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1.
Int J Mol Sci ; 22(5)2021 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-33673453

RESUMO

Advances in understanding disease pathogenesis correlates to modifications in gene expression within different tissues and organ systems. In depth knowledge about the dysregulation of gene expression profiles is fundamental to fully uncover mechanisms in disease development and changes in host homeostasis. The body of knowledge surrounding mammalian regulatory elements, specifically regulators of chromatin structure, transcriptional and translational activation, has considerably surged within the past decade. A set of key regulators whose function still needs to be fully elucidated are small non-coding RNAs (sncRNAs). Due to their broad range of unfolding functions in the regulation of gene expression during transcription and translation, sncRNAs are becoming vital to many cellular processes. Within the past decade, a novel class of sncRNAs called PIWI-interacting RNAs (piRNAs) have been implicated in various diseases, and understanding their complete function is of vital importance. Historically, piRNAs have been shown to be indispensable in germline integrity and stem cell development. Accumulating research evidence continue to reveal the many arms of piRNA function. Although piRNA function and biogenesis has been extensively studied in Drosophila, it is thought that they play similar roles in vertebrate species, including humans. Compounding evidence suggests that piRNAs encompass a wider functional range than small interfering RNAs (siRNAs) and microRNAs (miRNAs), which have been studied more in terms of cellular homeostasis and disease. This review aims to summarize contemporary knowledge regarding biogenesis, and homeostatic function of piRNAs and their emerging roles in the development of pathologies related to cardiomyopathies, cancer, and infectious diseases.


Assuntos
RNA Interferente Pequeno/metabolismo , Animais , Doenças Cardiovasculares/genética , Doenças Cardiovasculares/metabolismo , Doenças Transmissíveis/genética , Doenças Transmissíveis/metabolismo , Regulação da Expressão Gênica , Humanos , Neoplasias/genética , Neoplasias/metabolismo , RNA Interferente Pequeno/fisiologia
2.
Int J Mol Sci ; 21(24)2020 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-33322418

RESUMO

Trypanosoma cruzi dysregulates the gene expression profile of primary human cardiomyocytes (PHCM) during the early phase of infection through a mechanism which remains to be elucidated. The role that small non-coding RNAs (sncRNA) including PIWI-interacting RNA (piRNA) play in regulating gene expression during the early phase of infection is unknown. To understand how T. cruzi dysregulate gene expression in the heart, we challenged PHCM with T. cruzi trypomastigotes and analyzed sncRNA, especially piRNA, by RNA-sequencing. The parasite induced significant differential expression of host piRNAs, which can target and regulate the genes which are important during the early infection phase. An average of 21,595,866 (88.40%) of clean reads mapped to the human reference genome. The parasite induced 217 unique piRNAs that were significantly differentially expressed (q ≥ 0.8). Of these differentially expressed piRNAs, 6 were known and 211 were novel piRNAs. In silico analysis showed that some of the dysregulated known and novel piRNAs could target and potentially regulate the expression of genes including NFATC2, FOS and TGF-ß1, reported to play important roles during T. cruzi infection. Further evaluation of the specific functions of the piRNAs in the regulation of gene expression during the early phase of infection will enhance our understanding of the molecular mechanism of T. cruzi pathogenesis. Our novel findings constitute the first report that T. cruzi can induce differential expression of piRNAs in PHCM, advancing our knowledge about the involvement of piRNAs in an infectious disease model, which can be exploited for biomarker and therapeutic development.


