When the loop-mediated isothermal amplification(LAMP)assay is used for detecting target genes,DNA extraction is unnecessary in many cases.Simple pretreatment(e.g.heating)is enough to obtain rather sensitive responses....When the loop-mediated isothermal amplification(LAMP)assay is used for detecting target genes,DNA extraction is unnecessary in many cases.Simple pretreatment(e.g.heating)is enough to obtain rather sensitive responses.Even test samples without any pretreatment can be used as template.This feature suggests that LAMP is superior to PCR in developing point-of-care test strategies.In this study,using Stx1 gene from E.coli as model,we verified that viable cells,dead cells and extracellular DNA could function as template in the LAMP assay.In the incubation at 63℃,viable bacteria in the LAMP reaction mixture lysed completely within 2 min,providing DNA template for nucleic acid amplification.The Stx1 gene in diluted culture medium,spiked tap water,spiked seawater and real seawater all could be detected,with or without the step of DNA extraction.We found that the complex substances in real sample(e.g.natural seawater)exhibited considerable inhibitory effect on the sensitivity of the LAMP assay.These outcomes are meaningful for building a point-of-care strategy by employing the LAMP assay for environmental monitoring,bio-resource surveys,food safety,etc.in particular those based on environmental DNA.展开更多
DNA is the genetic material of various organisms. Extracellular DNA adsorbed or bound on surface-active particles in soils has been shown to persist for long periods against nucleases degradation and still retain the ...DNA is the genetic material of various organisms. Extracellular DNA adsorbed or bound on surface-active particles in soils has been shown to persist for long periods against nucleases degradation and still retain the ability to transform competent cells. This paper reviews some recent advances on the binding and transformation of extracellular DNA in soils,which is fundamental to understanding the nature of the soil, regulating biodiversity, and assessing the risk of releasing genetically engineered microorganisms (GEMs) as well as being helpful for development of the genetic evolutional theory of bacteria. Several influencing factors, such as soil pH, ionic strength, soil surface properties, and characteristics of the DNA polymer, are discussed. To date, the understanding of the type of molecular binding sites and the conformation of adsorbed and bound DNA to soil particles is still in its infancy.展开更多
AIM To demonstrate that specific bacteria might release bacterial extracellular DNA(e DNA) to exert immunomodulatory functions in the mouse small intestine.METHODS Extracellular DNA was extracted using phosphate buffe...AIM To demonstrate that specific bacteria might release bacterial extracellular DNA(e DNA) to exert immunomodulatory functions in the mouse small intestine.METHODS Extracellular DNA was extracted using phosphate buffered saline with 0.5 mmol/L dithiothreitol combined with two phenol extractions. TOTO-1 iodide, a cell-impermeant and high-affinity nucleic acid stain, was used to confirm the existence of e DNA in the mucus layers of the small intestineand colon in healthy Male C57 BL/6 mice. Composition difference of e DNA and intracellular DNA(i DNA) of the small intestinal mucus was studied by Illumina sequencing and terminal restriction fragment length polymorphism(T-RFLP). Stimulation of cytokine production by e DNA was studied in RAW264.7 cells in vitro.RESULTS TOTO-1 iodide staining confirmed existence of e DNA in loose mucus layer of the mouse colon and thin surface mucus layer of the small intestine. Illumina sequencing analysis and T-RFLP revealed that the composition of the e DNA in the small intestinal mucus was significantly different from that of the i DNA of the small intestinal mucus bacteria. Illumina Miseq sequencing showed that the e DNA sequences came mainly from Gram-negative bacteria of Bacteroidales S24-7. By contrast, predominant bacteria of the small intestinal flora comprised Grampositive bacteria. Both e DNA and i DNA were added to native or lipopolysaccharide-stimulated Raw267.4 macrophages, respectively. The e DNA induced significantly lower tumor necrosis factor-α/interleukin-10(IL-10) and IL-6/IL-10 ratios than i DNA, suggesting the predominance for maintaining immune homeostasis of the gut.CONCLUSION Our results indicated that degraded bacterial genomic DNA was mainly released by Gram-negative bacteria, especially Bacteroidales-S24-7 and Stenotrophomonas genus in gut mucus of mice. They decreased pro-inflammatory activity compared to total gut flora genomic DNA.展开更多
