Coaggregation among periodontal pathogens, emphasizing Bacteroides gingivalis –Actinomyces viscosus cohesion on a saliva-coated mineral surface

Teeth offer nonshedding surfaces on which a wide range of bacterial species accumulate as thick, cohesive plaques. Intergeneric coaggregation mediated by specific recognition between surface "cohesins" is thought to contribute to both the cohesiveness of plaque and the sequence in which bacteria colonize the tooth surface. There is some evidence that Gram-positive species, like the efficient tooth colonizer Actinomyces viscosus, enhance subsequent tooth colonization by the more virulent periodontal pathogen Bacteroides gingivalis. To study their mechanism of cohesion, we have developed an in vitro assay that measures the sequential binding of tritium-labeled B. gingivalis to A. viscosus adsorbed to saliva-coated hydroxyapatite beads, mimicking teeth (actinobeads). The assay yields equilibrium and kinetics data amenable to statistical analysis. The presence of A. viscosus significantly increased the number of B. gingivalis cells bound. Inhibition studies were conducted to test the sensitivity of binding to heat; to various saccharides and sugar amines; to proteolytic treatment of Bacteroides; and to incorporation of various chaotropic agents, increased KCl, and saliva in the suspension buffer. Heating the Bacteroides cells but not the actinobeads diminished Bacteroides adherence. Proteolysis and various saccharides had little, if any, effect. Among chaotropic agents, NaSCN and LiCl reduced numbers of cells bound by 40%, but tetramethylurea had no effect. Increasing the ionic concentration of KCl reduced binding by 50 to 60%. Diluted saliva showed a concentration-dependent inhibition of B. gingivalis adherence to actinobeads. To begin examining B. gingivalis surface molecules significant to these reactions, lipopolysaccharide was extracted by the phenol–water method and analyzed by biochemical assays and polyacrylamide gel electrophoresis. Both homologous and heterologous lipopolysaccharides partially inhibited binding in a concentration-dependent manner, but actual binding of B. gingivalis lipopolysaccharide to A. viscosus cells could not be demonstrated. While heat, lipopolysaccharide, chaotropic agents, and concentrated KCl interfered to a limited extent with B. gingivalis – A. viscosus interactions on a surface mimicking teeth, the actual mechanism of cohesion is currently obscure and under investigation. The relevance of in vitro coaggregation experiments to colonization by periodontal and other pathogens was discussed.

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CRL4-DCAF12 Ubiquitin Ligase Controls MOV10 RNA Helicase during Spermatogenesis and T Cell Activation

International Journal of Molecular Sciences ◽

10.3390/ijms22105394 ◽

2021 ◽

Vol 22 (10) ◽

pp. 5394

Author(s):

Tomas Lidak ◽

Nikol Baloghova ◽

Vladimir Korinek ◽

Radislav Sedlacek ◽

Jana Balounova ◽

...

Keyword(s):

T Cell ◽

T Cell Activation ◽

Ubiquitin Ligase ◽

Cell Activation ◽

Rna Helicase ◽

Dependent Manner ◽

Amino Acid Residues ◽

Risc Complex ◽

Wide Range

Multisubunit cullin-RING ubiquitin ligase 4 (CRL4)-DCAF12 recognizes the C-terminal degron containing acidic amino acid residues. However, its physiological roles and substrates are largely unknown. Purification of CRL4-DCAF12 complexes revealed a wide range of potential substrates, including MOV10, an “ancient” RNA-induced silencing complex (RISC) complex RNA helicase. We show that DCAF12 controls the MOV10 protein level via its C-terminal motif in a proteasome- and CRL-dependent manner. Next, we generated Dcaf12 knockout mice and demonstrated that the DCAF12-mediated degradation of MOV10 is conserved in mice and humans. Detailed analysis of Dcaf12-deficient mice revealed that their testes produce fewer mature sperms, phenotype accompanied by elevated MOV10 and imbalance in meiotic markers SCP3 and γ-H2AX. Additionally, the percentages of splenic CD4+ T and natural killer T (NKT) cell populations were significantly altered. In vitro, activated Dcaf12-deficient T cells displayed inappropriately stabilized MOV10 and increased levels of activated caspases. In summary, we identified MOV10 as a novel substrate of CRL4-DCAF12 and demonstrated the biological relevance of the DCAF12-MOV10 pathway in spermatogenesis and T cell activation.

