Substrata effects on bacterial biofilm development in a subsurface flow dairy waste treatment wetland

2003 ◽  
Vol 48 (8) ◽  
pp. 261-269 ◽  
Author(s):  
G. Silyn-Roberts ◽  
G. Lewis
Keyword(s):  
Population Structure ◽  
In Situ Hybridisation ◽  
Treatment Wetland ◽  
Bacterial Populations ◽  
Rock Types ◽  
Bacterial Adsorption ◽  
Adsorption Rates ◽  
Biofilm Development

Biofilm development on two distinct rock substrata was investigated both in vitro and in a subsurface flow wastewater treatment wetland in order to determine the effect of hydrophobicity on initial bacterial adsorption, tertiary biofilm development and microbial population structure. Two commonly used wetland rock types, slag (a hydrophobic by-product of the steel smelter industry) and greywacke (a more hydrophilic sedimentary rock) were evaluated. In vitro investigations of initial microbial adsorption trends showed that the more hydrophobic slag displayed rapid bacterial adsorption rates compared to greywacke. Mean microbial adsorption rates of a mixed wetland bacterial population over 5 hours, described using a first order kinetics model, were 1.3 × 10-12 m/sec for slag and consistently lower at 8.7 × 10-13 m/sec for greywacke. Pristine rock studs of the two substrata were also exposed to wetland microbial communities during a six week field trial using confocal scanning laser microscopy to determine tertiary biofilm structure and fluorescent in situ hybridisation to investigate bacterial populations. During the first five weeks of growth CSLM analysis revealed that 75% of biofilms on slag were thicker and had greater coverage compared with those grown on greywacke. After six weeks of growth over 50% of the tertiary biofilms were structurally very similar on both rock types and only 25% of those grown on slag were larger than those on greywacke. In situ hybridisation analysis of bacterial populations revealed very little difference in population structure between biofilms grown on slag and those grown on greywacke. Eubacteria were present as a very high proportion of total bacteria throughout biofilm development (74.3%). The beta subgroup was the most populous of the Proteobacteria (31.4%) followed by the gamma subgroup (13.4%)and the alpha subgroup (1.3%). The results of this study suggest that slag, as a more hydrophobic substratum, promotes the initial adsorption of bacteria during early biofilm growth and better supports mature biofilm structures when used in wetlands. This study has implications for the design and construction of wastewater treatment wetlands.

scholarly journals Cryptic roles of tetrathionate in the sulfur cycle of marine sediments: microbial drivers and indicators

2020 ◽  
Vol 17 (18) ◽  
pp. 4611-4631 ◽  
Author(s):  
Subhrangshu Mandal ◽  
Sabyasachi Bhattacharya ◽  
Chayan Roy ◽  
Moidu Jameela Rameez ◽  
Jagannath Sarkar ◽  
...  
Keyword(s):  
Sediment Cores ◽  
Sulfur Cycle ◽  
Growth Media ◽  
Pore Waters ◽  
Bacterial Populations ◽  
Metagenome Analysis ◽  
Minimum Zone ◽  
Chemolithotrophic Bacteria

Abstract. To explore the potential role of tetrathionate in the sedimentary sulfur cycle, population ecology of microorganisms capable of metabolizing this polythionate was revealed at 15–30 cm resolution along two, ∼3 m long, cores collected from 530 and 580 m below the sea level, off India's west coast, within the oxygen minimum zone (OMZ) of the Arabian Sea. Metagenome analysis along the cores revealed widespread occurrence of genes involved in the formation, oxidation, and reduction of tetrathionate; high diversity and relative abundance were also detected for bacteria that are known to render these metabolisms in vitro. Results of slurry culture of the sediment samples in thiosulfate- or tetrathionate-containing microbial growth media, data obtained via pure-culture isolation, and finally metatranscriptome analyses corroborated the in situ functionality of the tetrathionate-forming, tetrathionate-oxidizing, and tetrathionate-reducing microorganisms. Ion chromatography of pore waters revealed the presence of up to 11.1 µM thiosulfate in the two cores, whereas tetrathionate remained undetected in spectroscopic assay based on its reaction with cyanide. While thiosulfate oxidation by chemolithotrophic bacteria prevalent in situ is the apparent source of tetrathionate in this ecosystem, high biochemical and geochemical reactivity of this polythionate could be instrumental in its cryptic status in the sulfur cycle. Potential abiotic origin of tetrathionate in the sediment horizon explored could neither be ruled out nor confirmed from the geochemical information available. On the other hand, tetrathionate potentially present in the system can be either oxidized to sulfate or reduced back to thiosulfate/sulfide via chemolithotrophic oxidation and respiration by native bacterial populations, respectively. Up to 2.01 mM sulfide present in the sediment cores may also reduce tetrathionate abiotically to thiosulfate and elemental sulfur. However, in the absence of measured data for O2 or other oxyanions having possibilities of serving as electron acceptors, the biogeochemical modalities of the oxidative half of the tetrathionate cycle remained unresolved.

