Interrogation of the mammalian gut-brain axis using LC-MS/MS-based targeted metabolomics with in vitro bacterial and organoid cultures and in vivo gnotobiotic mouse models.

Interest in the communication between the gastrointestinal tract and central nervous system, known as the gut-brain axis, has prompted the development of quantitative analytical platforms to analyze microbe- and host-derived signals. This protocol enables investigations into connections between microbial colonization and intestinal and brain neurotransmitters and contains strategies for the comprehensive evaluation of metabolites in in vitro (organoids) and in vivo mouse model systems. Here we present an optimized workflow that includes procedures for preparing these gut-brain axis model systems: (stage 1) growth of microbes in defined media; (stage 2) microinjection of intestinal organoids; and (stage 3) generation of animal models including germ-free (no microbes), specific-pathogen-free (complete gut microbiota) and specific-pathogen-free re-conventionalized (germ-free mice associated with a complete gut microbiota from a specific-pathogen-free mouse), and Bifidobacterium dentium and Bacteroides ovatus mono-associated mice (germ-free mice colonized with a single gut microbe). We describe targeted liquid chromatography-tandem mass spectrometry-based metabolomics methods for analyzing microbially derived short-chain fatty acids and neurotransmitters from these samples. Unlike other protocols that commonly examine only stool samples, this protocol includes bacterial cultures, organoid cultures and in vivo samples, in addition to monitoring the metabolite content of stool samples. The incorporation of three experimental models (microbes, organoids and animals) enhances the impact of this protocol. The protocol requires 3 weeks of murine colonization with microbes and ~1-2 weeks for liquid chromatography-tandem mass spectrometry-based instrumental and quantitative analysis, and sample post-processing and normalization.

Unraveling the Metabolic Requirements of the Gut Commensal Bacteroides ovatus.

Background: Bacteroidetes are the most common bacterial phylum in the mammalian intestine and the effects of several Bacteroides spp. on multiple facets of host physiology have been previously described. Of the Bacteroides spp., Bacteroides ovatus has recently garnered attention due to its beneficial effects in the context of intestinal inflammation. In this study, we aimed to examine model host intestinal physiological conditions and dietary modifications to characterize their effects on B. ovatus growth. Methods and Results: Using Biolog phenotypic microarrays, we evaluated 62 primary carbon sources and determined that B. ovatus ATCC 8384 can use the following carbohydrates as primary carbon sources: 10 disaccharides, 4 trisaccharides, 4 polysaccharides, 4 polymers, 3 L-linked sugars, 6 D-linked sugars, 5 amino-sugars, 6 alcohol sugars, and 15 organic acids. Proteomic profiling of B. ovatus bacteria revealed that a significant portion of the B. ovatus proteome contains proteins important for metabolism. Among the proteins, we found glycosyl hydrolase (GH) familes GH2, GH5, GH20, GH 43, GH88, GH92, and GH95. We also identified multiple proteins with antioxidant properties and reasoned that these proteins may support B. ovatus growth in the GI tract. Upon further testing, we showed that B. ovatus grew robustly in various pH, osmolarity, bile, ethanol, and H2O2 concentrations; indicating that B. ovatus is a well-adapted gut microbe. Conclusion: Taken together, we have demonstrated that key host and diet-derived changes in the intestinal environment influence B. ovatus growth. These data provide the framework for future work toward understanding how diet and lifestyle interventions may promote a beneficial environment for B. ovatus growth.

Health disparity and COVID-19-A retrospective analysis.

BACKGROUND AND AIMS: According to the World Health Organization (WHO), more than 75.7 million confirmed cases of coronavirus disease 2019 (COVID-19), a global pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), have been reported so far. Researchers are working relentlessly to find effective solutions to this catastrophe, using genomic sequence-based investigation, immunological analysis, and more. The role of health disparity has also emerged as an intriguing factor that made a huge impact on the lives of people. METHODS: We analyzed various factors that triggered the health disparity in the United States of America along with the rate of COVID-19 morbidity and mortality. Furthermore, we have also focused on the State of Mississippi, which is suffering from an extreme health disparity. Data have been obtained from publicly available data sources including, Center for Disease Control and Prevention and Mississippi State Department of Health. Correlation analysis of the dataset has been performed using R software. RESULTS: Our analysis suggested that the COVID-19 infection rate per 100 000 people is directly correlated with the increasing number of the African American population in the United States. We have found a strong correlation between the obesity and the COVID-19 cases as well. All the counties in Mississippi demonstrate a strong correlation between a higher number of African American population to COVID-19 cases and obesity. Our data also indicate that a higher number of African American populations are facing socioeconomic disadvantages, which enhance their chances of becoming vulnerable to pre-existing ailments such as obesity, type-2 diabetes, and cardiovascular diseases. CONCLUSION: We proposed a possible explanation of increased COVID-19 infectivity in the African American population in the United States. This work has highlighted the intriguing factors that increased the health disparity at the time of the COVID-19 pandemic.

The metabolic profile of Bifidobacterium dentium reflects its status as a human gut commensal.