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184 Two types of anti-TIGIT antibodies with distinct binding epitope and functional activities

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0184 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A198-A198

Author(s):

Tingting Zhong ◽

Xinghua Pang ◽

Zhaoliang Huang ◽

Na Chen ◽

Xiaoping Jin ◽

...

Keyword(s):

T Cells ◽

Mixed Culture ◽

Cell Activity ◽

Cross Reactivity ◽

Binding Activity ◽

Jurkat Cells ◽

List Type ◽

Dependent Manner ◽

Vitro Assay

BackgroundTIGIT is an inhibitory receptor mainly expressed on natural killer (NK) cells, CD8+ T cells, CD4+ T cells and Treg cells. TIGIT competes with CD226 for binding with CD155. In cancers, CD155 has been reported to up-regulate on tumor cells, and TIGIT was found to increase on TILs.1 Activation of TIGIT/CD155 pathway would mediate immunosuppression in tumor; while blockade of TIGIT promotes anti-tumor immune response.MethodsAK126 and AK113 are two humanized anti-human TIGIT monoclonal antibodies developed by Akesobio. Binding activity of AK126 and AK113 to human TIGIT, and competitive binding activity with CD155 and CD112, were performed by using ELISA, Fortebio, and FACS assays. Cross-reactivity with cynomolgus monkey TIGIT and epitope binning were also tested by ELISA assay. In-vitro assay to investigate the activity to promote IL-2 secretion was performed in mixed-culture of Jurkat-TIGIT cells and THP-1 cells.ResultsAK126 and AK113 could specifically bind to human TIGIT with comparative affinity and effectively blocked the binding of human CD155 and CD112 to human TIGIT. X-ray crystal structure of TIGIT and PVR revealed the C’-C’’ loop and FG loop regions of TIGIT are the main PVR interaction regions.2 The only amino acid residue differences in these regions between human and monkey TIGIT are 70C and 73D. AK126 binds to both human and monkey TIGIT, AK113 binds only to monkey TIGIT. This suggests that these residues are required for AK113 binding to human TIGIT, but not required for AK126. Interestingly, results from cell-based assays indicated that AK126 and AK113 showed significantly different activity to induce IL-2 secretion in mixed-culture of Jurkat-TIGIT cells and THP-1 cells (figure 1A and B), in which AK126 had a comparable capacity of activity to 22G2, a leading TIGIT mAb developed by another company, to induce IL-2 secretion, while, AK113 showed a significantly higher capacity than 22G2 and AK126.Abstract 184 Figure 1Anti-TIGIT Antibodies Rescues IL-2 Production in Vitro T-Cell Activity Assay in a dose dependent manner. Jurkat-TIGIT cells (Jurkat cells engineered to over-express human TIGIT) were co-cultured with THP-1 cells, and stimulated with plate-bound anti-CD3 mAb in the presence of TIGIT ligand CD155 (A) or CD112 (B) with anti-TIGIT antibodies. After incubated for 48h at 37° C and 5.0% CO2, IL-2 levels were assessed in culture supernatants by ELISA. Data shown as mean with SEM for n = 2.ConclusionsWe discovered two distinct types of TIGIT antibodies with differences in both epitope binding and functional activity. The mechanism of action and clinical significance of these antibodies require further investigation.ReferencesSolomon BL, Garrido-Laguna I. TIGIT: a novel immunotherapy target moving from bench to bedside. Cancer Immunol Immunother 2018;67:1659–1667.Stengel KF, Harden-Bowles K, Yu X, et al. Structure of TIGIT immunoreceptor bound to poliovirus receptor reveals a cell-cell adhesion and signaling mechanism that requires cis-trans receptor clustering. Proc Natl Acad Sci USA 2012;109:5399–5404.

