scholarly journals Role of GDNF, GFRα1 and GFAP in a Bifidobacterium-Intervention Induced Mouse Model of Intestinal Neuronal Dysplasia

2022 ◽  
Vol 9 ◽  
Author(s):  
Wei Liu ◽  
Tingting Zhou ◽  
Jinqiu Tian ◽  
Xiaofang Yu ◽  
Chuantao Ren ◽  
...  
Keyword(s):  
Mouse Model ◽  
Colonic Motility ◽  
Gene Sequencing ◽  
Abdominal Circumference ◽  
Intestinal Neuronal Dysplasia ◽  
Rrna Gene ◽  
Activation Process ◽  
Altered Expression ◽  
Enteric Glial Cells ◽  
Neuronal Dysplasia

ObjectiveTo investigate the effects of glial cell-derived neurotrophic factor (GDNF), GDNF family receptor alpha 1 (GFRα1), and glial fibrillary acidic protein (GFAP) on colonic motility in a mouse model of intestinal neuronal dysplasia by intervention with Bifidobacterium and to explore the influence of Bifidobacterium on enteric glial cells (EGCs).MethodsWestern blotting and qRT-PCR were employed to detect the expression of GFRα1 and GFAP in colonic tissues of mice with or without Tlx2 mutations, and ELISA was used to detect the expression of GDNF in serum. IHC was used to detect the appearance of the ganglion cells. Subsequently, Tlx2 homozygous mutant (Tlx2−/−) mice were treated with Bifidobacterium. Colonic motility was measured before and after intervention by measuring the glass bead expelling time. The variations in abdominal circumference and GDNF, GFRα1, and GFAP expression were measured. In addition, 16SrRNA gene sequencing was performed to detect the abundance of the intestinal microbiota.ResultsThe mRNA and protein expression of GFRα1 and GFAP was decreased in the colonic tissues of Tlx2−/− mice and GDNF expression was decreased in serum compared with Tlx2+/− and WT mice. After confirming the colonization of Bifidobacterium by 16S rRNA gene sequencing, the expelling time and abdominal distension were ameliorated, and the expression of GFAP, GDNF, and GFRα1 was increased.ConclusionsThe expression of GDNF, GFRα1, and GFAP is associated with colonic motility. The altered expression of EGC-related factors suggested that Bifidobacterium may be involved in the EGC activation process. The amelioration of IND symptoms after intervention with Bifidobacterium prompted the elicitation of adjuvant therapy.

scholarly journals Functional lower airways genomic profiling of the microbiome to capture active microbial metabolism

2021 ◽  
pp. 2003434
Author(s):  
Imran Sulaiman ◽  
Benjamin G. Wu ◽  
Yonghua Li ◽  
Jun-Chieh Tsay ◽  
Maya Sauthoff ◽  
...  
Keyword(s):  
16S Rrna ◽  
Mouse Model ◽  
16S Rrna Gene ◽  
Gene Sequencing ◽  
16S Rrna Gene Sequencing ◽  
Short Chain Fatty Acids ◽  
Lower Airway ◽  
Rrna Gene ◽  
Lower Airways ◽  
Rrna Gene Sequencing

RationaleMicrobiome studies of the lower airway based on bacterial 16S rRNA gene sequencing assess microbial community structure but can only infer functional characteristics. Microbial products, such as short chain fatty acids (SCFAs), in the lower airways have significant impact on the host's immune tone. Thus, functional approaches to the analyses of the microbiome are necessary.MethodsHere we used upper and lower airway samples from a research bronchoscopy smoker cohort. In addition, we validated our results in an experimental mouse model.MeasurementsWe extended our microbiota characterisation beyond 16S rRNA gene sequencing with the use of whole genome (WGS) and RNA metatranscriptome sequencing. Short chain fatty acids (SCFA) were also measured in lower airway samples and correlated with each of the sequencing datasets. In the mouse model, 16S rRNA gene and RNA metatranscriptome sequencing were performed.Main ResultsFunctional evaluations of the lower airway microbiota using inferred metagenome, WGS and metatranscriptome were dissimilar. Comparison with measured levels of SCFAs shows that the inferred metagenome from the 16S rRNA gene sequencing data was poorly correlated, while better correlations were noted when SCFAs levels were compared with WGS and metatranscriptome. Modelling lower airway aspiration with oral commensals in a mouse model showed that the metatranscriptome most efficiently captures transient active microbial metabolism, which was overestimated by 16S rRNA gene sequencing.ConclusionsFunctional characterisation of the lower airway microbiota through metatranscriptome identify metabolically active organisms capable of producing metabolites with immunomodulatory capacity such as SCFAs.