Immunological mechanisms to establish embryo tolerance in early bovine pregnancy

2011 ◽  
Vol 23 (5) ◽  
pp. 619 ◽  
Author(s):  
A. E. Groebner ◽  
K. Schulke ◽  
J. C. Schefold ◽  
G. Fusch ◽  
F. Sinowatz ◽  
...  
Keyword(s):  
In Situ Hybridisation ◽  
Immunohistochemical Analysis ◽  
Kynurenine Pathway ◽  
Endometrial Cells ◽  
Maternal Rejection ◽  
Immunological Mechanisms ◽  
Bovine Pregnancy ◽  
Coculture Model

A well-balanced immunological interaction between mother and the semi-allogenic embryo is of particular importance. The objective of the present study was to analyse mechanisms of immune tolerance in bovine pregnancy during peri-implantation. Simmental heifers inseminated with either cryopreserved spermatozoa or seminal plasma were killed 12, 15 or 18 days after oestrus. Uteri were flushed for the recovery of conceptuses and the ipsilateral intercaruncular endometrium was sampled for gene expression analysis. Indoleamine 2,3-dioxygenase (IDO) mRNA, coding for the initial enzyme of the kynurenine pathway, was 18-fold (P < 0.001) more abundant in the endometrium of Day 18 pregnant v. non-pregnant animals. Tandem mass spectrometry revealed a decrease of endometrial l-tryptophan (P = 0.0008), but an increase of l-kynurenine concentration (P = 0.005) from Day 12 to Day 18, suggesting increasing IDO activity (P < 0.03). An in vitro coculture model of endometrial cells showed an induction of IDO expression following interferon-τ exposure primarily in stroma cells, which was confirmed by in situ hybridisation localising IDO mRNA mainly in deep stroma cells. Immunohistochemical analysis revealed fewer CD45-positive leucocytes in the zona basalis of pregnant animals. Elevated IDO activity may reduce the presence of leucocytes in the pregnant endometrium, providing a possible mechanism for protecting the semi-allogenic conceptus from maternal rejection.

scholarly journals Novel lncRNA UPLA1 mediates tumorigenesis and prognosis in lung adenocarcinoma

2020 ◽  
Vol 11 (11) ◽  
Author(s):  
Xiaoyang Han ◽  
Hua Jiang ◽  
Jianni Qi ◽  
Jiamei Li ◽  
Jinghan Yang ◽  
...  
Keyword(s):  
Lung Adenocarcinoma ◽  
In Situ Hybridisation ◽  
Vital Role ◽  
Luciferase Reporter ◽  
Fluorescence In Situ Hybridisation ◽  
Biological Functions ◽  
Pulldown Assay

