scholarly journals Bugs on Drugs: A Drosophila melanogaster Gut Model to Study In Vivo Antibiotic Tolerance of E. coli

2022 ◽  
Vol 10 (1) ◽  
pp. 119
Author(s):  
Bram Van den Bergh
Keyword(s):  
Drosophila Melanogaster ◽  
In Vitro Assays ◽  
Antibiotic Tolerance ◽  
In Vivo Models ◽  
Antibiotic Development ◽  
E Coli ◽  
Germ Free ◽  
Flexible System

With an antibiotic crisis upon us, we need to boost antibiotic development and improve antibiotics’ efficacy. Crucial is knowing how to efficiently kill bacteria, especially in more complex in vivo conditions. Indeed, many bacteria harbor antibiotic-tolerant persisters, variants that survive exposure to our most potent antibiotics and catalyze resistance development. However, persistence is often only studied in vitro as we lack flexible in vivo models. Here, I explored the potential of using Drosophila melanogaster as a model for antimicrobial research, combining methods in Drosophila with microbiology techniques: assessing fly development and feeding, generating germ-free or bacteria-associated Drosophila and in situ microscopy. Adult flies tolerate antibiotics at high doses, although germ-free larvae show impaired development. Orally presented E. coli associates with Drosophila and mostly resides in the crop. E. coli shows an overall high antibiotic tolerance in vivo potentially resulting from heterogeneity in growth rates. The hipA7 high-persistence mutant displays an increased antibiotic survival while the expected low persistence of ΔrelAΔspoT and ΔrpoS mutants cannot be confirmed in vivo. In conclusion, a Drosophila model for in vivo antibiotic tolerance research shows high potential and offers a flexible system to test findings from in vitro assays in a broader, more complex condition.

scholarly journals Probiotic and Functional Properties of Limosilactobacillus reuteri INIA P572

Nutrients ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 1860
Author(s):  
Patricia Diez-Echave ◽  
Izaskun Martín-Cabrejas ◽  
José Garrido-Mesa ◽  
Susana Langa ◽  
Teresa Vezza ◽  
...  
Keyword(s):  
In Vitro Assays ◽  
Immunomodulatory Activity ◽  
Probiotic Properties ◽  
Protective Effects ◽  
Mice Model ◽  
In Vivo Models ◽  
Food Borne ◽  
Gastrointestinal Conditions

Limosilactobacillus reuteri INIA P572 is a strain able to produce the antimicrobial compound reuterin in dairy products, exhibiting a protective effect against some food-borne pathogens. In this study, we investigated some probiotic properties of this strain such as resistance to gastrointestinal passage or to colonic conditions, reuterin production in a colonic environment, and immunomodulatory activity, using different in vitro and in vivo models. The results showed a high resistance of this strain to gastrointestinal conditions, as well as capacity to grow and produce reuterin in a human colonic model. Although the in vitro assays using the RAW 264.7 macrophage cell line did not demonstrate direct immunomodulatory properties, the in vivo assays using a Dextran Sulphate Sodium (DSS)-induced colitic mice model showed clear immunomodulatory and protective effects of this strain.

Comparison of Vibrio and Firefly Luciferases as Reporter Gene Systems for Use in Bacteria and Plants

1996 ◽  
Vol 23 (1) ◽  
pp. 75 ◽  
Author(s):  
SR Mudge ◽  
WR Lewis-Henderson ◽  
RG Birch
Keyword(s):  
Gene Expression ◽  
Reporter Gene ◽  
Ccd Camera ◽  
Reporter Gene Expression ◽  
In Vitro Assays ◽  
E Coli ◽  
Cell Extracts ◽  
Cooled Ccd

Luciferase genes from Vibrio harveyi (luxAB) and firefly (luc) were introduced into E. coli, Agrobacteriurn, Arabidopsis and tobacco. Transformed bacteria and plants were quantitatively assayed for luciferase activity using a range of in vitro and in vivo assay conditions. Both lux and luc proved efficient reporter genes in bacteria, although it is important to be aware that the sensitive assays may detect expression due to readthrough from distant promoters. LUX activity was undetectable by liquid nitrogen-cooled CCD camera assays on intact tissues of plants which showed strong luxAB expression by in vitro assays. The decanal substrate for the lux assay was toxic to many plant tissues, and caused chemiluminescence in untransformed Arabidopsis leaves. These are serious limitations to application of the lux system for sensitive, non-toxic assays of reporter gene expression in plants. In contrast, LUC activity was readily detectable in intact tissues of all plants with luc expression detectable by luminometer assays on cell extracts. Image intensities of luc-expressing leaves were commonly two to four orders of magnitude above controls under the CCD camera. Provided adequate penetration of the substrate luciferin is obtained, luc is suitable for applications requiring sensitive, non-toxic assays of reporter gene expression in plants.

