scholarly journals Characterization of aMycobacterium aviumsubsp.aviumOperon Associated with Virulence and Drug Detoxification

2014 ◽  
Vol 2014 ◽  
pp. 1-10 ◽  
Author(s):  
Mariana Noelia Viale ◽  
Kun Taek Park ◽  
Belén Imperiale ◽  
Andrea Karina Gioffre ◽  
María Alejandra Colombatti Olivieri ◽  
...  
Keyword(s):  
Mycobacterium Avium ◽  
Efflux Pump ◽  
Mice Model ◽  
Toxic Compounds ◽  
Drug Detoxification ◽  
Insight Into ◽  
Knockout Mutation

ThelprG-p55operon ofMycobacterium tuberculosisandMycobacterium bovisis involved in the transport of toxic compounds. P55 is an efflux pump that provides resistance to several drugs, while LprG is a lipoprotein that modulates the host's immune response against mycobacteria. The knockout mutation of this operon severely reduces the replication of both mycobacterial species during infection in mice and increases susceptibility to toxic compounds. In order to gain insight into the function of LprG in theMycobacterium aviumcomplex, in this study, we assayed the effect of the deletion oflprG gene in the D4ER strain ofMycobacterium aviumsubsp.avium. The replacement oflprG gene with a hygromycin cassette caused a polar effect on the expression ofp55. Also, a twofold decrease in ethidium bromide susceptibility was observed and the resistance to the antibiotics rifampicin, amikacin, linezolid, and rifabutin was impaired in the mutant strain. In addition, the mutation decreased the virulence of the bacteria in macrophagesin vitroand in a mice modelin vivo. These findings clearly indicate that functional LprG and P55 are necessary for the correct transport of toxic compounds and for the survival of MAAin vitroandin vivo.

scholarly journals Preclinical Evidence of Nanomedicine Formulation to Target Mycobacterium tuberculosis at Its Bone Marrow Niche

Pathogens ◽  
2020 ◽  
Vol 9 (5) ◽  
pp. 372 ◽  
Author(s):  
Jaishree Garhyan ◽  
Surender Mohan ◽  
Vinoth Rajendran ◽  
Rakesh Bhatnagar
Keyword(s):  
Bone Marrow ◽  
Mycobacterium Tuberculosis ◽  
In Vitro Release ◽  
Bone Marrow Niche ◽  
Mice Model ◽  
Latently Infected ◽  
Marrow Mesenchymal Stem Cells

One-third of the world’s population is estimated to be latently infected with Mycobacterium tuberculosis (Mtb). Recently, we found that dormant Mtb hides in bone marrow mesenchymal stem cells (BM-MSCs) post-chemotherapy in mice model and in clinical subjects. It is known that residual Mtb post-chemotherapy may be responsible for increased relapse rates. However, strategies for Mtb clearance post-chemotherapy are lacking. In this study, we engineered and formulated novel bone-homing PEGylated liposome nanoparticles (BTL-NPs) which actively targeted the bone microenvironment leading to Mtb clearance. Targeting of BM-resident Mtb was carried out through bone-homing liposomes tagged with alendronate (Ald). BTL characterization using TEM and DLS showed that the size of bone-homing isoniazid (INH) and rifampicin (RIF) BTLs were 100 ± 16.3 nm and 84 ± 18.4 nm, respectively, with the encapsulation efficiency of 69.5% ± 4.2% and 70.6% ± 4.7%. Further characterization of BTLs, displayed by sustained in vitro release patterns, increased in vivo tissue uptake and enhanced internalization of BTLs in RAW cells and CD271+BM-MSCs. The efficacy of isoniazid (INH)- and rifampicin (RIF)-loaded BTLs were shown using a mice model where the relapse rate of the tuberculosis was decreased significantly in targeted versus non-targeted groups. Our findings suggest that BTLs may play an important role in developing a clinical strategy for the clearance of dormant Mtb post-chemotherapy in BM cells.

scholarly journals Computational Insight into the Effect of Natural Compounds on the Destabilization of Preformed Amyloid-β(1-40) Fibrils

2018 ◽  
Author(s):  
Francesco Tavanti ◽  
Alfonso Pedone ◽  
Maria Cristina Menziani
Keyword(s):  
Therapeutic Strategy ◽  
Therapeutic Potential ◽  
Structure Stability ◽  
Insoluble Form ◽  
The Brain ◽  
Identification And Characterization ◽  
Insight Into