Screening and Molecular Characterization of Cellulase Producing Actinobacteria from Litchi Orchard

2019 ◽  
Vol 13 (1) ◽  
pp. 90-101
Author(s):  
Sanju Kumari ◽  
Utkarshini Sharma ◽  
Rohit Krishna ◽  
Kanak Sinha ◽  
Santosh Kumar
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Molecular Characterization ◽  
Biochemical Characterization ◽  
Evolutionary Relationship ◽  
Gene Sequencing ◽  
Cellulase Activity ◽  
16S Rrna Gene Sequencing ◽  
Rrna Gene ◽  
Rrna Gene Sequencing

Background: Cellulolysis is of considerable economic importance in laundry detergents, textile and pulp and paper industries and in fermentation of biomass into biofuels. Objective: The aim was to screen cellulase producing actinobacteria from the fruit orchard because of its requirement in several chemical reactions. Methods: Strains of actinobacteria were isolated on Sabouraud’s agar medium. Similarities in cultural and biochemical characterization by growing the strains on ISP medium and dissimilarities among them perpetuated to recognise nine groups of actinobacteria. Cellulase activity was measured by the diameter of clear zone around colonies on CMC agar and the amount of reducing sugar liberated from carboxymethyl cellulose in the supernatant of the CMC broth. Further, 16S rRNA gene sequencing and molecular characterization were placed before NCBI for obtaining recognition with accession numbers. Results: Prominent clear zones on spraying Congo Red were found around the cultures of strains of three groups SK703, SK706, SK708 on CMC agar plates. The enzyme assay for carboxymethylcellulase displayed extra cellulase activity in broth: 0.14, 0.82 and 0.66 µmol mL-1 min-1, respectively at optimum conditions of 35°C, pH 7.3 and 96 h of incubation. However, the specific cellulase activities per 1 mg of protein did not differ that way. It was 1.55, 1.71 and 1.83 μmol mL-1 min-1. The growing mycelia possessed short compact chains of 10-20 conidia on aerial branches. These morphological and biochemical characteristics, followed by their verification by Bergey’s Manual, categorically allowed the strains to be placed under actinobacteria. Further, 16S rRNA gene sequencing, molecular characterization and their evolutionary relationship through phylogenetics also confirmed the putative cellulase producing isolates of SK706 and SK708 subgroups to be the strains of Streptomyces. These strains on getting NCBI recognition were christened as Streptomyces glaucescens strain SK91L (KF527284) and Streptomyces rochei strain SK78L (KF515951), respectively. Conclusion: Conclusive evidence on the basis of different parameters established the presence of cellulase producing actinobacteria in the litchi orchard which can convert cellulose into fermentable sugar.

scholarly journals Much ado about nothing? Off-target amplification can lead to false-positive bacterial brain microbiome detection in healthy and Parkinson’s disease individuals

Microbiome ◽  
2021 ◽  
Vol 9 (1) ◽  
Author(s):  
Janis R. Bedarf ◽  
Naiara Beraza ◽  
Hassan Khazneh ◽  
Ezgi Özkurt ◽  
David Baker ◽  
...  
Keyword(s):  
Parkinson’S Disease ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
False Positive ◽  
Gene Sequencing ◽  
Bacterial Biomass ◽  
16S Rrna Gene Sequencing ◽  
Amplicon Sequencing ◽  
Rrna Gene ◽  
Rrna Gene Sequencing

Abstract Background Recent studies suggested the existence of (poly-)microbial infections in human brains. These have been described either as putative pathogens linked to the neuro-inflammatory changes seen in Parkinson’s disease (PD) and Alzheimer’s disease (AD) or as a “brain microbiome” in the context of healthy patients’ brain samples. Methods Using 16S rRNA gene sequencing, we tested the hypothesis that there is a bacterial brain microbiome. We evaluated brain samples from healthy human subjects and individuals suffering from PD (olfactory bulb and pre-frontal cortex), as well as murine brains. In line with state-of-the-art recommendations, we included several negative and positive controls in our analysis and estimated total bacterial biomass by 16S rRNA gene qPCR. Results Amplicon sequencing did detect bacterial signals in both human and murine samples, but estimated bacterial biomass was extremely low in all samples. Stringent reanalyses implied bacterial signals being explained by a combination of exogenous DNA contamination (54.8%) and false positive amplification of host DNA (34.2%, off-target amplicons). Several seemingly brain-enriched microbes in our dataset turned out to be false-positive signals upon closer examination. We identified off-target amplification as a major confounding factor in low-bacterial/high-host-DNA scenarios. These amplified human or mouse DNA sequences were clustered and falsely assigned to bacterial taxa in the majority of tested amplicon sequencing pipelines. Off-target amplicons seemed to be related to the tissue’s sterility and could also be found in independent brain 16S rRNA gene sequences. Conclusions Taxonomic signals obtained from (extremely) low biomass samples by 16S rRNA gene sequencing must be scrutinized closely to exclude the possibility of off-target amplifications, amplicons that can only appear enriched in biological samples, but are sometimes assigned to bacterial taxa. Sequences must be explicitly matched against any possible background genomes present in large quantities (i.e., the host genome). Using close scrutiny in our approach, we find no evidence supporting the hypothetical presence of either a brain microbiome or a bacterial infection in PD brains.

