scholarly journals Cultivatingin situtriggers growth of previously uncultivated microorganisms via a growth initiation factor in nature

2019 ◽  
Author(s):  
Dawoon Jung ◽  
Koshi Machida ◽  
Yoichi Nakao ◽  
Tomonori Kindaichi ◽  
Akiyoshi Ohashi ◽  
...  
Keyword(s):  
Growth Promotion ◽  
Initiation Factor ◽  
Natural Environments ◽  
Controlling System ◽  
Growth Initiation ◽  
Membrane Bound ◽  
Simple Growth ◽  
Specific Growth Rates ◽  
Sponge Extract

AbstractMost microorganisms resist cultivation under standard laboratory conditions. On the other hand, to cultivate microbes in a membrane-bound device incubated in nature (in situcultivation) is an effective approach. In the present study, we appliedin situcultivation to isolate diverse previously uncultivated marine sponge-associated microbes and comparatively analyzed this method’s efficiencies with those of the conventional method. Then, we attempted to clarify the key and unknown mechanism ofin situcultivation by focusing on growth triggering via growth initiation factor. We hypothesized that majority of environmental microorganisms are in nongrowing state and requiring “growth initiation factor” for the recovery and that can be provided from environments. Consequently, significantly more novel and diverse microbial types were isolated viain situcultivation than by standard direct plating (SDP). Next, the effect of the sponge extract on starvation recovery was compared between strains derived fromin situand SDP cultivation. Adding small amounts of the sponge extracts to the medium elevated the colony-formation efficiencies of thein situstrains at the starvation recovery step, while it showed no positive effect on that of SDP strains. Conversely, specific growth rates or carrying capacities of all tested strains were not positively affected. These results indicate that, 1) the sponge extract contains chemical compounds that facilitate starvation recovery, these substances selectively worked on thein situstrains, and 2) growth initiation factor in the sponge extract did not continuously promote growth activity but worked as triggers for regrowth (resuscitation from dormancy).ImportanceMost microbial species resist cultivation under laboratory condition. This is critical impediment for both academic and applied microbiology, and thus clarification of the mechanism of microbial uncultivability is highly demanded. Several evidences have been reported that to cultivate microbes in a membrane-bound device incubated in nature (in situcultivation) is an effective approach. However, the mechanism behind this approach has not been clarified. The present study shows the evidence that 1) initiating growth is a key for cultivating previously uncultivated microbes rather than simple growth promotion, and 2) growth initiation factor (signaling-like compounds) in natural environments stimulate microbial resuscitation from a nongrowing state. Since no study has focused on growth initiation for cultivation of previously uncultivated microorganisms, the discovery shown in the present study provides a new insight into microorganisms previously considered uncultivable and a microbial growth controlling system in nature.

scholarly journals In-Situ Synthesis of TiO2@GO Nanosheets for Polymers Degradation in a Natural Environment

Polymers ◽  
2021 ◽  
Vol 13 (13) ◽  
pp. 2158
Author(s):  
Yueqin Shi ◽  
Zhanyang Yu ◽  
Zhengjun Li ◽  
Xiaodong Zhao ◽  
Yongjun Yuan
Keyword(s):  
Natural Environment ◽  
New Technologies ◽  
Separation Efficiency ◽  
Basic Mechanism ◽  
Chemical Degradation ◽  
Natural Environments ◽  
Additional Energy ◽  
Photogenerated Electrons ◽  
Main Components

Plastic photodegradation naturally takes 300–500 years, and their chemical degradation typically needs additional energy or causes secondary pollution. The main components of global plastic are polymers. Hence, new technologies are urgently required for the effective decomposition of the polymers in natural environments, which lays the foundation for this study on future plastic degradation. This study synthesizes the in-situ growth of TiO2 at graphene oxide (GO) matrix to form the TiO2@GO photocatalyst, and studies its application in conjugated polymers’ photodegradation. The photodegradation process could be probed by UV-vis absorption originating from the conjugated backbone of polymers. We have found that the complete decomposition of various polymers in a natural environment by employing the photocatalyst TiO2@GO within 12 days. It is obvious that the TiO2@GO shows a higher photocatalyst activity than the TiO2, due to the higher crystallinity morphology and smaller size of TiO2, and the faster transmission of photogenerated electrons from TiO2 to GO. The stronger fluorescence (FL) intensity of TiO2@GO compared to TiO2 at the terephthalic acid aqueous solution indicates that more hydroxyl radicals (•OH) are produced for TiO2@GO. This further confirms that the GO could effectively decrease the generation of recombination centers, enhance the separation efficiency of photoinduced electrons and holes, and increase the photocatalytic activity of TiO2@GO. This work establishes the underlying basic mechanism of polymers photodegradation, which might open new avenues for simultaneously addressing the white pollution crisis in a natural environment.

