scholarly journals Revealing the in vivo growth and division patterns of mouse gut bacteria

2020 ◽  
Vol 6 (36) ◽  
pp. eabb2531
Author(s):  
Liyuan Lin ◽  
Qiuyue Wu ◽  
Jia Song ◽  
Yahui Du ◽  
Juan Gao ◽  
...  
Keyword(s):  
Amino Acid ◽  
Gut Microbiota ◽  
Growth Dynamics ◽  
Gut Bacteria ◽  
Metabolic Labeling ◽  
Taxonomic Identification ◽  
In Vivo Growth

Current techniques for studying gut microbiota are unable to answer some important microbiology questions, like how different bacteria grow and divide in the gut. We propose a method that integrates the use of sequential d-amino acid–based in vivo metabolic labeling with fluorescence in situ hybridization (FISH), for characterizing the growth and division patterns of gut bacteria. After sequentially administering two d-amino acid–based probes containing different fluorophores to mice by gavage, the resulting dual-labeled peptidoglycans provide temporal information on cell wall synthesis of gut bacteria. Following taxonomic identification with FISH probes, the growth and division patterns of the corresponding bacterial taxa, including species that cannot be cultured separately in vitro, are revealed. Our method offers a facile yet powerful tool for investigating the in vivo growth dynamics of the bacterial gut microbiota, which will advance our understanding of bacterial cytology and facilitate elucidation of the basic microbiology of this gut “dark matter.”

scholarly journals Ponticulin is an atypical membrane protein.

1994 ◽  
Vol 126 (6) ◽  
pp. 1421-1431 ◽  
Author(s):  
A L Hitt ◽  
T H Lu ◽  
E J Luna
Keyword(s):  
Plasma Membrane ◽  
Amino Acid ◽  
Plasma Membranes ◽  
Signal Sequence ◽  
Metabolic Labeling ◽  
Cortical Actin ◽  
Intact Cells ◽  
Membrane Spanning

We have cloned and sequenced ponticulin, a 17,000-dalton integral membrane glycoprotein that binds F-actin and nucleates actin assembly. A single copy gene encodes a developmentally regulated message that is high during growth and early development, but drops precipitously during cell streaming at approximately 8 h of development. The deduced amino acid sequence predicts a protein with a cleaved NH2-terminal signal sequence and a COOH-terminal glycosyl anchor. These predictions are supported by amino acid sequencing of mature ponticulin and metabolic labeling with glycosyl anchor components. Although no alpha-helical membrane-spanning domains are apparent, several hydrophobic and/or sided beta-strands, each long enough to traverse the membrane, are predicted. Although its location on the primary sequence is unclear, an intracellular domain is indicated by the existence of a discontinuous epitope that is accessible to antibody in plasma membranes and permeabilized cells, but not in intact cells. Such a cytoplasmically oriented domain also is required for the demonstrated role of ponticulin in binding actin to the plasma membrane in vivo and in vitro (Hitt, A. L., J. H. Hartwig, and E. J. Luna. 1994. Ponticulin is the major high affinity link between the plasma membrane and the cortical actin network in Dictyostelium. J. Cell Biol. 126:1433-1444). Thus, ponticulin apparently represents a new category of integral membrane proteins that consists of proteins with both a glycosyl anchor and membrane-spanning peptide domain(s).

scholarly journals Comparative activity of Ceftriaxone, Ciprofloxacin and Gentamicin as a function of bacterial growth rate probed by Escherichia coli chromosome replication in the mouse peritonitis model

2018 ◽  
Author(s):  
Maria Schei Haugan ◽  
Anders Løbner-Olesen ◽  
Niels Frimodt-Møller
Keyword(s):  
Growth Rate ◽  
Bacterial Growth ◽  
Growth Dynamics ◽  
Chromosome Replication ◽  
Bacterial Cells ◽  
Bacterial Growth Rate ◽  
Pre Treatment