One of the principal hallmarks of Alzheimer’s disease (AD) is related to the aggregation of amyloid-β fibrils in an insoluble form in the brain, also known as amyloidosis. Therefore, a prominent therapeutic strategy against AD consists either in blocking the amyloid aggregation and/or destroying the already formed aggregates. Natural products have shown significant therapeutic potential as amyloid inhibitors from in vitro studies as well as in vivo animal tests. In this study, the interaction of five natural biophenols (curcumin, dopamine, (-)-Epigallocatechin-3-gallate, Quercetin, and Rosmarinic acid) with the amyloid-β(1-40) fibrils has been studied through computational simulations. The results allowed the identification and characterization of the different binding modalities of each compounds and their consequences on fibril dynamics and aggregation. It emerges that the lateral aggregation of the fibrils is strongly influenced by the intercalation of the ligands, which modulate the double-layered structure stability.

scholarly journals Characterization of IS1245 for Strain Typing of Mycobacterium avium

1998 ◽  
Vol 36 (7) ◽  
pp. 1859-1863 ◽  
Author(s):  
Martine Pestel-Caron ◽  
Robert D. Arbeit
Keyword(s):  
Mycobacterium Avium ◽  
Strain Typing ◽  
Environmental Isolates ◽  
Pfge Analysis ◽  
Insertion Element ◽  
Copy Numbers ◽  
Immunodeficiency Virus

IS1245 is an insertion element widely prevalent among isolates of Mycobacterium avium. We used PvuII Southern blots to analyze IS1245 polymorphisms among 159M. avium isolates (141 clinical isolates from 40 human immunodeficiency virus-infected patients plus 18 epidemiologically related environmental isolates) that represented 40 distinct M. avium strains, as resolved by previous studies by pulsed-field gel electrophoresis (PFGE). All 40 strains carried DNA homologous to IS1245 and thus were typeable. Twenty-five (63%) strains had ≥10 copies of the element, 6 (15%) had 4 to 9 copies, and 9 (23%) had only 1 to 3 copies. Among the last group of nine strains (each of which was distinct by PFGE analysis), IS1245typing resolved only four patterns and thus provided poor discriminatory power. To evaluate the in vivo stability of IS1245, we analyzed 32 strains for which sets of 2 to 19 epidemiologically related isolates were available. For 19 (59%) of these sets, all isolates representing the same strain had indistinguishable IS1245 patterns. Within eight (25%) sets, one or more isolates had IS1245 patterns that differed by one or two fragments from the modal pattern for the isolates of that strain. Five (16%) sets included isolates whose patterns differed by three or more fragments; on the basis of IS1245 typing those isolates would have been designated distinct strains. IS1245 was stable during in vitro passage, suggesting that the variations observed represented natural translocations of the element. IS1245 provides a useful tool for molecular strain typing of M. avium but may have limitations for analyzing strains with low copy numbers or for resolving extended epidemiologic relationships.

scholarly journals Pulmonary Surfactant Promotes Virulence Gene Expression and Biofilm Formation inKlebsiella pneumoniae

2018 ◽  
Vol 86 (7) ◽  
Author(s):  
Graham G. Willsey ◽  
Sebastian Ventrone ◽  
Kristin C. Schutz ◽  
Aaron M. Wallace ◽  
John W. Ribis ◽  
...  
Keyword(s):  
Biofilm Formation ◽  
Pulmonary Surfactant ◽  
De Novo ◽  
Efflux Pump ◽  
Initial Point ◽  
Bacterial Survival ◽  
Content Type ◽  
Insight Into

