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3.
Children (Basel) ; 7(6)2020 May 26.
Article in English | MEDLINE | ID: mdl-32466620

ABSTRACT

Food allergies are common and estimated to affect 8% of children and 11% of adults in the United States. They pose a significant burden-physical, economic and social-to those affected. There is currently no available cure for food allergies. Emerging evidence suggests that the microbiome contributes to the development and manifestations of atopic disease. According to the hygiene hypothesis, children growing up with older siblings have a lower incidence of allergic disease compared with children from smaller families, due to their early exposure to microbes in the home. Research has also demonstrated that certain environmental exposures, such as a farming environment, during early life are associated with a diverse bacterial experience and reduced risk of allergic sensitization. Dysregulation in the homeostatic interaction between the host and the microbiome or gut dysbiosis appears to precede the development of food allergy, and the timing of such dysbiosis is critical. The microbiome affects food tolerance via the secretion of microbial metabolites (e.g., short chain fatty acids) and the expression of microbial cellular components. Understanding the biology of the microbiome and how it interacts with the host to maintain gut homeostasis is helpful in developing smarter therapeutic approaches. There are ongoing trials evaluating the benefits of probiotics and prebiotics, for the prevention and treatment of atopic diseases to correct the dysbiosis. However, the routine use of probiotics as an intervention for preventing allergic disease is not currently recommended. A new approach in microbial intervention is to attempt a more general modification of the gut microbiome, such as with fecal microbiota transplantation. Developing targeted bacterial therapies for food allergy may be promising for both the treatment and prevention of food allergy. Similarly, fecal microbiota transplantation is being explored as a potentially beneficial interventional approach. Overall, targeted bacterial therapies for food allergy may be promising for both the treatment and prevention of food allergy.

4.
ACS Infect Dis ; 5(10): 1738-1753, 2019 10 11.
Article in English | MEDLINE | ID: mdl-31373203

ABSTRACT

Emerging resistance to current antimalarial medicines underscores the importance of identifying new drug targets and novel compounds. Malaria parasites are purine auxotrophic and import purines via the Plasmodium falciparum equilibrative nucleoside transporter type 1 (PfENT1). We previously showed that PfENT1 inhibitors block parasite proliferation in culture. Our goal was to identify additional, possibly more optimal chemical starting points for a drug discovery campaign. We performed a high throughput screen (HTS) of GlaxoSmithKline's 1.8 million compound library with a yeast-based assay to identify PfENT1 inhibitors. We used a parallel progression strategy for hit validation and expansion, with an emphasis on chemical properties in addition to potency. In one arm, the most active hits were tested for human cell toxicity; 201 had minimal toxicity. The second arm, hit expansion, used a scaffold-based substructure search with the HTS hits as templates to identify over 2000 compounds; 123 compounds had activity. Of these 324 compounds, 175 compounds inhibited proliferation of P. falciparum parasite strain 3D7 with IC50 values between 0.8 and ∼180 µM. One hundred forty-two compounds inhibited PfENT1 knockout (pfent1Δ) parasite growth, indicating they also hit secondary targets. Thirty-two hits inhibited growth of 3D7 but not pfent1Δ parasites. Thus, PfENT1 inhibition was sufficient to block parasite proliferation. Therefore, PfENT1 may be a viable target for antimalarial drug development. Six compounds with novel chemical scaffolds were extensively characterized in yeast-, parasite-, and human-erythrocyte-based assays. The inhibitors showed similar potencies against drug sensitive and resistant P. falciparum strains. They represent attractive starting points for development of novel antimalarial drugs.


Subject(s)
Antimalarials/pharmacology , Biological Transport/drug effects , Cell Proliferation/drug effects , Drug Discovery , Plasmodium falciparum/drug effects , Purines/metabolism , Antimalarials/chemistry , Erythrocytes/drug effects , Gene Knockout Techniques , Hep G2 Cells/drug effects , High-Throughput Screening Assays , Humans , Malaria/parasitology , Malaria, Falciparum/parasitology , Nucleobase, Nucleoside, Nucleotide, and Nucleic Acid Transport Proteins/drug effects , Nucleobase, Nucleoside, Nucleotide, and Nucleic Acid Transport Proteins/genetics , Plasmodium falciparum/genetics , Plasmodium falciparum/growth & development , Plasmodium falciparum/metabolism , Protozoan Proteins/drug effects , Protozoan Proteins/genetics , Transcriptome , Yeasts/drug effects
5.
J Pediatric Infect Dis Soc ; 8(3): 265-268, 2019 Jul 01.
Article in English | MEDLINE | ID: mdl-30085250

ABSTRACT

We report here 18 cases of pediatric group A streptococcal toxic shock syndrome, associated clinical findings, and bacterial molecular genetic characteristics discovered through whole-genome sequencing. This comparative whole-genome sequencing revealed unique gene content (speK) and polymorphisms (dpiB) in emm87 group A Streptococcus, the relative contributions of which, in combination with the host response, in the development of streptococcal toxic shock syndrome remain to be elucidated.


