forward-genetics-screens-using-macrophages-to-identify-toxoplasma

Forward Genetics Screens Using Macrophages to Identify <em>Toxoplasma gondii </em>Genes Important for Resistance to IFN-&#947;-Dependent Cell Autonomous Immunity

Toxoplasma gondii, the causative agent of toxoplasmosis, is an obligate intracellular protozoan pathogen. The parasite invades and replicates within ...

P2Y(2) and P2Y(4) receptors are G protein-coupled receptors, activated by UTP and dinucleoside tetraphosphates, which are difficult to distinguish pharmacologically for lack of potent and selective ligands. We structurally varied phosphate and uracil moieties in analogues of pyrimidine nucleoside 5'-triphosphates and 5'-tetraphosphate esters. P2Y(4) receptor potency in phospholipase C stimulation in transfected 1321N1 human astrocytoma cells was enhanced in N(4)-alkyloxycytidine derivatives. OH groups on a terminal δ-glucose phosphoester of uridine 5'-tetraphosphate were inverted or substituted with H or F to probe H-bonding effects. N(4)-(Phenylpropoxy)-CTP 16 (MRS4062), Up(4)-[1]3'-deoxy-3'-fluoroglucose 34 (MRS2927), and N(4)-(phenylethoxy)-CTP 15 exhibit ≥10-fold selectivity for human P2Y(4) over P2Y(2) and P2Y(6) receptors (EC(50) values 23, 62, and 73 nM, respectively). δ-3-Chlorophenyl phosphoester 21 of Up(4) activated P2Y(2) but not P2Y(4) receptor. Selected nucleotides tested for chemical and enzymatic stability were much more stable than UTP. Agonist docking at CXCR4-based P2Y(2) and P2Y(4) receptor models indicated greater steric tolerance of N(4)-phenylpropoxy group at P2Y(4). Thus, distal structural changes modulate potency, selectivity, and stability of extended uridine tetraphosphate derivatives, and we report the first P2Y(4) receptor-selective agonists.

Pyrimidine nucleotides with 4-alkyloxyimino and terminal tetraphosphate δ-ester modifications as selective agonists of the P2Y(4) receptor.

We report the development of the multiplexed nanoflare, a nanoparticle agent that is capable of simultaneously detecting two distinct mRNA targets inside a living cell. These probes are spherical nucleic acid (SNA) gold nanoparticle (Au NP) conjugates consisting of densely packed and highly oriented oligonucleotide sequences, many of which are hybridized to a reporter with a distinct fluorophore label and each complementary to its corresponding mRNA target. When multiplexed nanoflares are exposed to their targets, they provide a sequence specific signal in both extra- and intracellular environments. Importantly, one of the targets can be used as an internal control, improving detection by accounting for cell-to-cell variations in nanoparticle uptake and background. Compared to single-component nanoflares, these structures allow one to determine more precisely relative mRNA levels in individual cells, improving cell sorting and quantification.

Multiplexed nanoflares: mRNA detection in live cells.

A series of flexible carbocyclic pyrimidine nucleosides has been designed and synthesized. In contrast to previously reported "fleximers" from our laboratory, these analogues have the connectivity of the heterocyclic base system "reversed", where the pyrimidine ring is attached to the sugar moiety, rather than the five membered imidazole ring. As was previously seen with the ribose fleximers, their inherent flexibility should allow them to adjust to enzyme binding site mutations, as well as increase the affinity for atypical enzymes. Preliminary biological screening has revealed surprising inhibition of adenosine deaminase, despite their lack of resemblance to adenosine.

"Reverse" carbocyclic fleximers: synthesis of a new class of adenosine deaminase inhibitors.

To discuss the techniques and mechanisms of retinal oximetry with a focus on utilization of retinal oximetry in the assessment of retinal oxygen saturation in glaucoma.

Retinal oxygen saturation and metabolism: how does it pertain to glaucoma? An update on the application of retinal oximetry in glaucoma.

Development of isoform-selective histone deacetylase (HDAC) inhibitors is important in elucidating the function of individual HDAC enzymes and their potential as therapeutic agents. Among the eleven zinc-dependent HDACs in humans, HDAC6 is structurally and functionally unique. Here, we show that a hydroxamic acid-based small-molecule N-hydroxy-4-(2-[(2-hydroxyethyl)(phenyl)amino]-2-oxoethyl)benzamide (HPOB) selectively inhibits HDAC6 catalytic activity in vivo and in vitro. HPOB causes growth inhibition of normal and transformed cells but does not induce cell death. HPOB enhances the effectiveness of DNA-damaging anticancer drugs in transformed cells but not normal cells. HPOB does not block the ubiquitin-binding activity of HDAC6. The HDAC6-selective inhibitor HPOB has therapeutic potential in combination therapy to enhance the potency of anticancer drugs.