Protective effects of Cathelicidin against glomerulonephritis through promotion of host defense

2014 ◽  
Vol 2 (3) ◽  
pp. 189-198
Author(s):  
Ajay H. Bahl ◽  
Wanda Lee
Keyword(s):  
Host Defense ◽  
Disulfide Bonds ◽  
Helical Conformation ◽  
Antimicrobial Protein ◽  
Protective Effects ◽  
In Vivo Models ◽  
E Coli ◽  
Risk Of Death

Cathelicidin-related antimicrobial peptides are a family of polypeptides found in lysosomes of macrophages and polymorphonuclear leukocytes (PMNs). Some of these peptides can assume an alpha-helical conformation, others contain one or two disulfide bonds, still others are Pro- and Arg-rich, or Trp-rich. Higher levels of human cathelicidin antimicrobial protein (hCAP18), which are up-regulated by vitamin D, appear to significantly reduce the risk of death from infection in dialysis patients. Using in vitro and in vivo models of kidney infection, we demonstrate key antimicrobial and host immunomodulatory properties of cathelicidins. To directly assess the role of endogenous cathelicidin in the development of glomerulonephritis, WT and mCRAMP KO mice were provided with 5% DSS to induce glomerulonephritis. Some mice groups were administered with E. coli DNA I.P. Our findings showed that mCRAMP KO mice develop more severe glomerulonephritis. These data demonstrate key roles for cathelicidins in host defense against glomerulonephritis and the potential to inform the development of synthetic analogues to modulate specific host-pathogen interactions as novel antimicrobial therapeutics.

scholarly journals A comparative study of GT27 Family N-acetylgalactosaminyltransferases

2021 ◽  
Author(s):  
Biprajit Sanyal
Keyword(s):  
Half Life ◽  
Homo Sapiens ◽  
Effective Means ◽  
Therapeutic Proteins ◽  
In Vitro Assays ◽  
Future Research ◽  
E Coli ◽  
Glycosylation Sites

Therapeutic proteins face short half lives in vivo. Their high costs and associated toxicity effects of increasing dosage warrant exploration of methods to increase serum half-life. These proteins can be produced with native or engineered glycosylation sites, which has been shown to be an effective means of prolonging serum half-life. Engineered E. coli represents an economical route of production. I have been able to produce, purify and test the activity of three N-acetylgalactosaminyltransferase isoform 2 in Escherichia coli and show glycosylation on peptides derived from Interleukin 29. I followed the activity of these enzymes on three candidate therapeutic proteins via lectin blotting. Data suggest the Homo sapiens orthologue of GalNAcT2 is the most efficient enzyme in the in vitro assays with all candidate therapeutic protein substrates displaying the Tn-antigen. Future research should investigate continuous assays for precise results as well as assaying native peptides as opposed to non-native ones.

scholarly journals In vitro methods for the study of microbial biofilms

2011 ◽  
Vol 1 (1) ◽  
pp. 031-040 ◽  
Keyword(s):  
Ferrite Nanoparticles ◽  
In Vitro Assays ◽  
Confocal Laser ◽  
Microbial Biofilm ◽  
In Vivo Models ◽  
Microbial Biofilms ◽  
Human Pathology ◽  
Biofilm Development

The emergence of microbial biofilm related infections (bacterial and fungal) has a significant impact for the human pathology in the entire world. The understanding of microbial infections related to the biofilm development on tissues or indwelling devices was possible by using different qualitative and quantitative in vitro assays, in continuous and discontinuous systems, as well as in vivo models. A necessary step for obtaining more standardized, reliable and comparable results among different laboratories is the simplification of the available techniques used for investigating the biofilm formation and properties, including the biofilms susceptibility to antibiotics. The aim of the present study was to exemplify a series of available methods for the investigation of in vitro microbial biofilms developed on inert substrata, as well as coated with ferrite nanoparticles, using as experimental model a Sacharomyces cerevisiae strain. Microbial biofilm architecture was directly examined by two microscopy techniques (inverted microscopy and confocal laser microscopy scanning). The in vitro study of the influence of suspended ferrite nanoparticles on planktonic cells growth, adherence and consecutive biofilm development on inert substrata was performed by using a simple microtiter method.