scholarly journals Competition between Metals for Binding to Methanobactin Enables Expression of Soluble Methane Monooxygenase in the Presence of Copper

2014 ◽  
Vol 81 (3) ◽  
pp. 1024-1031 ◽  
Author(s):  
Bhagyalakshmi Kalidass ◽  
Muhammad Farhan Ul-Haque ◽  
Bipin S. Baral ◽  
Alan A. DiSpirito ◽  
Jeremy D. Semrau
Keyword(s):  
Methane Monooxygenase ◽  
High Specificity ◽  
Copper Uptake ◽  
Content Type ◽  
Soluble Methane Monooxygenase ◽  
Sds Page ◽  
Genes Encoding ◽  
Key Factor ◽  
Membrane Bound

ABSTRACTIt is well known that copper is a key factor regulating expression of the two forms of methane monooxygenase found in proteobacterial methanotrophs. Of these forms, the cytoplasmic, or soluble, methane monooxygenase (sMMO) is expressed only at low copper concentrations. The membrane-bound, or particulate, methane monooxygenase (pMMO) is constitutively expressed with respect to copper, and such expression increases with increasing copper. Recent findings have shown that copper uptake is mediated by a modified polypeptide, or chalkophore, termed methanobactin. Although methanobactin has high specificity for copper, it can bind other metals, e.g., gold. Here we show that inMethylosinus trichosporiumOB3b, sMMO is expressed and active in the presence of copper if gold is also simultaneously present. Such expression appears to be due to gold binding to methanobactin produced byM. trichosporiumOB3b, thereby limiting copper uptake. Such expression and activity, however, was significantly reduced if methanobactin preloaded with copper was also added. Further, quantitative reverse transcriptase PCR (RT-qPCR) of transcripts of genes encoding polypeptides of both forms of MMO and SDS-PAGE results indicate that both sMMO and pMMO can be expressed when copper and gold are present, as gold effectively competes with copper for binding to methanobactin. Such findings suggest that under certain geochemical conditions, both forms of MMO may be expressed and activein situ. Finally, these findings also suggest strategies whereby field sites can be manipulated to enhance sMMO expression, i.e., through the addition of a metal that can compete with copper for binding to methanobactin.

scholarly journals Aptamers selected against the unglycosylated EGFRvIII ectodomain and delivered intracellularly reduce membrane-bound EGFRvIII and induce apoptosis

2009 ◽  
Vol 390 (2) ◽  
pp. 137-144 ◽  
Author(s):  
Yingmiao Liu ◽  
Chien-Tsun Kuan ◽  
Jing Mi ◽  
Xiuwu Zhang ◽  
Bryan M. Clary ◽  
...  
Keyword(s):  
Growth Factor Receptor ◽  
Rna Aptamers ◽  
Normal Brain ◽  
Post Translational Modification ◽  
Expression And Purification ◽  
Post Translational Modifications ◽  
Protein Expression And Purification ◽  
Epidermal Growth ◽  
Membrane Bound

Abstract Epidermal growth factor receptor variant III (EGFRvIII) is a glycoprotein uniquely expressed in glioblastoma, but not in normal brain tissues. To develop targeted therapies for brain tumors, we selected RNA aptamers against the histidine-tagged EGFRvIII ectodomain, using an Escherichia coli system for protein expression and purification. Representative aptamer E21 has a dissociation constant (Kd) of 33×10-9 m, and exhibits high affinity and specificity for EGFRvIII in ELISA and surface plasmon resonance assays. However, selected aptamers cannot bind the same protein expressed from eukaryotic cells because glycosylation, a post-translational modification present only in eukaryotic systems, significantly alters the structure of the target protein. By transfecting EGFRvIII aptamers into cells, we find that membrane-bound, glycosylated EGFRvIII is reduced and the percentage of cells undergoing apoptosis is increased. We postulate that transfected aptamers can interact with newly synthesized EGFRvIII, disrupt proper glycosylation, and reduce the amount of mature EGFRvIII reaching the cell surface. Our work establishes the feasibility of disrupting protein post-translational modifications in situ with aptamers. This finding is useful for elucidating the function of proteins of interest with various modifications, as well as dissecting signal transduction pathways.