Subject(s)
Genes, Bacterial/genetics , Shock, Septic/microbiology , Streptococcal Infections/microbiology , Streptococcus pyogenes/genetics , Antigens, Bacterial/blood , Antigens, Bacterial/genetics , Bacterial Outer Membrane Proteins/genetics , Child , Child, Preschool , Female , Genotyping Techniques , Humans , Male , Phylogeny , Polymorphism, Single Nucleotide , Retrospective Studies , Whole Genome Sequencing
6.
Front Pediatr ; 6: 390, 2018.
Article in English | MEDLINE | ID: mdl-30713837

ABSTRACT

Fanconi anemia (FA) is an inherited bone marrow failure and cancer predisposition disorder due to mutations in DNA repair pathways proteins (FANC). The dysfunctional proteins are unable to repair DNA breaks and cause genomic instability. Mutations in many of the 19 FANC genes are well characterized biochemically and clinically. Little is known about the FANCD2 gene which acts downstream of the FA-core proteins. Here we report a 11-year-old female previously diagnosed with FA and bone marrow failure. Gene sequencing demonstrated deletion of exons 2-18 and a pathologic missense mutation (c. 2444G>A, p. Arg815Gln) in FANCD2 (Chr3). Her medical history is significant for an episode of pneumococcal sepsis despite adequate vaccination. Repeated blood samples and immunophenotyping demonstrated severe lymphopenia. There were markedly low CD4+ T-cell counts with a low CD4:CD8 ratio. Changes in the composition of the B-cell population included significantly diminished absolute total B-cells, and decreased mature cells. There was no immunogenic response to vaccination against S. pneumoniae. The NK-cell count was unaffected and demonstrated normal spontaneous and stimulated cytotoxic response. Bone marrow analysis demonstrated hypocellularity without dysplasia. The clinical and laboratory features are suggestive of combined immune deficiency. FANCD2 may be involved in the transition of immature B and T cells to mature cells, a process that requires substantial DNA recombination not observed in NK cells. Additional genetic and biochemical evaluation is needed to further characterize the novel genetic and clinical findings.

7.
Mol Pharmacol ; 89(6): 678-85, 2016 06.
Article in English | MEDLINE | ID: mdl-27048953

ABSTRACT

Malaria is a critical public health issue in the tropical world, causing extensive morbidity and mortality. Infection by unicellular, obligate intracellular Plasmodium parasites causes malaria. The emergence of resistance to current antimalarial drugs necessitates the development of novel therapeutics. A potential novel drug target is the purine import transporter. Because Plasmodium parasites are purine auxotrophic, they must import purines from their host to fulfill metabolic requirements. They import purines via equilibrative nucleoside transporter 1 (ENT1) homologs. Recently, we used a yeast-based high-throughput screen to identify inhibitors of the P. falciparum ENT1 (PfENT1) that kill P. falciparum parasites in culture. P. berghei infection of mice is an animal model for human malaria. Because P. berghei ENT1 (PbENT1) shares only 60% amino acid sequence identity with PfENT1, we sought to characterize PbENT1 and its sensitivity to our PfENT1 inhibitors. We expressed PbENT1 in purine auxotrophic yeast and used radiolabeled substrate uptake to characterize its function. We showed that PbENT1 transports both purines and pyrimidines. It preferred nucleosides compared with nucleobases. Inosine (IC50 = 3.7 µM) and guanosine (IC50 = 21.3 µM) had the highest affinities. Our recently discovered PfENT1 inhibitors were equally effective against both PbENT1- and PfENT1-mediated purine uptake. The PfENT1 inhibitors are at least 10-fold more potent against PfENT1 than human hENT1. They kill P. berghei parasites in 24-hour ex vivo culture. Thus, the P. berghei murine malaria model may be useful to evaluate the efficacy of PfENT1 inhibitors in vivo and their therapeutic potential for treatment of malaria.