scholarly journals The growth response of rice (Oryza sativa L. var. FARO 44) in vitro after inoculation with bacterial isolates from a typical ferruginous ultisol

2021 ◽  
Vol 45 (1) ◽  
Author(s):  
Musa Saheed Ibrahim ◽  
Beckley Ikhajiagbe
Keyword(s):  
16S Rrna ◽  
Bacillus Cereus ◽  
Proteus Mirabilis ◽  
Growth Response ◽  
Gene Sequencing ◽  
16S Rrna Gene Sequencing ◽  
Rrna Gene ◽  
Rice Seedlings ◽  
Rrna Gene Sequencing

Abstract Background Rice forms a significant portion of food consumed in most household worldwide. Rice production has been hampered by soil factors such as ferruginousity which has limited phosphorus availability; an important mineral component for the growth and yield of rice. The presence of phosphate-solubilizing bacteria (PSB) in soils has been reported to enhance phosphate availability. In view of this, the present study employed three bacteria species (BCAC2, EMBF2 and BCAF1) that were previously isolated and proved P solubilization capacities as inocula to investigate the growth response of rice germinants in an in vitro setup. The bacteria isolates were first identified using 16S rRNA gene sequencing and then applied as inoculum. The inolula were prepared in three concentrations (10, 7.5 and 5.0 ml) following McFarland standard. Viable rice (var. FARO 44) seeds were sown in petri dishes and then inoculated with the three inocula at the different concentrations. The setup was studied for 28 days. Results 16S rRNA gene sequencing identified the isolates as: isolate BCAC2= Bacillus cereus strain GGBSU-1, isolate BCAF1= Proteus mirabilis strain TL14-1 and isolate EMBF2= Klebsiella variicola strain AUH-KAM-9. Significant improvement in rice germination, morphology, physiology and biomass parameters in the bacteria-inoculated setups was observed compared to the control. Germination percentage after 4 days was 100 % in the inoculated rice germinants compared to 65% in the control (NiS). Similarly, inoculation with the test isolates enhanced water-use efficiency by over 40%. The rice seedlings inoculated with Bacillus cereus strain GGBSU-1 (BiS) showed no signs of chlorosis and necrosis throughout the study period as against those inoculated with Proteus mirabilis strain TL14-1 (PiS) and Klebsiella variicola strain AUH-KAM-9 (KiS). Significant increase in chlorophyll-a, chlorophyll-b and alpha amylase was observed in the rice seedlings inoculated with BiS as against the NiS. Conclusion Inoculating rice seeds with Bacillus cereus strain GGBSU-1, Proteus mirabilis strain TL14-1 and Klebsiella variicola strain AUH-KAM-9 in an in vitro media significantly improved growth parameters of the test plant. Bacillus cereus strain GGBSU-1 showed higher efficiency due to a more improved growth properties observed.

scholarly journals Effect of Yuzu (Citrus junos) Seed Limonoids and Spermine on Intestinal Microbiota and Hypothalamic Tissue in the Sandhoff Disease Mouse Model

2021 ◽  
Vol 9 (1) ◽  
pp. 17
Author(s):  
Mayumi Minamisawa ◽  
Takuma Suzumura ◽  
Sudeep Bose ◽  
Tetsuyuki Taniai ◽  
Gota Kawai ◽  
...  
Keyword(s):  
Mouse Model ◽  
Intestinal Microbiota ◽  
Neuronal Degeneration ◽  
Sandhoff Disease ◽  
Short Chain Fatty Acids ◽  
Rrna Gene ◽  
Wild Type ◽  
Degrading Bacteria ◽  
Citrus Junos ◽  
Hypothalamic Tissue