The migrating TdT<sup>+</sup> thymocytes can die in other tissues, promoting the surrounding cells’ renewing likes holocrine secretion does. To clarify the role of TdT-enzyme for this function of progenit...The migrating TdT<sup>+</sup> thymocytes can die in other tissues, promoting the surrounding cells’ renewing likes holocrine secretion does. To clarify the role of TdT-enzyme for this function of progenitor lymphocytes, their extracellular media with its components included by living cells analyzed <em>in vitro</em> before and after<em> in vivo</em> irradiation of donor rats. The nucleoid with DNase-sensitive (free) DNA and TdT activity discovered in extracellular media conditioned preliminary by spontaneous apoptotic death of a minor part of the thymocyte’s suspension <em>in vitro</em>. The penetration of labeled products of non-template synthesis with free DNA’ primers from media into cells by pinocytosis confirmed by exogenous polymeric DNA marked artificially. The DNA penetration into cells follows an increase of the cell’s viability and acceleration of spontaneous intracellular DNA-synthesis controlled with labeled thymidine uptake. Both phenomena are typical for either the lowest initial concentration of intact cells or their preliminary irradiation <em>in vivo</em>. The data point to possible involvement of apoptotic decay of TdT<sup>+</sup> cells in the reutilization of the extracellular DNA fragments for reparation/regeneration of surrounding living cells.展开更多
Biofilms, the preferred bacterial mode of living and survival, are employed by most microorganisms—which tend to attach to surfaces—to gain physical support, increase nutrient utilization and availability, and augme...Biofilms, the preferred bacterial mode of living and survival, are employed by most microorganisms—which tend to attach to surfaces—to gain physical support, increase nutrient utilization and availability, and augment their resistance against anti-bacterial agents. Rhodococcus ruber (C208) has been shown to form a dense biofilm on polyethylene surfaces while degrading them. Bacterial biofilms comprise bacterial cells embedded in self-secreted extracellular polymeric substances (EPS) whose main components are polysaccharides, proteins and nucleic acids. Revealing the roles of these components will enable further insight into biofilm development and, therefore, the EPS structure-function relationship. The current study focuses on contribution of extracellular DNA to biofilm formation and stability. This was approached by investigating the influence of nucleases on biofilm formation via degradation of their corresponding substrates within the biofilm of C208. RNase application to cultures of C208 decreased biofilm formation. Degradation of biofilm DNA by DNase reduced early-stage biofilm formation by 20% -25% but had no significant effect on established, mature biofilm. Likewise, the addition of DNA to cultures significantly enhanced early-stage biofilm formation by 50% -100%. RAPD-PCR analysis revealed different band patterns from intra-cellular DNA and extra-cellular DNA and also between the supernatant and biofilm fractions of extra-cellular DNA, indicating that perhaps only certain DNA molecules are utilized as part of the biofilm.展开更多
With increasing environmental application,biochar(BC)will inevitably interact with and impact environmental behaviors of widely distributed extracellular DNA(eDNA),which however still remains to be studied.Herein,the ...With increasing environmental application,biochar(BC)will inevitably interact with and impact environmental behaviors of widely distributed extracellular DNA(eDNA),which however still remains to be studied.Herein,the adsorption/desorption and the degradation by nucleases of eDNA on three aromatized BCs pyrolyzed at 700℃were firstly investigated.The results show that the eDNA was irreversibly adsorbed by aromatized BCs and the pseudo-second-order and Freundlich models accurately described the adsorption process.Increasing solution ionic strength or decreasing pH below 5.0 significantly increased the eDNA adsorption on BCs.However,increasing pH from 5.0 to 10.0 faintly decreased eDNA adsorption.Electrostatic interaction,Ca ion bridge interaction,andπ-πinteraction between eDNA and BC could dominate the eDNA adsorption,while ligand exchange and hydrophobic interactions were minor contributors.The presence of BCs provided a certain protection to eDNA against degradation by DNase I.BC-bound eDNA could be partly degraded by nuclease,while BC-bound nuclease completely lost its degradability.These findings are of fundamental significance for the potential application of biochar in eDNA dissemination management and evaluating the environmental fate of eDNA.展开更多