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Strong antihemolytic and antioxidant properties of aqueous extract from Algerian Hammada elegans (Bge.) Botsch (Chenopodiaceae)

Current Enzyme Inhibition ◽

10.2174/1573408017666210419115739 ◽

2021 ◽

Vol 17 ◽

Author(s):

Brahim Asseli ◽

Reguia Mahfoudi ◽

Amar Djeridane ◽

Mohamed Yousfi

Keyword(s):

Free Radical ◽

Aqueous Extract ◽

Radical Scavenging Activity ◽

Condensed Tannin ◽

Antioxidant Properties ◽

Radical Scavenging ◽

Total Phenolic ◽

Dependent Manner ◽

Vitro Assay

Background: Research on medicinal plant antioxidants has emerged as a potential therapeutic to prevent free radical generated damage in the human body. Hammada elegans Botsch (popularly known as “Ajram”) is a xerophytic plant widely found in Laghouat region, but there are only a few reports about the biological or chemical properties of these species. Hence, the aim of this study is to investigate the antioxidant and the antihemolytic activities of hexanic, acetonic, methanolic and aqueous extracts of aerial parts of Algerian Hammada elegans Botsch by employing different in vitro assay systems. Methods: The total phenolic content, the flavonoid content and the condensed tannin amount were analyzed using Folin-Ciocalteu, aluminum chloride and vanillin assays, respectively. The in vitro antioxidant capacity of extracts was assessed by CUPRAC, iron chelating, ABTS•+and antihemolytic assays, and was expressed as EC50 values. Results: Among the analyzed extracts, the aqueous extract had the highest phenolic, flavonoid and tannin contents. Also, this extract displayed the highest antioxidant capacities compared to the other extracts and standards. Its EC50 value for ABTS radical-scavenging activity was 0.265 ± 0.003 mg/L. Moreover, this extract showed high iron (II) chelating ability (EC50 = 0.958 ± 0.001 mg/L), and good antioxidant activity in the cupric ion reducing activity (CUPRAC) in a concentration dependent manner (EC50 were 0.709 ± 0.002 mg/L). Additionally, this extract had the best antihemolytic activity against AAPH-induced hemolysis (EC50=0.090 ± 0.004 mg/L). Conclusion: Our study revealed that the aqueous extract of Hammada elegans Botsch, is a potential source of antioxidants which possess a high protective effect of membrane against free radical.

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In vitro analysis of a transcription termination site for RNA polymerase II.

Molecular and Cellular Biology ◽

10.1128/mcb.10.11.5782 ◽

1990 ◽

Vol 10 (11) ◽

pp. 5782-5795 ◽

Cited By ~ 19

Author(s):

D K Wiest ◽

D K Hawley

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Dna Sequences ◽

Nuclear Extract ◽

Transcription Unit ◽

Dependent Manner ◽

Wide Range ◽

Vitro Analysis

Transcription from the adenovirus major late (ML) promoter has previously been shown to pause or terminate prematurely in vivo and in vitro at a site within the first intron of the major late transcription unit. We are studying the mechanism of elongation arrest at this site in vitro to define the DNA sequences and proteins that determine the elongation behavior of RNA polymerase II. Our assay system consists of a nuclear extract prepared from cultured human cells. With standard reaction conditions, termination is not observed downstream of the ML promoter. However, in the presence of Sarkosyl, up to 80% of the transcripts terminate 186 nucleotides downstream of the start site. Using this assay, we showed that the DNA sequences required to promote maximal levels of termination downstream of the ML promoter reside within a 65-base-pair region and function in an orientation-dependent manner. To test whether elongation complexes from the ML promoter were functionally homogeneous, we determined the termination efficiency at each of two termination sites placed in tandem. We found that the behavior of the elongation complexes was different at these sites, with termination being greater at the downstream site over a wide range of Sarkosyl concentrations. This result ruled out a model in which the polymerases that read through the first site were stably modified to antiterminate. We also demonstrated that the ability of the elongation complexes to respond to the ML termination site was promoter specific, as the site did not function efficiently downstream of a heterologous promoter. Taken together, the results presented here are not consistent with the simplest class of models that have been proposed previously for the mechanism of Sarkosyl-induced termination.