scholarly journals INTEGRINS MEDIATE PLACENTAL EXTRACELLULAR VESICLE TRAFFICKING TO LUNG AND LIVER IN VIVO

2020 ◽  
Author(s):  
Sean L. Nguyen ◽  
Soo Hyun Ahn ◽  
Jacob W. Greenberg ◽  
Benjamin W. Collaer ◽  
Dalen W. Agnew ◽  
...  
Keyword(s):  
Immune Cells ◽  
Extracellular Vesicle ◽  
Dependent Manner ◽  
Cellular Phenotype ◽  
Reporter Mouse ◽  
Membrane Bound ◽  
First Time

ABSTRACTMembrane-bound extracellular vesicles (EVs) mediate intercellular communication in all organisms, and those produced by placental mammals have become increasingly recognized as significant mediators of fetal-maternal communication. Here, we aimed to identify maternal cells targeted by placental EVs and elucidate the mechanisms by which they traffic to these cells. Exogenously administered pregnancy-associated EVs traffic specifically to the lung; further, placental EVs associate with lung interstitial macrophages and liver Kupffer cells in an integrin-dependent manner. Localization of EV to maternal lungs was confirmed in unmanipulated pregnancy using a transgenic reporter mouse model, which also provided in situ and in vitro evidence that fetally-derived EVs, rarely, may cause genetic alteration of maternal cells. These results provide for the first time direct in vivo evidence for targeting of placental EVs to maternal immune cells, and further, evidence that EVs can alter cellular phenotype.

Development of a Direct In Situ PCR Method for Detection of Specific Bacteria in Natural Environments

1998 ◽  
Vol 64 (4) ◽  
pp. 1536-1540 ◽  
Author(s):  
Katsuji Tani ◽  
Ken Kurokawa ◽  
Masao Nasu
Keyword(s):  
Alkaline Phosphatase ◽  
Single Cell ◽  
Natural Environments ◽  
Single Cell Level ◽  
Bacterial Cells ◽  
Cell Level ◽  
In Situ Pcr ◽  
Glass Slides ◽  
Pcr Products

ABSTRACT We applied HNPP (2-hydroxy-3-naphthoic acid-2′-phenylanilide phosphate) to direct in situ PCR for the routine detection of specific bacterial cells at the single-cell level. PCR was performed on glass slides with digoxigenin-labeled dUTP. The digoxigenin-labeled PCR products were detected with alkaline phosphatase-labeled antidigoxigenin antibody and HNPP which was combined with Fast Red TR. A bright red fluorescent signal was produced from conversion to HNP (dephosphorylated form) by alkaline phosphatase. We used the ECOL DNA primer set for amplification of ribosomal DNA of Escherichia coli to identify cells specifically at the single-cell level in a bacterial mixture. High-contrast images were obtained under an epifluorescence microscope with in situ PCR. By image analysis,E. coli cells in polluted river water also were detected.

scholarly journals Stimulation of ovarian follicle growth after AMPK inhibition

Reproduction ◽  
2017 ◽  
Vol 153 (5) ◽  
pp. 683-694 ◽  
Author(s):  
Xiaowei Lu ◽  
Song Guo ◽  
Yuan Cheng ◽  
Jae-hong Kim ◽  
Yi Feng ◽  
...  
Keyword(s):  
Growth Factor ◽  
Protein Kinase ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Mrna Levels ◽  
Follicle Growth ◽  
Compound C ◽  
Old Mice ◽  
Stimulation Of

Previous studies showed that the protein kinase B (Akt)–mammalian target of rapamycin (mTOR) and Hippo signaling Yes-associated protein (YAP) pathways play important roles in promoting follicle growth. Additionally, other studies demonstrated that 5′ adenosine monophosphate-activated protein kinase (AMPK) is an upstream regulatory element of mTOR and YAP. Here, we used AMPK inhibitor (Compound C) toin vitrocultured ovaries from 10-day-old mice followed byin vivografting into adult hosts or toin situtreated ovaries of 3-week-old mice by intrabursal injection followed by gonadotropin stimulation. We found that the phosphorylation of ovarian mTOR and downstream proteins (ribosomal protein S6 (S6) and eukaryotic translation initiation factor 4B (eIF4B)) was upregulated following Compound C administration, whereas tuberous sclerosis complex 2 (TSC2) phosphorylation was downregulated. Additionally, treatment with Compound C increased hypoxia-inducible factor 1-alpha (Hif1a), vascular endothelial growth factor A (Vegfa), VEGF receptor 2 (Vegfr2) and connective tissue growth factor (Ctgf) mRNA levels. Furthermore, treatment of 10-day-old mice with Compound C promoted the growth of preantral and antral follicles accompanied by enhanced angiogenesis.In situintrabursal injection with Compound C, followed by controlled ovarian hyperstimulation, increased the number of ovulated oocytes in 3-week-old mice, and these oocytes could be successfully fertilized, leading to the delivery of healthy pups. Our results demonstrated that treatment with AMPK inhibitor resulted in the activation of the mTOR signaling pathway, increases inCtgfexpression in mouse ovaries, stimulation of follicle development and promotion of ovarian angiogenesis for ovary growth.