AbstractWith the development of molecular biotechnology and sequencing techniques, long non-coding RNAs (lncRNAs) have been shown to play a vital role in a variety of cancers including lung cancer. In our previous study, we used RNA sequencing and high-content screening proliferation screening data to identify lncRNAs that were significantly associated with tumour biological functions such as LINC01426. Herein, based on previous work, we report a novel lncRNA UPLA1 (upregulation promoting LUAD-associated transcript-1), which has not been explored or reported in any previous studies. Our results showed that UPLA1 is highly expressed and regulates important biological functions in lung adenocarcinoma. In vitro experiments revealed that UPLA1 promoted the migration, invasion, and proliferation abilities, and is related to cell cycle arrest, in lung adenocarcinoma cells. Moreover, the upregulation of UPLA1 significantly improved the growth of tumours in vivo. We identified that UPLA1 was mainly located in the nucleus using fluorescence in situ hybridisation, and that it promoted Wnt/β-catenin signalling by binding to desmoplakin using RNA pulldown assay and mass spectrometry. Additionally, luciferase reporter assay revealed that YY1 is the transcription factor of UPLA1 and suppressed the expression of UPLA1 as a transcriptional inhibitor. This finding provides important evidence regarding the two roles of YY1 in cancer. Furthermore, in situ hybridisation assay results showed that UPLA1 was closely related to the prognosis and tumour, node, metastasis (TNM) stage of lung adenocarcinoma. In summary, our results suggest that the novel lncRNA UPLA1 promotes the progression of lung adenocarcinoma and may be used as a prognostic marker, and thus, has considerable clinical significance.

A toddler SHIME® model to study microbiota of young children

2020 ◽  
Vol 367 (16) ◽  
Author(s):  
Pauline Bondue ◽  
Sarah Lebrun ◽  
Bernard Taminiau ◽  
Nadia Everaert ◽  
Gisele LaPointe ◽  
...  
Keyword(s):  
Young Children ◽  
Short Chain Fatty Acid ◽  
Amplicon Sequencing ◽  
Bacterial Populations ◽  
16S Rrna Amplicon Sequencing ◽  
Biofilm Development ◽  
Stabilization Period ◽  
Gut Microbiota Composition

ABSTRACT The ‘first 1000 days of life’ determine the gut microbiota composition and can have long-term health consequences. In this study, the simulator of the human intestinal microbial ecosystem (SHIME®) model, which represents the main functional sections of the digestive tract, was chosen to study the microbiota of young children. The aim of this study was to reproduce the digestive process of toddlers and their specific colonic environment. The ascending, transverse and descending colons of SHIME® model were inoculated with feces from three donors aged between 1 and 2 years-old, in three separate runs. For each run, samples from colon vessels were collected at days 14, 21 and 28 after microbiota stabilization period. Short chain fatty acid concentrations determined by HPLC showed that microbiota obtained in SHIME® model shared characteristics between adults and infants. In addition, microbial diversity and bacterial populations determined by 16S rRNA amplicon sequencing were specific to each colon vessel. In conclusion, the SHIME® model developed in this study seemed well adapted to evaluate prebiotic and probiotic impact on the specific microbiota of toddlers, or medicine and endocrine disruptor metabolism. Moreover, this study is the first to highlight some biofilm development in in vitro gastrointestinal modelling systems.

Localisation of mRNA for collagenase in osteocytic, bone surface and chondrocytic cells but not osteoclasts

1995 ◽  
Vol 108 (6) ◽  
pp. 2221-2230
Author(s):  
K. Fuller ◽  
T.J. Chambers
Keyword(s):  
Bone Resorption ◽  
Bone Cells ◽  
In Situ Hybridisation ◽  
Cysteine Proteinases ◽  
Bone Surface ◽  
Matrix Metalloproteinase 1 ◽  
Osteoclastic Resorption ◽  
Stimulation Of

Osteoclasts resorb the extracellular matrix of bone by secreting protons and enzymes into a circumpherentially sealed compartment between the osteoclast and the bone surface. Although the lysosomal cysteine proteinases play a major role in matrix degradation by osteoclasts, collagenase (matrix metalloproteinase-1, EC 3.4.24.7) is also required for osteoclastic bone resorption, and may be directly involved in collagen degradation in the hemivacuole. We assessed the effects of inhibitors of cysteine proteinases and collagenase on bone resorption by osteoclasts isolated from rodent bone. We found that while inhibition of cysteine proteinases strongly suppressed osteoclastic resorption, inhibitors of collagenase were without effect on the number, size, or demineralised fringe of excavations. We could find no evidence of expression of mRNA for collagenase in rat osteoclasts by in situ hybridisation, but found that it was expressed by chondrocytes, bone surface cells and osteocytes adjacent to osteoclasts. The distribution of these cells, and the correlation between increased collagenase production and increased stimulation of osteoclastic resorption in vitro by bone cells, suggests that these cells might be involved in the regulation of bone resorption in situ, and that collagenase production might play a role in this process.