BACKGROUND: Bifidobacteria are commensal microbes of the mammalian gastrointestinal tract. In this study, we aimed to identify the intestinal colonization mechanisms and key metabolic pathways implemented by Bifidobacterium dentium. RESULTS: B. dentium displayed acid resistance, with high viability over a pH range from 4 to 7; findings that correlated to the expression of Na+/H+ antiporters within the B. dentium genome. B. dentium was found to adhere to human MUC2+ mucus and harbor mucin-binding proteins. Using microbial phenotyping microarrays and fully-defined media, we demonstrated that in the absence of glucose, B. dentium could metabolize a variety of nutrient sources. Many of these nutrient sources were plant-based, suggesting that B. dentium can consume dietary substances. In contrast to other bifidobacteria, B. dentium was largely unable to grow on compounds found in human mucus; a finding that was supported by its glycosyl hydrolase (GH) profile. Of the proteins identified in B. dentium by proteomic analysis, a large cohort of proteins were associated with diverse metabolic pathways, indicating metabolic plasticity which supports colonization of the dynamic gastrointestinal environment. CONCLUSIONS: Taken together, we conclude that B. dentium is well adapted for commensalism in the gastrointestinal tract.

Revascularization of a Nonvital, Immature Permanent Tooth Using Amniotic Membrane: A Novel Approach.

AIM: To evaluate the clinical and radiological results of a revascularization treatment done in a nonvital, immature permanent tooth using the amniotic membrane. CASE DESCRIPTION: A 12-year-old boy reported with a complaint of pain in the lower left back tooth since 1 month due to dental caries. On clinical examination, the mandibular left second premolar was tender on percussion and discolored due to dental caries. Radiographic examination of the same tooth revealed open apex, thin root dentinal walls, and periodontal ligament widening. An access opening was prepared, necrotic pulp extirpated completely followed by thorough irrigation. After drying the canal, closed dressing with the 3-mix antibacterial paste was given for 15 days. After 15 days, the paste was removed and the amniotic membrane was placed inside the canal extending 1 mm beyond the apex and mineral tri-oxide aggregate was placed over this followed by sealing of the cavity. The patient was followed up at 1, 3, and 6 months for clinical and radiographic evaluation. CONCLUSION: After 6 months, clinically the tooth found asymptomatic. Radiographic examination showed continued root elongation, closure in the periapical opening, thick root dentinal walls with narrowing of the canal space, and normal periradicular architecture. CLINICAL SIGNIFICANCE: Amniotic membrane can be used as a scaffold for revascularization in nonvital immature teeth. HOW TO CITE THIS ARTICLE: NB Nagaveni, P Poornima, et al. Revascularization of a Nonvital, Immature Permanent Tooth Using Amniotic Membrane: A Novel Approach. Int J Clin Pediatr Dent 2019;12(2):150-152.

Comparative Analysis of CPP-ACP, Tricalcium Phosphate, and Hydroxyapatite on Assessment of Dentinal Tubule Occlusion on Primary Enamel Using SEM: An In Vitro Study.

BACKGROUND: Over the last few decades, fluoride in various forms has been proved to reduce caries and dentinal hypersensitivity in both the primary and permanent dentitions. Recently, newer materials containing calcium and phosphate ions, tricalcium phosphate, and hydroxyapatite has received much attention. AIM: The aim of the study was to compare CPP-ACP, tricalcium phosphate, and hydroxyapatite in relation to the assessment of dentine tubule occlusion on primary enamel using scanning electron microscope (SEM). MATERIALS AND METHODS: Forty freshly extracted noncarious primary molars were randomly divided into 4 groups (I-IV) with 10 sections in each group-group I: negative control, group II: CPP-ACP, group III: tricalcium phosphate, group IV: hydroxyapatite. To assess tubule occlusion, twenty dentin sections of 2-mm thickness were obtained from the cervical third of sound primary molars. Each section was processed to simulate the hypersensitive dentin and the test agents were brushed over the sections with an electric toothbrush and observed under a SEM for calculation of the percentage of occluded tubules. RESULTS: Groups II and IV showed a greater percentage of tubule occlusion than group III. An intergroup comparison of tubule occlusion potential of groups II and IV was not significant. CONCLUSION: Hydroxyapatite showed significantly higher dentinal tubule occlusion when compared to CPP-ACP and tricalcium phosphate. HOW TO CITE THIS ARTICLE: Shah R, Bajaj M. Comparative Analysis of CPP-ACP, Tricalcium Phosphate, and Hydroxyapatite on Assessment of Dentinal Tubule Occlusion on Primary Enamel Using SEM: An In Vitro Study. Int J Clin Pediatr Dent 2019;12(5):371-374.

Comparison of CPP-ACP, Tri-Calcium Phosphate and Hydroxyapatite on Remineralization of Artificial Caries Like Lesions on Primary Enamel -An in vitro Study.

OBJECTIVES: To compare CPP-ACP, Tri-calcium phosphate and Hydroxyapatite on remineralization of artificial caries like lesions on primary enamel. STUDY DESIGN: Ten extracted Primary molars coated with nail varnish, leaving a window of 2×4 mm on buccal and lingual surface were immersed in demineralizing solution for 96 hours and sectioned longitudinally to obtain 40 sections (4 sections per tooth) and were randomly divided into 4 groups (A to D) n=10; Group A: negative control, Group B: CPP-ACP, Group C: Tri-calcium phosphate, Group D: Hydroxyapatite. Sections were subjected to pH cycling for 10 days and were evaluated by polarized light microscope before and after treatment. RESULTS: Intra group comparison of demineralization and remineralization was done by paired t-test. One way ANOVA was used for multiple group comparisons followed by post HOC TUKEY'S Test for group wise comparisons. Remineralization was found more with Group D followed by Group B, C and A. CONCLUSION: Hydroxyapatite showed better remineralization when compared to CPP-ACP and Tri-calcium phosphate.