude of dental undergraduates toward it.

Boswellic acid induces epigenetic alterations by modulating DNA methylation in colorectal cancer cells.

Accumulating evidence suggests that chemopreventive effects of some dietary polyphenols may in part be mediated by their ability to influence epigenetic mechanisms in cancer cells. Boswellic acids, derived from the plant Boswellia serrata, have long been used for the treatment of various inflammatory diseases due to their potent anti-inflammatory activities. Recent preclinical studies have also suggested that this compound has anti-cancer potential against various malignancies. However, the precise molecular mechanisms underlying their anti-cancer effects remain elusive. Herein, we report that boswellic acids modulate DNA methylation status of several tumor suppressor genes in colorectal cancer (CRC) cells. We treated RKO, SW48 and SW480 CRC cell lines with the active principle present in boswellic acids, acetyl-keto-ß-boswellic acid (AKBA). Using genome-wide DNA methylation and gene expression microarray analyses, we discovered that AKBA induced a modest genome-wide demethylation that permitted simultaneous re-activation of the corresponding tumor suppressor genes. The quantitative methylation-specific PCR and RT-PCR validated the gene demethylation and re-expression in several putative tumor suppressor genes including SAMD14 and SMPD3. Furthermore, AKBA inhibited DNMT activity in CRC cells. Taken together, these results lend further support to the growing notion that anti-cancer effect of boswellic acids may in part be due to its ability to demethylate and reactivate methylation-silenced tumor suppressor genes. These results suggest that not only boswellic acid might be a promising epigenetic modulator in the chemoprevention and treatment of CRC, but also provide a rationale for future investigations on the usefulness of such botanicals for epigenetic therapy in other human malignancies.