ABSTRACTThe interactions betweenKlebsiella pneumoniaeand the host environment at the site of infection are largely unknown. Pulmonary surfactant serves as an initial point of contact for inhaled bacteria entering the lung and is thought to contain molecular cues that aid colonization and pathogenesis. To gain insight into this ecological transition, we characterized the transcriptional response ofK. pneumoniaeMGH 78578 to purified pulmonary surfactant. This work revealed changes within theK. pneumoniaetranscriptome that likely contribute to host colonization, adaptation, and virulencein vivo. Notable transcripts expressed under these conditions include genes involved in capsule synthesis, lipopolysaccharide modification, antibiotic resistance, biofilm formation, and metabolism. In addition, we tested the contributions of other surfactant-induced transcripts toK. pneumoniaesurvival using engineered isogenic KPPR1 deletion strains in a murine model of acute pneumonia. In these infection studies, we identified the MdtJI polyamine efflux pump and the ProU glycine betaine ABC transporter to be significant mediators ofK. pneumoniaesurvival within the lung and confirmed previous evidence for the importance ofde novoleucine synthesis to bacterial survival during infection. Finally, we determined that pulmonary surfactant promoted type 3 fimbria-mediated biofilm formation inK. pneumoniaeand identified two surfactant constituents, phosphatidylcholine and cholesterol, that drive this response. This study provides novel insight into the interactions occurring betweenK. pneumoniaeand the host at an important infection site and demonstrates the utility of purified lung surfactant preparations for dissecting host-lung pathogen interactionsin vitro.

scholarly journals Bacille Calmette-Guérin vaccine reprograms human neonatal lipid metabolism in vitro and in vivo

2021 ◽  
Author(s):  
Joann Diray-Arce ◽  
Asimenia Angelidou ◽  
Kristoffer Jarlov Jensen ◽  
Maria Giulia Conti ◽  
Rachel S. Kelly ◽  
...  
Keyword(s):  
Metabolic Pathways ◽  
Early Life ◽  
Mycobacterial Antigen ◽  
Bacille Calmette Guerin ◽  
Tlr Agonist ◽  
Induced Changes ◽  
Insight Into

SummaryVaccines have generally been developed with limited insight into their molecular impact. While systems vaccinology, including metabolomics, enables new characterization of vaccine mechanisms of action, these tools have yet to be applied to infants at high risk of infection and receive the most vaccines. Bacille Calmette-Guérin (BCG) protects infants against disseminated tuberculosis (TB) and TB-unrelated infections via incompletely understood mechanisms. We employed mass spectrometry-based metabolomics of blood plasma to profile BCG-induced infant responses in Guinea Bissau in vivo and the U.S. in vitro. BCG selectively altered plasma lipid pathways, including lysophospholipids. BCG-induced lysophosphatidylcholines (LPCs) correlated with both TLR agonist- and purified protein derivative (PPD, mycobacterial antigen)-induced blood cytokine production in vitro, raising the possibility that LPCs contribute to BCG immunogenicity. Analysis of an independent newborn cohort from The Gambia demonstrated shared vaccine-induced metabolites such as phospholipids and sphingolipids. BCG-induced changes to the plasma lipidome and LPCs may contribute to its immunogenicity and inform the discovery and development of early life vaccines.HighlightsNeonatal BCG immunization generates distinct metabolic shifts in vivo and in vitro across multiple independent cohorts.BCG induces prominent changes in concentrations of plasma lysophospholipids (LPLs)BCG induced changes in plasma lysophosphatidylcholines (LPCs) correlate with BCG effects on TLR agonist- and mycobacterial antigen-induced cytokine responses.Characterization of vaccine-induced changes in metabolism may define predictive signatures of vaccine responses and inform early life vaccine development.Abstract FigureGraphical abstract:BCG vaccination perturbs metabolic pathways in vivo and in vitro.Vaccines have traditionally been developed empirically, with limited insight into their impact on molecular pathways. Metabolomics provides a new approach to characterizing vaccine mechanisms but has not yet been applied to human newborns, who are at the highest risk of infection and receive the most vaccines. Bacille Calmette-Guérin (BCG) prevents disseminated mycobacterial disease in children and can induce broad protection to reduce mortality due to non-TB infections. Underlying mechanisms are incompletely characterized. Employing mass spectrometry-based metabolomics, we demonstrate that early BCG administration alters the human neonatal plasma metabolome, especially lipid metabolic pathways such as lysophosphatidylcholines (LPCs), both in vivo and in vitro. Plasma LPCs correlated with both innate TLR-mediated and PPD antigen-induced cytokine responses suggesting that BCG-induced lipids might contribute to the immunogenicity of this vaccine. Vaccine-induced metabolic changes may provide fresh insights into vaccine immunogenicity and inform the discovery and development of early life vaccines.

scholarly journals Antimalarial activities of ( Z )-2-(nitroheteroarylmethylene)-3(2 H )-benzofuranone derivatives: In vitro , in vivo assessment and β-hematin formation inhibition activity