Fly-on-a-Chip: Microfluidics for Drosophila melanogaster Studies

2019 ◽  
Vol 11 (12) ◽  
pp. 425-443 ◽  
Author(s):  
Alireza Zabihihesari ◽  
Arthur J Hilliker ◽  
Pouya Rezai
Keyword(s):  
Drosophila Melanogaster ◽  
Developmental Stages ◽  
Model Organism ◽  
Fruit Fly ◽  
In Vitro Assays ◽  
Behavioral Studies ◽  
And Behavior ◽  
Manual Manipulation

Abstract The fruit fly or Drosophila melanogaster has been used as a promising model organism in genetics, developmental and behavioral studies as well as in the fields of neuroscience, pharmacology, and toxicology. Not only all the developmental stages of Drosophila, including embryonic, larval, and adulthood stages, have been used in experimental in vivo biology, but also the organs, tissues, and cells extracted from this model have found applications in in vitro assays. However, the manual manipulation, cellular investigation and behavioral phenotyping techniques utilized in conventional Drosophila-based in vivo and in vitro assays are mostly time-consuming, labor-intensive, and low in throughput. Moreover, stimulation of the organism with external biological, chemical, or physical signals requires precision in signal delivery, while quantification of neural and behavioral phenotypes necessitates optical and physical accessibility to Drosophila. Recently, microfluidic and lab-on-a-chip devices have emerged as powerful tools to overcome these challenges. This review paper demonstrates the role of microfluidic technology in Drosophila studies with a focus on both in vivo and in vitro investigations. The reviewed microfluidic devices are categorized based on their applications to various stages of Drosophila development. We have emphasized technologies that were utilized for tissue- and behavior-based investigations. Furthermore, the challenges and future directions in Drosophila-on-a-chip research, and its integration with other advanced technologies, will be discussed.

scholarly journals Systems Biology Analysis Reveals Eight SLC22 Transporter Subgroups, Including OATs, OCTs, and OCTNs

2020 ◽  
Vol 21 (5) ◽  
pp. 1791 ◽  
Author(s):  
Darcy C. Engelhart ◽  
Jeffry C. Granados ◽  
Da Shi ◽  
Milton H. Saier Jr. ◽  
Michael E. Baker ◽  
...  
Keyword(s):  
Cyclic Nucleotides ◽  
Signaling Molecules ◽  
In Vitro Assays ◽  
Specific Substrate ◽  
In Vivo Models ◽  
Using Data ◽  
Carnitine Derivatives ◽  
Metabolite Network

The SLC22 family of OATs, OCTs, and OCTNs is emerging as a central hub of endogenous physiology. Despite often being referred to as “drug” transporters, they facilitate the movement of metabolites and key signaling molecules. An in-depth reanalysis supports a reassignment of these proteins into eight functional subgroups, with four new subgroups arising from the previously defined OAT subclade: OATS1 (SLC22A6, SLC22A8, and SLC22A20), OATS2 (SLC22A7), OATS3 (SLC22A11, SLC22A12, and Slc22a22), and OATS4 (SLC22A9, SLC22A10, SLC22A24, and SLC22A25). We propose merging the OCTN (SLC22A4, SLC22A5, and Slc22a21) and OCT-related (SLC22A15 and SLC22A16) subclades into the OCTN/OCTN-related subgroup. Using data from GWAS, in vivo models, and in vitro assays, we developed an SLC22 transporter-metabolite network and similar subgroup networks, which suggest how multiple SLC22 transporters with mono-, oligo-, and multi-specific substrate specificity interact to regulate metabolites. Subgroup associations include: OATS1 with signaling molecules, uremic toxins, and odorants, OATS2 with cyclic nucleotides, OATS3 with uric acid, OATS4 with conjugated sex hormones, particularly etiocholanolone glucuronide, OCT with neurotransmitters, and OCTN/OCTN-related with ergothioneine and carnitine derivatives. Our data suggest that the SLC22 family can work among itself, as well as with other ADME genes, to optimize levels of numerous metabolites and signaling molecules, involved in organ crosstalk and inter-organismal communication, as proposed by the remote sensing and signaling theory.