The effect of limonoids and spermine (Spm) extracted from yuzu (Citrus junos) seeds on the gut and the brain in a mouse model with Sandhoff disease (SD) was investigated. Wild-type and SD mice were fed a normal diet, or a diet supplemented with limonoid, Spm, or limonoid + Spm for 14–18 weeks, and then 16S rRNA gene amplicon sequencing with extracted DNA from their feces was executed. For SD control mice, intestinal microbiota was mostly composed of Lactobacillus and linked to dysbiosis. For SD and wild-type mice fed with limonoids + Spm or limonoids alone, intestinal microbiota was rich in mucin-degrading bacteria, including Bacteroidetes, Verrucomicrobia, and Firmicutes, and displayed a higher production of short-chain fatty acids and immunoglobulin A. Additionally, SD mice fed with limonoids + Spm or limonoids alone had less inflammation in hypothalamic tissues and displayed a greater number of neurons. Administration of limonoids and/or Spm improved the proportions of beneficial intestinal microbiota to host health and reduced neuronal degeneration in SD mice. Yuzu seed limonoids and Spermine may help to maintain the homeostasis of intestinal microbiota and hypothalamic tissue in the SD mouse model.

scholarly journals Gut microbiota accelerates cisplatin-induced acute liver injury associated with robust inflammation and oxidative stress in mice

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Shenhai Gong ◽  
Yinglin Feng ◽  
Yunong Zeng ◽  
Huanrui Zhang ◽  
Meiping Pan ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
16S Rrna ◽  
Gut Microbiota ◽  
Gene Sequencing ◽  
16S Rrna Gene Sequencing ◽  
Rrna Gene ◽  
Metabolomic Analysis ◽  
Rrna Gene Sequencing ◽  
And Oxidative Stress

Abstract Background Gut microbiota has been reported to be disrupted by cisplatin, as well as to modulate chemotherapy toxicity. However, the precise role of intestinal microbiota in the pathogenesis of cisplatin hepatotoxicity remains unknown. Methods We compared the composition and function of gut microbiota between mice treated with and without cisplatin using 16S rRNA gene sequencing and via metabolomic analysis. For understanding the causative relationship between gut dysbiosis and cisplatin hepatotoxicity, antibiotics were administered to deplete gut microbiota and faecal microbiota transplantation (FMT) was performed before cisplatin treatment. Results 16S rRNA gene sequencing and metabolomic analysis showed that cisplatin administration caused gut microbiota dysbiosis in mice. Gut microbiota ablation by antibiotic exposure protected against the hepatotoxicity induced by cisplatin. Interestingly, mice treated with antibiotics dampened the mitogen-activated protein kinase pathway activation and promoted nuclear factor erythroid 2-related factor 2 nuclear translocation, resulting in decreased levels of both inflammation and oxidative stress in the liver. FMT also confirmed the role of microbiota in individual susceptibility to cisplatin-induced hepatotoxicity. Conclusions This study elucidated the mechanism by which gut microbiota mediates cisplatin hepatotoxicity through enhanced inflammatory response and oxidative stress. This knowledge may help develop novel therapeutic approaches that involve targeting the composition and metabolites of microbiota.

scholarly journals Comparison between 16S rRNA and shotgun sequencing data for the taxonomic characterization of the gut microbiota

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Francesco Durazzi ◽  
Claudia Sala ◽  
Gastone Castellani ◽  
Gerardo Manfreda ◽  
Daniel Remondini ◽  
...  
Keyword(s):  
16S Rrna ◽  
Gut Microbiota ◽  
16S Rrna Gene ◽  
Gene Sequencing ◽  
16S Rrna Gene Sequencing ◽  
Shotgun Sequencing ◽  
Rrna Gene ◽  
Metagenomic Sequencing ◽  
Experimental Conditions ◽  
Rrna Gene Sequencing

AbstractIn this paper we compared taxonomic results obtained by metataxonomics (16S rRNA gene sequencing) and metagenomics (whole shotgun metagenomic sequencing) to investigate their reliability for bacteria profiling, studying the chicken gut as a model system. The experimental conditions included two compartments of gastrointestinal tracts and two sampling times. We compared the relative abundance distributions obtained with the two sequencing strategies and then tested their capability to distinguish the experimental conditions. The results showed that 16S rRNA gene sequencing detects only part of the gut microbiota community revealed by shotgun sequencing. Specifically, when a sufficient number of reads is available, Shotgun sequencing has more power to identify less abundant taxa than 16S sequencing. Finally, we showed that the less abundant genera detected only by shotgun sequencing are biologically meaningful, being able to discriminate between the experimental conditions as much as the more abundant genera detected by both sequencing strategies.

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