Expression and regulation of Lhx6 and Lhx7, a novel subfamily of LIM homeodomain encoding genes, suggests a role in mammalian head development

Development ◽  
1998 ◽  
Vol 125 (11) ◽  
pp. 2063-2074 ◽  
Author(s):  
M. Grigoriou ◽  
A.S. Tucker ◽  
P.T. Sharpe ◽  
V. Pachnis
Keyword(s):  
Basal Forebrain ◽  
In Situ Hybridisation ◽  
Branchial Arch ◽  
Tooth Formation ◽  
Head Development ◽  
Expression Studies ◽  
Overlapping Domains ◽  
Encoding Genes

LIM-homeobox containing (Lhx) genes encode trascriptional regulators which play critical roles in a variety of developmental processes. We have identified two genes belonging to a novel subfamily of mammalian Lhx genes, designated Lhx6 and Lhx7. Whole-mount in situ hybridisation showed that Lhx6 and Lhx7 were expressed during mouse embryogenesis in overlapping domains of the first branchial arch and the basal forebrain. More specifically, expression of Lhx6 and Lhx7 was detected prior to initiation of tooth formation in the presumptive oral and odontogenic mesenchyme of the maxillary and mandibular processes. During tooth formation, expression was restricted to the mesenchyme of individual teeth. Using explant cultures, we have shown that expression of Lhx6 and Lhx7 in mandibular mesenchyme was under the control of signals derived from the overlying epithelium; such signals were absent from the epithelium of the non-odontogenic second branchial arch. Furthermore, expression studies and bead implantation experiments in vitro have provided strong evidence that Fgf8 is primarily responsible for the restricted expression of Lhx6 and Lhx7 in the oral aspect of the maxillary and mandibular processes. In the telencephalon, expression of both genes was predominantly localised in the developing medial ganglionic eminences, flanking a Fgf8-positive midline region. We suggest that Fgf8 and Lhx6 and Lhx7 are key components of signalling cascades which determine morphogenesis and differentiation in the first branchial arch and the basal forebrain.

scholarly journals Comparative prebiotic activity of mixtures of cereal grain polysaccharides

AMB Express ◽  
2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Suzanne Harris ◽  
Andrea Monteagudo-Mera ◽  
Ondrej Kosik ◽  
Dimitris Charalampopoulos ◽  
Peter Shewry ◽  
...  
Keyword(s):  
Wheat Flour ◽  
In Situ Hybridisation ◽  
Short Chain Fatty Acids ◽  
Fluorescence In Situ Hybridisation ◽  
In Vitro Fermentation ◽  
Bacterial Populations ◽  
Prebiotic Activity ◽  
Cereal Grain ◽  
Main Components

AbstractThe main components of the non-starch polysaccharide (NSP) fraction of wheat flour are arabinoxylan (AX) and β-glucan. These are also present in other cereal grains, but their proportions vary with AX being the major component in wheat and rye and β-glucan in barley and oats. Therefore, it was hypothesised that these NSPs could act synergistically when fermented in vitro at the ratios present in the major foods consumed, resulting in increased prebiotic activity. AX and β-glucan were therefore tested in in vitro fermentation studies to assess their prebiotic activity when used individually and/or in different ratios. Short-chain fatty-acids (SCFAs) produced from in vitro fermentation were measured using HPLC and bacterial populations were measured using flow cytometry with fluorescence in situ hybridisation (Flow-FISH). Fermentation of AX alone resulted in a significant bifidogenic activity and increased concentrations of SCFAs, mainly acetate, after 8–24 h of fermentation, however β-glucan alone did not show prebiotic activity. The greatest prebiotic activity, based on concentration of total SCFAs and increases in total bacteria as well as beneficial Bifidobacterium and Clostridium coccoides/Eubacterium groups, was observed when AX and β-glucan were combined at a 3:1 ratio, which corresponds to their ratios in wheat flour which is major source of cereal fibre in the diet. This indicates that the population of bacteria in the human GI tract may be modulated by the composition of the fibre in the diet, to maximise the prebiotic potential.