Subject(s)
Antimalarials/pharmacology , Equilibrative Nucleoside Transporter 1/metabolism , Plasmodium berghei/metabolism , Adenosine/metabolism , Animals , Female , Humans , Inhibitory Concentration 50 , Mice , Saccharomyces cerevisiae/metabolism , Substrate Specificity/drug effects , Time Factors , Tritium/metabolism , Uridine/metabolism
8.
Int J Parasitol Drugs Drug Resist ; 6(1): 1-11, 2016 Apr.
Article in English | MEDLINE | ID: mdl-26862473

ABSTRACT

Infection with Plasmodium falciparum and vivax cause most cases of malaria. Emerging resistance to current antimalarial medications makes new drug development imperative. Ideally a new antimalarial drug should treat both falciparum and vivax malaria. Because malaria parasites are purine auxotrophic, they rely on purines imported from the host erythrocyte via Equilibrative Nucleoside Transporters (ENTs). Thus, the purine import transporters represent a potential target for antimalarial drug development. For falciparum parasites the primary purine transporter is the P. falciparum Equilibrative Nucleoside Transporter Type 1 (PfENT1). Recently we identified potent PfENT1 inhibitors with nanomolar IC50 values using a robust, yeast-based high throughput screening assay. In the current work we characterized the Plasmodium vivax ENT1 (PvENT1) homologue and its sensitivity to the PfENT1 inhibitors. We expressed a yeast codon-optimized PvENT1 gene in Saccharomyces cerevisiae. PvENT1-expressing yeast imported both purines ([(3)H]adenosine) and pyrimidines ([(3)H]uridine), whereas wild type (fui1Δ) yeast did not. Based on radiolabel substrate uptake inhibition experiments, inosine had the lowest IC50 (3.8 µM), compared to guanosine (14.9 µM) and adenosine (142 µM). For pyrimidines, thymidine had an IC50 of 183 µM (vs. cytidine and uridine; mM range). IC50 values were higher for nucleobases compared to the corresponding nucleosides; hypoxanthine had a 25-fold higher IC50 than inosine. The archetypal human ENT1 inhibitor 4-nitrobenzylthioinosine (NBMPR) had no effect on PvENT1, whereas dipyridamole inhibited PvENT1, albeit with a 40 µM IC50, a 1000-fold less sensitive than human ENT1 (hENT1). The PfENT1 inhibitors blocked transport activity of PvENT1 and the five known naturally occurring non-synonymous single nucleotide polymorphisms (SNPs) with similar IC50 values. Thus, the PfENT1 inhibitors also target PvENT1. This implies that development of novel antimalarial drugs that target both falciparum and vivax ENT1 may be feasible.


Subject(s)
Antimalarials/pharmacology , Drug Discovery , Equilibrative Nucleoside Transporter 1/antagonists & inhibitors , Nucleobase, Nucleoside, Nucleotide, and Nucleic Acid Transport Proteins/antagonists & inhibitors , Nucleobase, Nucleoside, Nucleotide, and Nucleic Acid Transport Proteins/metabolism , Plasmodium falciparum/drug effects , Plasmodium vivax/drug effects , Protozoan Proteins/antagonists & inhibitors , Protozoan Proteins/metabolism , Adenosine/pharmacology , Dipyridamole/pharmacology , Equilibrative Nucleoside Transporter 1/genetics , Guanosine/pharmacology , Humans , Inhibitory Concentration 50 , Inosine/pharmacology , Malaria, Falciparum/drug therapy , Malaria, Falciparum/parasitology , Malaria, Vivax/drug therapy , Malaria, Vivax/parasitology , Malaria, Vivax/prevention & control , Nucleobase, Nucleoside, Nucleotide, and Nucleic Acid Transport Proteins/genetics , Plasmodium falciparum/metabolism , Plasmodium vivax/genetics , Polymorphism, Single Nucleotide , Protozoan Proteins/genetics , Purines/metabolism , Purines/pharmacology , Pyrimidines/metabolism , Saccharomyces cerevisiae/genetics , Uridine/pharmacology
9.
ACS Chem Biol ; 10(3): 775-83, 2015 Mar 20.
Article in English | MEDLINE | ID: mdl-25602169