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A Possible Role of Allatostatin C in Inhibiting Ecdysone Biosynthesis Revealed in the Mud Crab Scylla paramamosain

Frontiers in Marine Science ◽

10.3389/fmars.2021.740251 ◽

2021 ◽

Vol 8 ◽

Author(s):

An Liu ◽

Wenyuan Shi ◽

Dongdong Lin ◽

Haihui Ye

Keyword(s):

Scylla Paramamosain ◽

Dependent Manner ◽

Mud Crab ◽

Methyl Farnesoate ◽

Independent Manner ◽

Wide Range ◽

Negative Effect

C-type allatostatins (C-type ASTs) are a family of structurally related neuropeptides found in a wide range of insects and crustaceans. To date, the C-type allatostatin receptor in crustaceans has not been deorphaned, and little is known about its physiological functions. In this study, we aimed to functionally define a C-type ASTs receptor in the mud crab, Scylla paramamosian. We showed that C-type ASTs receptor can be activated by ScypaAST-C peptide in a dose-independent manner and by ScypaAST-CCC peptide in a dose-dependent manner with an IC50 value of 6.683 nM. Subsequently, in vivo and in vitro experiments were performed to investigate the potential roles of ScypaAST-C and ScypaAST-CCC peptides in the regulation of ecdysone (20E) and methyl farnesoate (MF) biosynthesis. The results indicated that ScypaAST-C inhibited biosynthesis of 20E in the Y-organ, whereas ScypaAST-CCC had no effect on the production of 20E. In addition, qRT-PCR showed that both ScypaAST-C and ScypaAST-CCC significantly decreased the level of expression of the MF biosynthetic enzyme gene in the mandibular organ, suggesting that the two neuropeptides have a negative effect on the MF biosynthesis in mandibular organs. In conclusion, this study provided new insight into the physiological roles of AST-C in inhibiting ecdysone biosynthesis. Furthermore, it was revealed that AST-C family peptides might inhibit MF biosynthesis in crustaceans.

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Effects of polymethoxylated flavone metabolites on apoB100 secretion and MTP activity in Huh7.5 cells

Current Bioactive Compounds ◽

10.2174/1573407218666211230140952 ◽

2021 ◽

Vol 18 ◽

Author(s):

Danielle R. Gonçalves ◽

Thais B. Cesar ◽

John A. Manthey ◽

Paulo I. Costa

Keyword(s):

Lipid Synthesis ◽

Dependent Manner ◽

Transfer Protein ◽

Low Concentrations ◽

Wide Range ◽

Cell Assays ◽

Polymethoxylated Flavones ◽

High Concentrations

Background: Citrus polymethoxylated flavones (PMFs) reduce the synthesis of liver lipoproteins in animal and in vitro cell assays, but few studies have evaluated the direct effects of their metabolites on this highly regulated process. Objective: To investigate the effects of representative metabolites of PMF on the secretion of liver lipoproteins using the mammalian cell Huh7.5. Method: In this study, the influences of three PMFs and five previously isolated PMF metabolites on hepatic apoB-100 secretion and microsomal transfer protein (MTP) activity were evaluated. Tangeretin (TAN), nobiletin (NOB) and 3,5,6,7,8,3′,4′-heptamethoxyflavone (HMF), and their glucuronides (TAN-Gluc, NOB-Gluc and HMF-Gluc) and oxidatively demethylated metabolites (TAN-OH, NOB-OH, HMF-OH) were incubated with Huh7.5 cells to measure their inhibitory effects on lipid synthesis. Results: The results showed that TAN, HMF and TAN-OH reduced the secretion of apoB-100 in a dose-dependent manner, while NOB and the other tested metabolites showed no inhibition. MTP activity in the Huh7.5 cells was significantly reduced in the presence of low concentrations of TAN, and in high concentrations of NOB-OH. This study also showed that PMFs and PMF metabolites produced a wide range of effects on apoB-100 secretion and MTP activity. Conclusion: The results suggest that while PMFs and their metabolites control dyslipidemia in vivo, the inhibition of MTP activity cannot be the only pathway influenced by these compounds.