AbstractCommonly used antibiotics exert their effect predominantly on rapidly growing bacterial cells, yet growth dynamics taking place during infection in a complex host environment remain largely unknown. Hence, means to measure in situ bacterial growth rate is essential to predict the outcome of antibacterial treatment. We have recently validated chromosome replication as readout for in situ bacterial growth rate during Escherichia coli infection in the mouse peritonitis model. By the use of two complementary methods (qPCR and fluorescence microscopy) for differential genome origin and terminus copy number quantification, we demonstrated the ability to track bacterial growth rate, both on a population average and on a single-cell level; from one single biological specimen. Here, we asked whether the in situ growth rate could predict antibiotic treatment effect during infection in the same model. Parallel in vitro growth experiments were conducted as proof-of-concept. Our data demonstrate that the activity of commonly used antibiotics Ceftriaxone and Gentamicin correlated with pre-treatment bacterial growth rate; both drugs performing better during rapid growth than during slow growth. Conversely, Ciprofloxacin was less sensitive to bacterial growth rate, both in a homogenous in vitro bacterial population and in a more heterogeneous in vivo bacterial population. The method serves as a platform to test any antibiotic’s dependency upon active in situ bacterial growth. Improved insight into this relationship in vivo could ultimately prove helpful in evaluating future antibacterial strategies.ImportanceMost antibiotics in clinical use exert their effect predominantly on rapidly growing bacterial cells, yet there is a lack of insight into bacterial growth dynamics taking place during infection in vivo. We have applied inexpensive and easily accessible methods for extraction of in situ bacterial growth rate from bacterial chromosome replication during experimental murine infection. This approach not only allows for a better understanding of bacterial growth dynamics taking place during the course of infection, but also serves as a platform to test the activity of different antibiotics as a function of pre-treatment in situ growth rate. The method has the advantage that bacterial growth rate can be probed from a single biological sample, with the potential for extension into clinical use in pre-treatment infected biological specimens. A better understanding of commonly used antibiotics’ level of dependency upon bacterial growth, combined with measurements of in situ bacterial growth rate in infected clinical specimens, could prove helpful in evaluating future antibacterial treatment regimens.

scholarly journals Bruceae Fructus Oil Inhibits Triple-Negative Breast Cancer by Restraining Autophagy: Dependence on the Gut Microbiota-Mediated Amino Acid Regulation

2021 ◽  
Vol 12 ◽  
Author(s):  
Jiyan Su ◽  
Xiaohong Chen ◽  
Yuanjie Xiao ◽  
Dan Li ◽  
Muxia Li ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Amino Acid ◽  
Gut Microbiota ◽  
Triple Negative Breast Cancer ◽  
Tumor Suppression ◽  
Triple Negative ◽  
Critical Role ◽  
Amino Acid Profile

Triple-negative breast cancer (TNBC) has been acknowledged as an aggressive disease with worst prognosis, which requires endeavor to develop novel therapeutic agents. Bruceae fructus oil (BO), a vegetable oil derived from the fruit of Brucea javanica (L.) Merr., is an approved marketable drug for the treatment of cancer in China for several decades. Despite that the anti–breast cancer activity of several quassinoids derived from B. javanica has been found, it was the first time that the potential of BO against TNBC was revealed. Although BO had no cytotoxicity on TNBC cell lines in vitro, the oral administration of BO exhibited a gut microbiota–dependent tumor suppression without toxicity on the non-targeted organs in vivo. By metagenomics and untargeted metabolomics, it was found that BO not only altered the composition and amino acid metabolism function of gut microbiota but also regulated the host’s amino acid profile, which was in accordance with the metabolism alternation in gut microbiota. Moreover, the activity of mTOR in tumor was promoted by BO treatment as indicated by the phosphorylation of 4E-binding protein 1 (4E-BP1) and ribosomal protein S6, and hyper-autophagy was consequently restrained. By contrast, the failure of tumor suppression by BO under pseudo germ-free (PGF) condition came with indistinctive changes in autophagy and mTOR activity, implying the critical role of the gut microbiota in BO’s anticancer activity. The present study highlighted a promising application of BO against breast cancer with novel efficacy and safety.

scholarly journals Resistance and Vulnerability of Honeybee (Apis mellifera) Gut Bacteria to Commonly Used Pesticides

2021 ◽  
Vol 12 ◽  
Author(s):  
Ana Cuesta-Maté ◽  
Justinn Renelies-Hamilton ◽  
Per Kryger ◽  
Annette Bruun Jensen ◽  
Veronica M. Sinotte ◽  
...  
Keyword(s):  
Oxalic Acid ◽  
Gut Microbiota ◽  
Gut Microbiome ◽  
Gut Bacteria ◽  
Sequence Variant ◽  
Long Term Effects ◽  
Network Analyses ◽  
Honeybee Health