scholarly journals Virulence of the emerging pathogen, Burkholderia pseudomallei, depends upon the O-linked oligosaccharyltransferase, PglL

2020 ◽  
Vol 15 (4) ◽  
pp. 241-257
Author(s):  
Samuel J Willcocks ◽  
Carmen Denman ◽  
Felipe Cia ◽  
Elizabeth McCarthy ◽  
Jon Cuccui ◽  
...  
Keyword(s):  
Burkholderia Pseudomallei ◽  
Galleria Mellonella ◽  
In Vitro Assays ◽  
In Vivo Models ◽  
Bacterial Fitness ◽  
Isogenic Mutants ◽  
Type Strains ◽  
Broad Role

Aim: We sought to characterize the contribution of the O-OTase, PglL, to virulence in two Burkholderia spp. by comparing isogenic mutants in Burkholderia pseudomallei with the related species, Burkholderia thailandensis. Materials & methods: We utilized an array of in vitro assays in addition to Galleria mellonella and murine in vivo models to assess virulence of the mutant and wild-type strains in each Burkholderia species. Results: We found that pglL contributes to biofilm and twitching motility in both species. PglL uniquely affected morphology; cell invasion; intracellular motility; plaque formation and intergenus competition in B. pseudomallei. This mutant was attenuated in the murine model, and extended survival in a vaccine-challenge experiment. Conclusion: Our data support a broad role for pglL in bacterial fitness and virulence, particularly in B. pseudomallei.

scholarly journals Huzhangoside A Suppresses Tumor Growth through Inhibition of Pyruvate Dehydrogenase Kinase Activity

Cancers ◽  
2019 ◽  
Vol 11 (5) ◽  
pp. 712 ◽  
Author(s):  
Choong-Hwan Kwak ◽  
Jung-Hee Lee ◽  
Eun-Yeong Kim ◽  
Chang Woo Han ◽  
Keuk-Jun Kim ◽  
...  
Keyword(s):  
Tumor Growth ◽  
Pyruvate Dehydrogenase ◽  
Malignant Tumors ◽  
Aerobic Glycolysis ◽  
Pyruvate Dehydrogenase Complex ◽  
Pyruvate Dehydrogenase Kinase ◽  
In Vitro Assays ◽  
In Vivo Models

Aerobic glycolysis is one of the important metabolic characteristics of many malignant tumors. Pyruvate dehydrogenase kinase (PDHK) plays a key role in aerobic glycolysis by phosphorylating the E1α subunit of pyruvate dehydrogenase (PDH). Hence, PDHK has been recognized as a molecular target for cancer treatment. Here, we report that huzhangoside A (Hu.A), a triterpenoid glycoside compound isolated from several plants of the Anemone genus, acts as a novel PDHK inhibitor. Hu.A was found to decrease the cell viability of human breast cancer MDA-MB-231, hepatocellular carcinoma Hep3B, colon cancer HT-29, DLD-1, and murine lewis lung carcinoma LLC cell lines. The activity of PDHK1 was decreased by Hu.A in both in vitro assays and in vivo assays in DLD-1 cells. Hu.A significantly increased the oxygen consumption and decreased the secretory lactate levels in DLD-1 cells. In addition, Hu.A interacted with the ATP-binding pocket of PDHK1 without affecting the interaction of PDHK1 and pyruvate dehydrogenase complex (PDC) subunits. Furthermore, Hu.A significantly induced mitochondrial reactive oxygen species (ROS) and depolarized the mitochondrial membrane potential in DLD-1 cells. Consistently, when Hu.A was intraperitoneally injected into LLC allograft mice, the tumor growth was significantly decreased. In conclusion, Hu.A suppressed the growth of tumors in both in vitro and in vivo models via inhibition of PDHK activity.

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