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RNA G-Quadruplex Structures Mediate Gene Regulation in Bacteria

mBio ◽

10.1128/mbio.02926-19 ◽

2020 ◽

Vol 11 (1) ◽

Cited By ~ 4

Author(s):

Xiaolong Shao ◽

Weitong Zhang ◽

Mubarak Ishaq Umar ◽

Hei Yuen Wong ◽

Zijing Seng ◽

...

Keyword(s):

Gene Regulation ◽

Cell Envelope ◽

Bacterial Species ◽

Virulence Gene ◽

Content Type ◽

Cellular Processes ◽

Wide Range ◽

G Quadruplex ◽

Eukaryotic Organisms

ABSTRACT Guanine (G)-rich sequences in RNA can fold into diverse RNA G-quadruplex (rG4) structures to mediate various biological functions and cellular processes in eukaryotic organisms. However, the presence, locations, and functions of rG4s in prokaryotes are still elusive. We used QUMA-1, an rG4-specific fluorescent probe, to detect rG4 structures in a wide range of bacterial species both in vitro and in live cells and found rG4 to be an abundant RNA secondary structure across those species. Subsequently, to identify bacterial rG4 sites in the transcriptome, the model Escherichia coli strain and a major human pathogen, Pseudomonas aeruginosa, were subjected to recently developed high-throughput rG4 structure sequencing (rG4-seq). In total, 168 and 161 in vitro rG4 sites were found in E. coli and P. aeruginosa, respectively. Genes carrying these rG4 sites were found to be involved in virulence, gene regulation, cell envelope synthesis, and metabolism. More importantly, biophysical assays revealed the formation of a group of rG4 sites in mRNAs (such as hemL and bswR), and they were functionally validated in cells by genetic (point mutation and lux reporter assays) and phenotypic experiments, providing substantial evidence for the formation and function of rG4s in bacteria. Overall, our study uncovers important regulatory functions of rG4s in bacterial pathogenicity and metabolic pathways and strongly suggests that rG4s exist and can be detected in a wide range of bacterial species. IMPORTANCE G-quadruplex in RNA (rG4) mediates various biological functions and cellular processes in eukaryotic organisms. However, the presence, locations, and functions of rG4 are still elusive in prokaryotes. Here, we found that rG4 is an abundant RNA secondary structure across a wide range of bacterial species. Subsequently, the transcriptome-wide rG4 structure sequencing (rG4-seq) revealed that the model E. coli strain and a major human pathogen, P. aeruginosa, have 168 and 161 in vitro rG4 sites, respectively, involved in virulence, gene regulation, cell envelope, and metabolism. We further verified the regulatory functions of two rG4 sites in bacteria (hemL and bswR). Overall, this finding strongly suggests that rG4s play key regulatory roles in a wide range of bacterial species.

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Phenolic Acids from Lycium barbarum Leaves: In Vitro and In Silico Studies of the Inhibitory Activity against Porcine Pancreatic α-Amylase

Processes ◽

10.3390/pr8111388 ◽

2020 ◽

Vol 8 (11) ◽

pp. 1388

Author(s):

Luna Pollini ◽

Alessandra Riccio ◽

Cristina Juan ◽

Carmela Tringaniello ◽

Federica Ianni ◽

...

Keyword(s):

Phenolic Acids ◽

Inhibitory Activity ◽

Leaf Extract ◽

Inhibitory Effect ◽

Dependent Manner ◽

Lycium Barbarum ◽

Plant Materials ◽

Vitro Assay ◽

In Silico Studies

Nowadays, bioactive compounds from vegetable food and waste are of great interest for their inhibitory potential against digestive enzymes. In the present study, the inhibitory activity of methanolic extract from Lycium barbarum leaves on porcine pancreas α-amylase has been studied. The α-amylase inhibitory activity of the constituent phenolic acids was also investigated. The leaves were extracted by ultrasound-assisted method, one of the most efficient techniques for bioactive extraction from plant materials, and then the phenolic acids were identified by Accurate-Mass Quadrupole Time-of-Flight (Q-TOF) Liquid Chromatography/Mass Spectrometry (LC/MS). Chlorogenic and salicylic acids were the most abundant phenolic acids in L. barbarum leaf extract. The inhibitory effect against α-amylase, determined for individual compounds by in vitro assay, was higher for chlorogenic, salicylic, and caffeic acids. L. barbarum leaf extract showed an appreciable α-amylase inhibitory effect in a concentration-dependent manner. Docking studies of the considered phenolic acids into the active site of α-amylase suggested a conserved binding mode that is mainly stabilized through H-bonds and π-π stacking interactions.