文摘When the loop-mediated isothermal amplification(LAMP)assay is used for detecting target genes,DNA extraction is unnecessary in many cases.Simple pretreatment(e.g.heating)is enough to obtain rather sensitive responses.Even test samples without any pretreatment can be used as template.This feature suggests that LAMP is superior to PCR in developing point-of-care test strategies.In this study,using Stx1 gene from E.coli as model,we verified that viable cells,dead cells and extracellular DNA could function as template in the LAMP assay.In the incubation at 63℃,viable bacteria in the LAMP reaction mixture lysed completely within 2 min,providing DNA template for nucleic acid amplification.The Stx1 gene in diluted culture medium,spiked tap water,spiked seawater and real seawater all could be detected,with or without the step of DNA extraction.We found that the complex substances in real sample(e.g.natural seawater)exhibited considerable inhibitory effect on the sensitivity of the LAMP assay.These outcomes are meaningful for building a point-of-care strategy by employing the LAMP assay for environmental monitoring,bio-resource surveys,food safety,etc.in particular those based on environmental DNA.
文摘DNA is the genetic material of various organisms. Extracellular DNA adsorbed or bound on surface-active particles in soils has been shown to persist for long periods against nucleases degradation and still retain the ability to transform competent cells. This paper reviews some recent advances on the binding and transformation of extracellular DNA in soils,which is fundamental to understanding the nature of the soil, regulating biodiversity, and assessing the risk of releasing genetically engineered microorganisms (GEMs) as well as being helpful for development of the genetic evolutional theory of bacteria. Several influencing factors, such as soil pH, ionic strength, soil surface properties, and characteristics of the DNA polymer, are discussed. To date, the understanding of the type of molecular binding sites and the conformation of adsorbed and bound DNA to soil particles is still in its infancy.
基金Supported by China Postdoctoral Science Foundation,No.172774Fund of Key Laboratory of Carbohydrate Chemistry and Biotechnology,Ministry of Education,Jiangnan University,No.KLCCB-KF201603National Natural Science Foundation of China,No.31201805
文摘AIM To demonstrate that specific bacteria might release bacterial extracellular DNA(e DNA) to exert immunomodulatory functions in the mouse small intestine.METHODS Extracellular DNA was extracted using phosphate buffered saline with 0.5 mmol/L dithiothreitol combined with two phenol extractions. TOTO-1 iodide, a cell-impermeant and high-affinity nucleic acid stain, was used to confirm the existence of e DNA in the mucus layers of the small intestineand colon in healthy Male C57 BL/6 mice. Composition difference of e DNA and intracellular DNA(i DNA) of the small intestinal mucus was studied by Illumina sequencing and terminal restriction fragment length polymorphism(T-RFLP). Stimulation of cytokine production by e DNA was studied in RAW264.7 cells in vitro.RESULTS TOTO-1 iodide staining confirmed existence of e DNA in loose mucus layer of the mouse colon and thin surface mucus layer of the small intestine. Illumina sequencing analysis and T-RFLP revealed that the composition of the e DNA in the small intestinal mucus was significantly different from that of the i DNA of the small intestinal mucus bacteria. Illumina Miseq sequencing showed that the e DNA sequences came mainly from Gram-negative bacteria of Bacteroidales S24-7. By contrast, predominant bacteria of the small intestinal flora comprised Grampositive bacteria. Both e DNA and i DNA were added to native or lipopolysaccharide-stimulated Raw267.4 macrophages, respectively. The e DNA induced significantly lower tumor necrosis factor-α/interleukin-10(IL-10) and IL-6/IL-10 ratios than i DNA, suggesting the predominance for maintaining immune homeostasis of the gut.CONCLUSION Our results indicated that degraded bacterial genomic DNA was mainly released by Gram-negative bacteria, especially Bacteroidales-S24-7 and Stenotrophomonas genus in gut mucus of mice. They decreased pro-inflammatory activity compared to total gut flora genomic DNA.