ABSTRACT

Equilibrative transporters are potential drug targets; however, most functional assays involve radioactive substrate uptake that is unsuitable for high-throughput screens (HTS). We developed a robust yeast-based growth assay that is potentially applicable to many equilibrative transporters. As proof of principle, we applied our approach to Equilibrative Nucleoside Transporter 1 of the malarial parasite Plasmodium falciparum (PfENT1). PfENT1 inhibitors might serve as novel antimalarial drugs since PfENT1-mediated purine import is essential for parasite proliferation. To identify PfENT1 inhibitors, we screened 64 560 compounds and identified 171 by their ability to rescue the growth of PfENT1-expressing fui1Δ yeast in the presence of a cytotoxic PfENT1 substrate, 5-fluorouridine (5-FUrd). In secondary assays, nine of the highest activity compounds inhibited PfENT1-dependent growth of a purine auxotrophic yeast strain with adenosine as the sole purine source (IC50 0.2-2 µM). These nine compounds completely blocked [(3)H]adenosine uptake into PfENT1-expressing yeast and erythrocyte-free trophozoite-stage parasites (IC50 5-50 nM), and inhibited chloroquine-sensitive and -resistant parasite proliferation (IC50 5-50 µM). Wild-type (WT) parasite IC50 values were up to 4-fold lower compared to PfENT1-knockout (pfent1Δ) parasites. pfent1Δ parasite killing showed a delayed-death phenotype not observed with WT. We infer that, in parasites, the compounds inhibit both PfENT1 and a secondary target with similar efficacy. The secondary target identity is unknown, but its existence may reduce the likelihood of parasites developing resistance to PfENT1 inhibitors. Our data support the hypothesis that blocking purine transport through PfENT1 may be a novel and compelling approach for antimalarial drug development.


Subject(s)
Antimalarials/pharmacology , High-Throughput Screening Assays , Nucleobase, Nucleoside, Nucleotide, and Nucleic Acid Transport Proteins/antagonists & inhibitors , Plasmodium falciparum/drug effects , Protozoan Proteins/antagonists & inhibitors , Small Molecule Libraries/pharmacology , Trophozoites/drug effects , Adenosine/metabolism , Antimalarials/chemistry , Axenic Culture , Biological Transport/drug effects , Gene Deletion , Gene Expression , Genetic Complementation Test , Nucleobase, Nucleoside, Nucleotide, and Nucleic Acid Transport Proteins/genetics , Nucleobase, Nucleoside, Nucleotide, and Nucleic Acid Transport Proteins/metabolism , Nucleoside Transport Proteins/genetics , Nucleoside Transport Proteins/metabolism , Plasmodium falciparum/growth & development , Plasmodium falciparum/metabolism , Protozoan Proteins/genetics , Protozoan Proteins/metabolism , Saccharomyces cerevisiae/drug effects , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Small Molecule Libraries/chemistry , Structure-Activity Relationship , Trophozoites/growth & development , Trophozoites/metabolism , Uridine/analogs & derivatives , Uridine/pharmacology
10.
Ann N Y Acad Sci ; 1342: 19-28, 2015 Apr.
Article in English | MEDLINE | ID: mdl-25424653

ABSTRACT

Infection with Plasmodium species parasites causes malaria. Plasmodium parasites are purine auxotrophs. In all life cycle stages, they require purines for RNA and DNA synthesis and other cellular metabolic processes. Purines are imported from the host erythrocyte by equilibrative nucleoside transporters (ENTs). They are processed via purine salvage pathway enzymes to form the required purine nucleotides. The Plasmodium falciparum genome encodes four putative ENTs (PfENT1-4). Genetic, biochemical, and physiologic evidence suggest that PfENT1 is the primary purine transporter supplying the purine salvage pathway. Protein mass spectrometry shows that PfENT1 is expressed in all parasite stages. PfENT1 knockout parasites are not viable in culture at purine concentrations found in human blood (<10 µM). Thus, PfENT1 is a potential target for novel antimalarial drugs, but no PfENT1 inhibitors have been identified to test the hypothesis. Identifying inhibitors of PfENT1 is an essential step to validate PfENT1 as a potential antimalarial drug target.