Agricultural and apicultural practices expose honeybees to a range of pesticides that have the potential to negatively affect their physiology, neurobiology, and behavior. Accumulating evidence suggests that these effects extend to the honeybee gut microbiome, which serves important functions for honeybee health. Here we test the potential effects of the pesticides thiacloprid, acetamiprid, and oxalic acid on the gut microbiota of honeybees, first in direct in vitro inhibition assays and secondly in an in vivo caged bee experiment to test if exposure leads to gut microbiota community changes. We found that thiacloprid did not inhibit the honeybee core gut bacteria in vitro, nor did it affect overall community composition or richness in vivo. Acetamiprid did also not inhibit bacterial growth in vitro, but it did affect community structure within bees. The eight bacterial genera tested showed variable levels of susceptibility to oxalic acid in vitro. In vivo, treatment with this pesticide reduced amplicon sequence variant (ASV) richness and affected gut microbiome composition, with most marked impact on the common crop bacteria Lactobacillus kunkeei and the genus Bombella. We conducted network analyses which captured known associations between bacterial members and illustrated the sensitivity of the microbiome to environmental stressors. Our findings point to risks of honeybee exposure to oxalic acid, which has been deemed safe for use in treatment against Varroa mites in honeybee colonies, and we advocate for more extensive assessment of the long-term effects that it may have on honeybee health.

scholarly journals In Situ Growth Rates and Biofilm Development of Pseudomonas aeruginosa Populations in Chronic Lung Infections

2007 ◽  
Vol 190 (8) ◽  
pp. 2767-2776 ◽  
Author(s):  
Lei Yang ◽  
Janus A. J. Haagensen ◽  
Lars Jelsbak ◽  
Helle Krogh Johansen ◽  
Claus Sternberg ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Growth Rates ◽  
Growth Dynamics ◽  
Chronic Infections ◽  
Lung Infections ◽  
New Perspective ◽  
Biofilm Development

ABSTRACT The growth dynamics of bacterial pathogens within infected hosts are a fundamental but poorly understood feature of most infections. We have focused on the in situ distribution and growth characteristics of two prevailing and transmissible Pseudomonas aeruginosa clones that have caused chronic lung infections in cystic fibrosis (CF) patients for more than 20 years. We used fluorescence in situ hybridization (FISH) directly on sputum specimens to examine the spatial distribution of the infecting P. aeruginosa cells. Mucoid variants were present in sputum as cell clusters surrounded by an extracellular matrix, whereas nonmucoid variants were present mainly as dispersed cells. To obtain estimates of the growth rates of P. aeruginosa in CF lungs, we used quantitative FISH to indirectly measure growth rates of bacteria in sputum samples (reflecting the in vivo lung conditions). The concentration of rRNA in bacteria isolated from sputa was measured and correlated with the rRNA contents of the same bacteria growing in vitro at defined rates. The results showed that most cells were actively growing with doubling times of between 100 and 200 min, with some growing even faster. Only a small stationary-phase subpopulation seemed to be present in sputa. This was found for both mucoid and nonmucoid variants despite their different organizations in sputum. The results suggest that the bacterial population may be confronted with selection forces that favor optimized growth activities. This scenario constitutes a new perspective on the adaptation and evolution of P. aeruginosa during chronic infections in CF patients in particular and on long-term infections in general.

scholarly journals Polyamine-stimulated phosphorylation of prostatic spermine-binding protein is mediated only by cyclic AMP-independent protein kinases

1985 ◽  
Vol 230 (2) ◽  
pp. 293-302 ◽  
Author(s):  
S A Goueli ◽  
A T Davis ◽  
R A Hiipakka ◽  
S Liao ◽  
K Ahmed
Keyword(s):  
Amino Acid ◽  
Protein Kinase ◽  
Cyclic Amp ◽  
Protein Kinases ◽  
Binding Protein ◽  
Regulatory Control ◽  
Independent Protein

Spermine-binding protein (a rat ventral prostatic protein with high affinity for spermine) was phosphorylated in situ through the action of intrinsic cellular protein kinase(s), suggesting it to be a phosphoprotein in vivo. The purified protein served as a substrate in a number of cyclic AMP-independent protein kinase reactions in vitro, but not for cyclic AMP-dependent, Ca2+ + calmodulin-dependent or Ca2+ + phospholipid-dependent protein kinases. Available data indicate that at least one of the cyclic AMP-independent protein kinases (cytosolic protein kinase C2) may be physiologically relevant in mediating the phosphorylation of this protein. The phosphorylation reaction was stimulated several-fold in the presence of spermine. Spermidine was somewhat less effective, whereas putrescine, cadaverine and 1,6-hexanediamine were minimally active. Phospho amino acid analysis of 32P-labelled spermine-binding protein indicated that phosphoserine was the only labelled phospho amino acid. Spermine-binding protein did not undergo autophosphorylation, or modify the stimulative effect of spermine on the phosphorylation of other substrates such as non-histone proteins. In situ the phosphorylation of spermine-binding protein in tissue from castrated rats was markedly diminished as compared with the normal. Since the phosphorylation of spermine-binding protein appears to be mediated by cyclic AMP-independent protein kinase(s) whose activity in the prostate is under androgenic control, it is suggested that androgen-dependent modulation of the protein kinase(s) exerts a regulatory control (via phosphorylation-dephosphorylation) on the spermine-binding activity and stability of this protein in vivo. Further, since this protein is a substrate for only the cyclic AMP-independent protein kinases, it could serve as a tool for the investigation of such kinases.