Effect of membrane perturbing treatments on the membrane-bound peptidases ofStreptococcus cremorisHP

1979 ◽  
Vol 46 (3) ◽  
pp. 473-484 ◽  
Author(s):  
Fred A. Exterkate
Keyword(s):  
Intact Cells ◽  
Irreversible Inhibition ◽  
Alkaline Conditions ◽  
Membrane Bound ◽  
Functional Peptide ◽  
Solvent Molecules ◽  
And Storage ◽  
No Activation ◽  
Proline Iminopeptidase

SummaryThe effects of solubilization, treatment with organic solvents and storage under alkaline conditions on membrane-associated peptidases of intact cells ofStreptococcus cremorisHP were studied. Differences in the response of the peptidase activities towards these membrane perturbing treatments were observed. Pyrrolidonecarboxylylpeptidase (PCP) and an endopeptidase (P50) showed 50% irreversible inhibition at the same concentration of each solvent tested. An amino- and proline iminopeptidase activity and the endopeptidase P37were in this respect much more sensitive to the action of the solvents. Within a homologous series of n-alkanols irreversible inhibition of PCP showed a dependence on the hydrophobicity of the solvent molecules. Only P37activity was increased considerably upon solubilization of the enzyme. Similar levels of activation were found upon treatment of cells with 3% (v/v) n-butanol at 25 °C or storage at 30 °C at an alkaline pH. Optimal activity of P50during n-butanol treatment was at 25 °C using a concentration of 5% (v/v), but no activation was observed upon solubilization. The results are discussed in terms of enzyme–lipid interaction and accessibility of the enzymes in situ. It is concluded that the enzymes apparently occupy different positions within the membrane although they may together constitute a functional peptide-hydrolysing unit.

scholarly journals From Genome to Function: Systematic Analysis of the Soil BacteriumBacillus subtilis

2001 ◽  
Vol 2 (1) ◽  
pp. 22-24 ◽  
Author(s):  
Colin R. Harwood ◽  
Samuel G. Crawshaw ◽  
Anil Wipat
Keyword(s):  
Functional Analysis ◽  
Bacillus Subtilis ◽  
Genome Sequence ◽  
Water Sources ◽  
Natural Environments ◽  
Positive Bacterium ◽  
Systematic Analysis ◽  
Analysis Programme ◽  
Analytical Approaches

Bacillus subtilisis a sporulating Gram-positive bacterium that lives primarily in the soil and associated water sources. Whilst this bacterium has been studied extensively in the laboratory, relatively few studies have been undertaken to study its activity in natural environments. The publication of theB. subtilisgenome sequence and subsequent systematic functional analysis programme have provided an opportunity to develop tools for analysing the role and expression ofBacillusgenesin situ. In this paper we discuss analytical approaches that are being developed to relate genes to function in environments such as the rhizosphere.

scholarly journals Targeting of c-myc and β-globin coding sequences to cytoskeletal-bound polysomes by c-myc 3′ untranslated region

1994 ◽  
Vol 298 (1) ◽  
pp. 143-148 ◽  
Author(s):  
J Hesketh ◽  
G Campbell ◽  
M Piechaczyk ◽  
J M Blanchard
Keyword(s):  
In Situ Hybridization ◽  
Untranslated Region ◽  
Mrna Localization ◽  
Eukaryotic Cells ◽  
Beta Globin ◽  
Globin Mrna ◽  
Directional Information ◽  
Coding Sequences ◽  
Membrane Bound

The influence of the 3′ untranslated region on mRNA localization was investigated by measuring the distribution of myc, beta-globin and hybrid myc-globin mRNAs between free, cytoskeletal-bound and membrane-bound polysomes in cells transfected with either control or chimeric gene constructs. c-myc sequences and beta-globin-coding sequences linked to the myc 3′ untranslated region were present at greatest enrichment in cytoskeletal-bound polysomes. beta-Globin mRNA and myc-coding sequences linked to the beta-globin 3′ untranslated region were recovered largely in the free polysomes. In situ hybridization confirmed that replacement of the c-myc 3′ untranslated region by that of globin caused a relocalization of the mRNA. The results suggest that mRNA localization in differentiated eukaryotic cells depends on a mechanism that involves directional information in the 3′ untranslated region of mRNAs.

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