Subject(s)
Antimalarials/metabolism , Drug Delivery Systems/trends , Drug Discovery/trends , Nucleobase, Nucleoside, Nucleotide, and Nucleic Acid Transport Proteins/metabolism , Plasmodium falciparum/metabolism , Protozoan Proteins/metabolism , Purines/metabolism , Animals , Antimalarials/administration & dosage , Humans , Malaria/drug therapy , Malaria/metabolism , Nucleobase, Nucleoside, Nucleotide, and Nucleic Acid Transport Proteins/antagonists & inhibitors , Parasites/drug effects , Parasites/metabolism , Plasmodium falciparum/drug effects , Protozoan Proteins/antagonists & inhibitors
11.
PLoS One ; 4(3): e4722, 2009.
Article in English | MEDLINE | ID: mdl-19266099

ABSTRACT

Microvesicles are plasma membrane-derived vesicles released into the extracellular environment by a variety of cell types. Originally characterized from platelets, microvesicles are a normal constituent of human plasma, where they play an important role in maintaining hematostasis. Microvesicles have been shown to transfer proteins and RNA from cell to cell and they are also believed to play a role in intercellular communication. We characterized the RNA and protein content of embryonic stem cell microvesicles and show that they can be engineered to carry exogenously expressed mRNA and protein such as green fluorescent protein (GFP). We demonstrate that these engineered microvesicles dock and fuse with other embryonic stem cells, transferring their GFP. Additionally, we show that embryonic stem cells microvesicles contain abundant microRNA and that they can transfer a subset of microRNAs to mouse embryonic fibroblasts in vitro. Since microRNAs are short (21-24 nt), naturally occurring RNAs that regulate protein translation, our findings open up the intriguing possibility that stem cells can alter the expression of genes in neighboring cells by transferring microRNAs contained in microvesicles. Embryonic stem cell microvesicles may be useful therapeutic tools for transferring mRNA, microRNAs, protein, and siRNA to cells and may be important mediators of signaling within stem cell niches.


Subject(s)
Cell-Derived Microparticles/genetics , Embryonic Stem Cells/ultrastructure , MicroRNAs/metabolism , Animals , Biological Transport/genetics , Cell Communication/genetics , Cell-Derived Microparticles/metabolism , Embryonic Stem Cells/metabolism , Green Fluorescent Proteins , Mice , MicroRNAs/analysis
12.
Mol Microbiol ; 58(4): 1074-86, 2005 Nov.
Article in English | MEDLINE | ID: mdl-16262791

ABSTRACT

Rheb GTPase is a key player in the control of growth, cell cycle and nutrient uptake that is conserved from yeast to humans. To further our understanding of the Rheb pathway, we sought to identify hyperactivating mutations in the Schizosaccharomyces pombe Rheb, Rhb1. Hyperactive forms of Rhb1 were found to result from single amino acid changes at valine-17, serine-21, lysine-120 or asparagine-153. Expression of these mutants confers resistance to canavanine and thialysine, phenotypes which are similar to phenotypes exhibited by cells lacking the Tsc1/Tsc2 complex that negatively regulates Rhb1. The thialysine-resistant phenotype of the hyperactive Rhb1 mutants is suppressed by a second mutation in the effector domain. Purified mutant proteins exhibit dramatically decreased binding of GDP, while their GTP binding is not drastically affected. In addition, some of the mutant proteins show significantly decreased GTPase activities. Thus the hyperactivating mutations are expected to result in an increase in the GTP-bound/GDP-bound ratio of Rhb1. By using the hyperactive mutant, Rhb1(K120R), we have been able to demonstrate that Rhb1 interacts with Tor2, one of the two S. pombe TOR (Target of Rapamycin) proteins. These fission yeast results provide the first evidence for a GTP-dependent association of Rheb with Tor.


Subject(s)
Amino Acid Substitution , GTP Phosphohydrolases/genetics , GTP Phosphohydrolases/metabolism , Schizosaccharomyces pombe Proteins/genetics , Schizosaccharomyces pombe Proteins/metabolism , Schizosaccharomyces/enzymology , Amino Acid Sequence , Antifungal Agents/pharmacology , Canavanine/pharmacology , Cell Cycle Proteins/metabolism , Cysteine/analogs & derivatives , Cysteine/pharmacology , Drug Resistance, Fungal , GTP Phosphohydrolases/analysis , GTP Phosphohydrolases/chemistry , GTP Phosphohydrolases/isolation & purification , Guanosine Diphosphate/metabolism , Guanosine Triphosphate/metabolism , Molecular Sequence Data , Mutation , Phosphatidylinositol 3-Kinases/metabolism , Protein Binding , Protein Synthesis Inhibitors/pharmacology , Schizosaccharomyces pombe Proteins/chemistry , Schizosaccharomyces pombe Proteins/isolation & purification , Sequence Homology, Amino Acid
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