Development of a histone deacetylase 6 inhibitor and its biological effects.

The purpose of this review is to discuss the evolution of nanotechnology and its potential diagnostic and therapeutic applications in the field of ophthalmology, particularly as it pertains to glaucoma. We reviewed literature using MEDLINE and PubMed databases with the following search terms: glaucoma, nanotechnology, nanomedicine, nanoparticles, ophthalmology and liposomes. We also reviewed pertinent references from articles found in this search. A brief history of nanotechnology and nanomedicine will be covered, followed by a discussion of the advantages and concerns of using this technology in the field of glaucoma. We will look at various studies concerning the development of nanomedicine, its potential applications in ocular drug delivery, diagnostic and imaging modalities and, surgical techniques. In particular, the challenges of assuring safety and efficacy of nanomedicine will be examined. We conclude that nanotechnology offers a novel approach to expanding diagnostic, imaging and surgical modalities in glaucoma and may contribute to the knowledge of disease pathogenesis at a molecular level. However, more research is needed to better elucidate the mechanism of cellular entry, the potential for nanoparticle cytotoxicity and the assurance of clinical efficacy.

Nanotechnology and glaucoma: a review of the potential implications of glaucoma nanomedicine.

Comparison of intraocular pressure in glaucoma subjects treated with Xalatan versus generic latanoprost.

Glaucoma is the leading cause of blindness in individuals of African descent (AD). While open-angle glaucoma (OAG) disproportionately affects individuals of AD compared with persons of European descent (ED), the physiological mechanisms behind this disparity are largely unknown. The more rapid progression and greater severity of the disease in persons of AD further raise the concern for identifying these underlying differences in disease pathophysiology between AD and ED glaucoma patients. Ocular structural differences between AD and ED patients, including larger optic disc area, cup:disc ratio and thinner corneas, have been found. AD individuals are also disproportionately affected by systemic vascular diseases, including hypertension, cardiovascular disease, stroke and diabetes mellitus. Abnormal ocular blood flow has been implicated as a risk factor for glaucoma, and pilot research is beginning to identify localized ocular vascular differences between AD and ED OAG patients. Given the known systemic vascular deficits and the relationship between glaucoma and ocular blood flow, exploring these concepts in terms of glaucoma risk factors may have a significant impact in elucidating the mechanisms behind the disease disparity in the AD population.

Vascular considerations in glaucoma patients of African and European descent.

Open angle glaucoma (OAG) is a severe ocular disease characterized by progressive and irreversible vision loss. While elevated intraocular pressure (IOP) is a well-established risk factor for OAG, the progression of OAG in many cases, despite IOP treatment, suggests that other risk factors must play significant roles in the development of the disease. For example, various structural properties of the eye, ocular blood flow properties, and systemic conditions have been identified as risk factors for OAG. Ethnicity has also been indicated as a relevant factor that affects the incidence and prevalence of OAG; in fact, OAG is the leading cause of blindness among people of African descent. Numerous clinical studies have been designed to examine the possible correlation and causation between OAG and these factors; however, these studies are met with the challenge of isolating the individual role of multiple interconnected factors. Over the last decade, various mathematical modeling approaches have been implemented in combination with clinical studies in order to provide a mechanical and hemodynamical description of the eye in relation to the entire human body and to assess the contribution of single risk factors to the development of OAG. This review provides a summary of the clinical evidence of ocular structural differences, ocular vascular differences and systemic vascular differences among people of African and European descent, describes the mathematical approaches that have been proposed to study ocular mechanics and hemodynamics while discussing how they could be used to investigate the relevance to OAG of racial disparities, and outlines possible new directions of research.

Mathematical modeling approaches in the study of glaucoma disparities among people of African and European descents.

The in vitro evaluation of thieno[3,2-d]pyrimidines identified halogenated compounds 1 and 2 with antiproliferative activity against three different cancer cell lines. A structure activity relationship study indicated the necessity of the chlorine at the C4-position for biological activity. The two most active compounds 1 and 2 were found to induce apoptosis in the leukemia L1210 cell line. Additionally, the compounds were screened against a variety of other microbial targets and as a result, selective activity against several fungi was also observed. The synthesis and preliminary biological results are reported herein.

Antiproliferative activities of halogenated thieno[3,2-d]pyrimidines.