scholarly journals Amino acid availability in poultry—in vitro and in vivo measurements

1999 ◽  
Vol 50 (5) ◽  
pp. 889 ◽  
Author(s):  
V. Ravindran ◽  
Wayne L. Bryden
Keyword(s):  
Amino Acid ◽  
Protein Quality ◽  
Poultry Feed ◽  
In Vitro Assays ◽  
Total Amino Acid ◽  
Amino Acid Availability ◽  
Amino Acid Contents ◽  
In Vivo Growth

Methodology to evaluate the protein quality or amino acid availability in feed ingredients for poultry using in vitro (enzymic, chemical, or microbiological assays), indirect in vivo (plasma amino acid assays), or direct in vivo (growth or digestibility assays) measurements has been reviewed. The specific applications and limitations of these methods are examined. In vitro assays are useful in providing information on heat damage in selected protein sources under defined conditions, and on relative ranking of different samples, but they cannot form the basis of practical feed formulations. While growth assays remain the only direct means of confirming nutritional relevance of values obtained by other procedures, in vivo digestibility assays appear to be most useful, at present, to estimate amino acid availability. Amino acid digestibility assays in poultry should be based on the analysis of digesta from the terminal ileum rather than excreta, because of the variable and modifying effects of hindgut microflora. Techniques used to estimate endogenous amino acid losses in poultry are discussed. The needs for correction of endogenous losses in amino acid digestibility calculations and the relative merits of apparent and true digestible amino acid systems are still being debated. It is, however, clear that both digestible amino systems are superior to the total amino acid system currently employed to formulate practical diets. Digestible amino acid values are likely to form the basis of poultry feed formulations in the future. In particular, there is an urgent need for more precise information on the variation in digestible amino acid contents of locally grown ingredients and on the factors causing this variation (e.g. variety, location, season, agronomic practices, processing, etc.).

In vitro and in vivo polysaccharides assembly: ultrastructural and cytochemical study of growing plant cell wall components.

1978 ◽  
Vol 36 (2) ◽  
pp. 434-435
Author(s):  
D. Reis ◽  
B. Vian ◽  
J. C. Roland
Keyword(s):  
Cell Wall ◽  
Self Assembly ◽  
Plant Cell Wall ◽  
Higher Plants ◽  
Three Dimensional ◽  
Cytochemical Study ◽  
Wall Components

Wall morphogenesis in higher plants is a problem still open to controversy. Until now the possibility of a transmembrane control and the involvement of microtubules were mostly envisaged. Self-assembly processes have been observed in the case of walls of Chlamydomonas and bacteria. Spontaneous gelling interactions between xanthan and galactomannan from Ceratonia have been analyzed very recently. The present work provides indications that some processes of spontaneous aggregation could occur in higher plants during the formation and expansion of cell wall.Observations were performed on hypocotyl of mung bean (Phaseolus aureus) for which growth characteristics and wall composition have been previously defined.In situ, the walls of actively growing cells (primary walls) show an ordered three-dimensional organization (fig. 1). The wall is typically polylamellate with multifibrillar layers alternately transverse and longitudinal. Between these layers intermediate strata exist in which the orientation of microfibrils progressively rotates. Thus a progressive change in the morphogenetic activity occurs.

Evaluation of Structure-Controlled Silk Fibroin Coatings for Orthopedic Infection and In-Situ Osteogenesis: In Vitro and In Vivo

2020 ◽  
Author(s):  
Wenhao Zhou ◽  
Teng Zhang ◽  
Jianglong Yan ◽  
QiYao Li ◽  
Panpan Xiong ◽  
...  
Keyword(s):  
Silk Fibroin ◽  
Orthopedic Infection
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