2021 ◽  
Author(s):  
Latifeh Navidpour ◽  
Kelly Chibale ◽  
Somayeh Esmaeili ◽  
Azadeh Ghiaee ◽  
Narges Hadjesfandiari ◽  
...  
Keyword(s):  
Drug Resistant ◽  
Inhibition Activity ◽  
Mice Model ◽  
Kb Cells ◽  
Cytotoxic Effects ◽  
Reference Drug ◽  
Insight Into ◽  
In Vivo Assessment

A series of ( Z )-2-(nitroheteroarylmethylene)-3(2 H )-benzofuranones possessing nitroheteroaryls groups of nitroimidazole, nitrofuran and nitrothiophene moieties was screened for antiplasmodium activity against drug-sensitive (3D7) as well as a chloroquine (CQ) and multi-drug resistant (K1) strains of P. falciparum 5-Nitroimidazole and 4-nitroimidazole analogs were highly selective and active against resistant parasites, while 5-nitrofuran and 5-nitrothiophene derivatives were more potent against the 3D7 strain than the K1 strain. Among the synthetic analogues, ( Z )-6-chloro-2-(1-methyl-5-nitroimidazol-2-ylmethylene)-3(2 H )-benzofuranone ( 5h ) exhibited the highest activity (IC 50 : 0.654 nM) against K1 strain and ( Z )-7-methoxy-2-(5-nitrothiophen-2-ylmethylene)-3(2 H )-benzofuranone ( 10g ) showed the highest activity (IC 50 : 0.28 μM) against the 3D7 strain in comparison with CQ (IC 50 s of 3.13 and 206.3 nM against 3D7 and K1 strains, respectively). The more active compounds with IC 50 s lower than 5 μg/mL (∼20 μM) were further studied for their cytotoxicity responses using KB cells. From these studies, 5-nitroimidazole, 4-nitroimidazole and 5-nitrofuran analogues were shown to be cytotoxic against KB cells, while 5-nitrothiophene analogues were shown to have the least cytotoxic effects. To gain some insight into their potential contributing mechanism of action, three derivatives 10e , 10g and 10h (from nitrothiophene subgroup possessing 6-methoxy, 7-methoxy and 6,7-dimethoxy substituents on their benzofuranone moieties, respectively) showing the least toxicity and highest selectivity indices were assessed for their β-hematin formation inhibition activity. 10g demonstrated the highest inhibition activity (IC 50 : 10.78 μM) in comparison with CQ (IC 50 : 2.63 μM) as the reference drug. Finally, these three analogues ( 10e , 10g and 10h ) were further evaluated for their in vivo activity against P. berghei /albino mice model (Peter’s test). Tested analogues were shown to be active, reducing the percentage of erythrocytes that contained parasites by 53.4, 48.8 and 32.4%, respectively.

Rapid, Refined, and Robust Method for Expression, Purification, and Characterization of Recombinant Human Amyloid-beta M1-42

2019 ◽  
Author(s):  
Priya Prakash ◽  
Travis Lantz ◽  
Krupal P. Jethava ◽  
Gaurav Chopra
Keyword(s):  
Amyloid Beta ◽  
Western Blots ◽  
Force Microscopy ◽  
E Coli ◽  
Atomic Force ◽  
Set Up ◽  
Short Time

Amyloid plaques found in the brains of Alzheimer’s disease (AD) patients primarily consists of amyloid beta 1-42 (Ab42). Commercially, Ab42 is synthetized using peptide synthesizers. We describe a robust methodology for expression of recombinant human Ab(M1-42) in Rosetta(DE3)pLysS and BL21(DE3)pLysS competent E. coli with refined and rapid analytical purification techniques. The peptide is isolated and purified from the transformed cells using an optimized set-up for reverse-phase HPLC protocol, using commonly available C18 columns, yielding high amounts of peptide (~15-20 mg per 1 L culture) in a short time. The recombinant Ab(M1-42) forms characteristic aggregates similar to synthetic Ab42 aggregates as verified by western blots and atomic force microscopy to warrant future biological use. Our rapid, refined, and robust technique to purify human Ab(M1-42) can be used to synthesize chemical probes for several downstream in vitro and in vivo assays to facilitate AD research.

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