We have recently shown that upregulation of glutamate transporter 1 (GLT1) in the brain is associated in part with reduction in ethanol intake in alcohol-preferring (P) male rats. In this study, we investigated the effects of a synthetic compound, (R)-(-)-5-methyl-1-nicotinoyl-2-pyrazoline (MS-153), known to activate GLT1 on ethanol consumption as well as GLT1 expression and certain signaling pathways in P rats. P rats were given 24-h concurrent access to 15 and 30% ethanol, water and food for 5 weeks. On week 6, P rats received MS-153 at a dose of 50 mg/kg (i.p.) or a vehicle (i.p.) for 5 consecutive days. We also tested the effect of MS-153 on daily sucrose (10%) intake. Our studies revealed a significant decrease in ethanol intake at the dose of 50 mg/kg MS-153 from Day 1 through 14. In addition, MS-153 at dose of 50 mg/kg did not induce any significant effect on sucrose intake. Importantly, we found that MS-153 upregulated the GLT1 level in the nucleus accumbens (NAc) but not in the prefrontal cortex (PFC). In accordance, we found upregulation of nuclear NFkB-65 level in NAc in MS-153-treated group, however, IkBα was downregulated in MS-153-treated group in NAc. We did not find any changes in NFkB-65 and IkBα levels in PFC. Interestingly, we revealed that p-Akt was downregulated in ethanol vehicle treated groups in the NAc; this downregulation was reversed by MS-153 treatment. We did not observe any significant differences in glutamate aspartate transporter (GLAST) expression among all groups. These findings reveal MS-153 as a GLT1 modulator that may have potential as a therapeutic drug for the treatment of alcohol dependence.

Effects of MS-153 on chronic ethanol consumption and GLT1 modulation of glutamate levels in male alcohol-preferring rats.

Evidence suggests that vascular abnormalities play a role in the pathogenesis of open-angle glaucoma (OAG) in some patients. This study aims to assess changes in retrobulbar and retinal blood flow over time in patients with glaucoma and examine their relationship to glaucomatous progression, as determined by retinal and optic nerve structure.

The role of retrobulbar and retinal circulation on optic nerve head and retinal nerve fibre layer structure in patients with open-angle glaucoma over an 18-month period.

Open-angle glaucoma is a multifactorial optic neuropathy characterized by progressive loss of retinal ganglion cells and their axons. It is an irreversible disease with no established cure. The only currently approved treatment is aimed at lowering intraocular pressure, the most significant risk factor known to date. However, it is now clear that there are other risk factors involved in glaucoma's pathophysiology. To achieve future improvements in glaucoma management, new approaches to therapies and novel targets must be developed. Such therapies may include new tissue targets for lowering intraocular pressure, molecules influencing ocular hemodynamics, and treatments providing neuroprotection of retinal ganglion cells. Furthermore, novel drug delivery systems are in development that may improve patient compliance, increase bioavailability, and decrease adverse side effects.

Article	Year
Pyrimidine nucleotides with 4-alkyloxyimino and terminal tetraphosphate δ-ester modifications as selective agonists of the P2Y(4) receptor. Journal of medicinal chemistry\| PubMed ID: 21528910	2011
Multiplexed nanoflares: mRNA detection in live cells. Analytical chemistry\| PubMed ID: 22288418	2012
"Reverse" carbocyclic fleximers: synthesis of a new class of adenosine deaminase inhibitors. Nucleosides, nucleotides & nucleic acids\| PubMed ID: 23473101	2013
Retinal oxygen saturation and metabolism: how does it pertain to glaucoma? An update on the application of retinal oximetry in glaucoma. European journal of ophthalmology\| PubMed ID: 23640511	2013
Development of a histone deacetylase 6 inhibitor and its biological effects. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 24023063	2013
Nanotechnology and glaucoma: a review of the potential implications of glaucoma nanomedicine. The British journal of ophthalmology\| PubMed ID: 24246373	2014
Comparison of intraocular pressure in glaucoma subjects treated with Xalatan versus generic latanoprost. Acta ophthalmologica\| PubMed ID: 24373426	2014
Vascular considerations in glaucoma patients of African and European descent. Acta ophthalmologica\| PubMed ID: 24460758	2014
Mathematical modeling approaches in the study of glaucoma disparities among people of African and European descents. Journal of coupled systems and multiscale dynamics\| PubMed ID: 24501718	2013
Antiproliferative activities of halogenated thieno[3,2-d]pyrimidines. Bioorganic & medicinal chemistry\| PubMed ID: 24631358	2014
Effects of MS-153 on chronic ethanol consumption and GLT1 modulation of glutamate levels in male alcohol-preferring rats. Frontiers in behavioral neuroscience\| PubMed ID: 25400560	2014
The role of retrobulbar and retinal circulation on optic nerve head and retinal nerve fibre layer structure in patients with open-angle glaucoma over an 18-month period. The British journal of ophthalmology\| PubMed ID: 25467967	2014
Novel therapies for open-angle glaucoma. F1000prime reports\| PubMed ID: 25580256	2014

Nathaniel Kim

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