Molecular cloning, characterisation and expression of two catalase genes from peach

2004 ◽  
Vol 31 (4) ◽  
pp. 349 ◽  
Author(s):  
Francesca Bagnoli ◽  
Susanna Danti ◽  
Valentina Magherini ◽  
Radiana Cozza ◽  
Anna M. Innocenti ◽  
...  
Keyword(s):  
Stress Responses ◽  
Seasonal Changes ◽  
Prunus Persica ◽  
Vascular Tissue ◽  
In Situ Hybridisation ◽  
Leaf Tissue ◽  
Amino Acid Sequences ◽  
Cdna Clones

Two cDNA clones encoding catalase (Cat1 and Cat2) from peach [Prunus persica (L.) Batsch] were identified, that show homologies to other plant catalases. The nucleotide sequences of the two coding regions showed 88% identity to each other. The amino acid sequences predicted from the two full-length clones showed the highest homology to a catalase from cotton and Nicotiana plumbaginifolia L. and included C-terminal tri-peptides typical of those used to target proteins to peroxisomes. Southern hybridisation analysis suggested the existence of two catalase genes in peach. The expression of Cat1 and Cat2 was determined in seeds, vegetative tissue, leaves during the seasonal cycle and in leaves in response to light / dark treatments. Cat1 had high levels of expression only in leaf tissue and was responsive to light and seasonal changes. Cat2 had high levels of expression in in vitro shoots and was also responsive to seasonal changes, but not to light. In situ hybridisations to leaf tissue indicated that the expression of Cat1 was localised mainly in palisade cells, while Cat2 mRNA was present in the vascular tissue. The results of the expression analysis and in situ hybridisation suggest a role for Cat1 in photorespiration and for Cat2 in stress responses.

Development of a fermentation system to model sessile bacterial populations in the human colon

Biofilms ◽  
2004 ◽  
Vol 1 (1) ◽  
pp. 13-19 ◽  
Author(s):  
H. M. Probert ◽  
G. R. Gibson
Keyword(s):  
Culture Fluid ◽  
Human Colon ◽  
Healthy Human ◽  
Bacterial Populations ◽  
Fermentation System ◽  
Colonic Microflora ◽  
Osmotic Effect ◽  
Planktonic Bacteria

A fermentation system was designed to model the human colonic microflora in vitro. The system provided a framework of mucin beads to encourage the adhesion of bacteria, which was encased within a dialysis membrane. The void between the beads was inoculated with faeces from human donors. Water and metabolites were removed from the fermentation by osmosis using a solution of polyethylene glycol (PEG). The system was concomitantly inoculated alongside a conventional single-stage chemostat. Three fermentations were carried out using inocula from three healthy human donors.Bacterial populations from the chemostat and biofilm system were enumerated using fluorescence in situ hybridization. The culture fluid was also analysed for its short-chain fatty acid (SCFA) content. A higher cell density was achieved in the biofilm fermentation system (taking into account the contribution made by the bead-associated bacteria) as compared with the chemostat, owing to the removal of water and metabolites. Evaluation of the bacterial populations revealed that the biofilm system was able to support two distinct groups of bacteria: bacteria growing in association with the mucin beads and planktonic bacteria in the culture fluid. Furthermore, distinct differences were observed between populations in the biofilm fermenter system and the chemostat, with the former supporting higher populations of clostridia and Escherichia coli. SCFA levels were lower in the biofilm system than in the chemostat, as in the former they were removed via the osmotic effect of the PEG. These experiments demonstrated the potential usefulness of the biofilm system for investigating the complexity of the human colonic microflora and the contribution made by sessile bacterial populations.

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