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Taurolidine Acts on Bacterial Virulence Factors and Does Not Induce Resistance in Periodontitis-Associated Bacteria—An In-Vitro Study

Antibiotics ◽

10.3390/antibiotics9040166 ◽

2020 ◽

Vol 9 (4) ◽

pp. 166

Author(s):

Sabrina Radakovic ◽

Nicola Andreoli ◽

Simon Schmid ◽

Sandor Nietzsche ◽

Jürg Zumbrunn ◽

...

Keyword(s):

Virulence Factors ◽

Bacterial Species ◽

Rare Event ◽

In Vitro Study ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Minimal Inhibitory Concentrations ◽

Development Of Resistance ◽

Subinhibitory Concentrations

The aims of the present study were: (a) to determine the mechanism of action of taurolidine against bacterial species associated with periodontal disease, and (b) to evaluate the potential development of resistance against taurolidine as compared with minocycline. After visualizing the mode of action of taurolidine by transmission electron micrographs, the interaction with most important virulence factors (lipopolysaccharide (LPS), Porphyromonas gingivalis gingipains, Aggregatibacter actinomycetemcomitans leukotoxin), was analyzed. Then, 14 clinical isolates from subgingival biofilm samples were transferred on agar plates containing subinhibitory concentrations of taurolidine or minocycline up to 50 passages. Before and after each 10 passages, minimal inhibitory concentrations (MICs) were determined. Increasing MICs were screened for efflux mechanism. Taurolidine inhibited in a concentration-dependent manner the activities of LPS and of the arginine-specific gingipains; however, an effect on A. actinomycetemcomitans leukotoxin was not detected. One P. gingivalis strain developed a resistance against taurolidine, which was probably linked with efflux mechanisms. An increase of MIC values of minocycline occurred in five of the 14 included strains after exposure to subinhibitory concentrations of the antibiotic. The present results indicate that: (a) taurolidine interacts with LPS and gingipains, and (b) development of resistance seems to be a rare event when using taurolidine.

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Defining the core essential genome ofPseudomonas aeruginosa

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1900570116 ◽

2019 ◽

Vol 116 (20) ◽

pp. 10072-10080 ◽

Cited By ~ 31

Author(s):

Bradley E. Poulsen ◽

Rui Yang ◽

Anne E. Clatworthy ◽

Tiantian White ◽

Sarah J. Osmulski ◽

...

Keyword(s):

Bacterial Species ◽

Growth Conditions ◽

Essential Genes ◽

The Core ◽

Transposon Insertion ◽

Wide Range ◽

Transposon Insertion Sequencing ◽

Antibiotic Discovery

Genomics offered the promise of transforming antibiotic discovery by revealing many new essential genes as good targets, but the results fell short of the promise. While numerous factors contributed to the disappointing yield, one factor was that essential genes for a bacterial species were often defined based on a single or limited number of strains grown under a single or limited number of in vitro laboratory conditions. In fact, the essentiality of a gene can depend on both the genetic background and growth condition. We thus developed a strategy for more rigorously defining the core essential genome of a bacterial species by studying many pathogen strains and growth conditions. We assessed how many strains must be examined to converge on a set of core essential genes for a species. We used transposon insertion sequencing (Tn-Seq) to define essential genes in nine strains ofPseudomonas aeruginosaon five different media and developed a statistical model,FiTnEss, to classify genes as essential versus nonessential across all strain–medium combinations. We defined a set of 321 core essential genes, representing 6.6% of the genome. We determined that analysis of four strains was typically sufficient inP. aeruginosato converge on a set of core essential genes likely to be essential across the species across a wide range of conditions relevant to in vivo infection, and thus to represent attractive targets for novel drug discovery.

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