文摘The migrating TdT<sup>+</sup> thymocytes can die in other tissues, promoting the surrounding cells’ renewing likes holocrine secretion does. To clarify the role of TdT-enzyme for this function of progenitor lymphocytes, their extracellular media with its components included by living cells analyzed <em>in vitro</em> before and after<em> in vivo</em> irradiation of donor rats. The nucleoid with DNase-sensitive (free) DNA and TdT activity discovered in extracellular media conditioned preliminary by spontaneous apoptotic death of a minor part of the thymocyte’s suspension <em>in vitro</em>. The penetration of labeled products of non-template synthesis with free DNA’ primers from media into cells by pinocytosis confirmed by exogenous polymeric DNA marked artificially. The DNA penetration into cells follows an increase of the cell’s viability and acceleration of spontaneous intracellular DNA-synthesis controlled with labeled thymidine uptake. Both phenomena are typical for either the lowest initial concentration of intact cells or their preliminary irradiation <em>in vivo</em>. The data point to possible involvement of apoptotic decay of TdT<sup>+</sup> cells in the reutilization of the extracellular DNA fragments for reparation/regeneration of surrounding living cells.
文摘Biofilms, the preferred bacterial mode of living and survival, are employed by most microorganisms—which tend to attach to surfaces—to gain physical support, increase nutrient utilization and availability, and augment their resistance against anti-bacterial agents. Rhodococcus ruber (C208) has been shown to form a dense biofilm on polyethylene surfaces while degrading them. Bacterial biofilms comprise bacterial cells embedded in self-secreted extracellular polymeric substances (EPS) whose main components are polysaccharides, proteins and nucleic acids. Revealing the roles of these components will enable further insight into biofilm development and, therefore, the EPS structure-function relationship. The current study focuses on contribution of extracellular DNA to biofilm formation and stability. This was approached by investigating the influence of nucleases on biofilm formation via degradation of their corresponding substrates within the biofilm of C208. RNase application to cultures of C208 decreased biofilm formation. Degradation of biofilm DNA by DNase reduced early-stage biofilm formation by 20% -25% but had no significant effect on established, mature biofilm. Likewise, the addition of DNA to cultures significantly enhanced early-stage biofilm formation by 50% -100%. RAPD-PCR analysis revealed different band patterns from intra-cellular DNA and extra-cellular DNA and also between the supernatant and biofilm fractions of extra-cellular DNA, indicating that perhaps only certain DNA molecules are utilized as part of the biofilm.
基金supported by the National Natural Science Foundation of China(Nos.21976158,21525728,and 21677129)。
文摘With increasing environmental application,biochar(BC)will inevitably interact with and impact environmental behaviors of widely distributed extracellular DNA(eDNA),which however still remains to be studied.Herein,the adsorption/desorption and the degradation by nucleases of eDNA on three aromatized BCs pyrolyzed at 700℃were firstly investigated.The results show that the eDNA was irreversibly adsorbed by aromatized BCs and the pseudo-second-order and Freundlich models accurately described the adsorption process.Increasing solution ionic strength or decreasing pH below 5.0 significantly increased the eDNA adsorption on BCs.However,increasing pH from 5.0 to 10.0 faintly decreased eDNA adsorption.Electrostatic interaction,Ca ion bridge interaction,andπ-πinteraction between eDNA and BC could dominate the eDNA adsorption,while ligand exchange and hydrophobic interactions were minor contributors.The presence of BCs provided a certain protection to eDNA against degradation by DNase I.BC-bound eDNA could be partly degraded by nuclease,while BC-bound nuclease completely lost its degradability.These findings are of fundamental significance for the potential application of biochar in eDNA dissemination management and evaluating the environmental fate of eDNA.