Assuntos
RNA Interferente Pequeno/metabolismo , Trypanosoma cruzi/metabolismo , Animais , Doença de Chagas/metabolismo , Humanos , Miócitos Cardíacos/metabolismo
3.
Int J Mol Sci ; 21(14)2020 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-32664627

RESUMO

The protozoan parasite Trypanosoma cruzi is the causative agent of Chagas disease. This neglected tropical disease causes severe morbidity and mortality in endemic regions. About 30% of T. cruzi infected individuals will present with cardiac complications. Invasive trypomastigotes released from infected cells can be carried in the vascular endothelial system to infect neighboring and distant cells. During the process of cellular infection, the parasite induces host cells, to increase the levels of host thrombospondin-1 (TSP-1), to facilitate the process of infection. TSP-1 plays important roles in the functioning of vascular cells, including vascular endothelial cells with important implications in cardiovascular health. Many signal transduction pathways, including the yes-associated protein 1 (YAP)/transcriptional coactivator, with PDZ-binding motif (TAZ) signaling, which are upstream of TSP-1, have been linked to the pathophysiology of heart damage. The molecular mechanisms by which T. cruzi signals, and eventually infects, heart endothelial cells remain unknown. To evaluate the importance of TSP-1 expression in heart endothelial cells during the process of T. cruzi infection, we exposed heart endothelial cells prepared from Wild Type and TSP-1 Knockout mouse to invasive T. cruzi trypomastigotes at multiple time points, and evaluated changes in the hippo signaling cascade using immunoblotting and immunofluorescence assays. We found that the parasite turned off the hippo signaling pathway in TSP-1KO heart endothelial cells. The levels of SAV1 and MOB1A increased to a maximum of 2.70 ± 0.23 and 5.74 ± 1.45-fold at 3 and 6 h, respectively, in TSP-1KO mouse heart endothelial cells (MHEC), compared to WT MHEC, following a parasite challenge. This was accompanied by a significant continuous increase in the nuclear translocation of downstream effector molecule YAP, to a maximum mean nuclear fluorescence intensity of 10.14 ± 0.40 at 6 h, compared to wild type cells. Furthermore, we found that increased nuclear translocated YAP significantly colocalized with the transcription co-activator molecule pan-TEAD, with a maximum Pearson's correlation coefficient of 0.51 ± 0.06 at 6 h, compared to YAP-Pan-TEAD colocalization in the WT MHEC, which decreased significantly, with a minimum Pearson's correlation coefficient of 0.30 ± 0.01 at 6 h. Our data indicate that, during the early phase of infection, upregulated TSP-1 is essential for the regulation of the hippo signaling pathway. These studies advance our understanding of the molecular interactions occurring between heart endothelial cells and T. cruzi, in the presence and absence of TSP-1, providing insights into processes linked to parasite dissemination and pathogenesis.


Assuntos
Transporte Ativo do Núcleo Celular/fisiologia , Células Endoteliais/parasitologia , Mioblastos/parasitologia , Miocárdio/citologia , Proteínas de Protozoários/fisiologia , Trombospondina 1/fisiologia , Trypanosoma cruzi/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Células Endoteliais/metabolismo , Técnicas de Inativação de Genes , Camundongos , Mioblastos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Ratos , Transdução de Sinais/fisiologia , Trombospondina 1/deficiência , Transativadores/fisiologia
4.
Artigo em Inglês | MEDLINE | ID: mdl-28461309

RESUMO

Within the past few decades, the incidence and complexity of human fungal infections have increased, and therefore, the need for safer and more efficient, broad-spectrum antifungal agents is high. In the study described here, we characterized the new tetrazole-based drug candidate VT-1598 as an inhibitor of sterol 14α-demethylase (CYP51B) from the filamentous fungus Aspergillus fumigatus VT-1598 displayed a high affinity of binding to the enzyme in solution (dissociation constant, 13 ± 1 nM) and in the reconstituted enzymatic reaction was revealed to have an inhibitory potency stronger than the potencies of all other simultaneously tested antifungal drugs, including fluconazole, voriconazole, ketoconazole, and posaconazole. The X-ray structure of the VT-1598/A. fumigatus CYP51 complex was determined and depicts the distinctive binding mode of the inhibitor in the enzyme active site, suggesting the molecular basis of the improved drug potency and broad-spectrum antifungal activity. These data show the formation of an optimized hydrogen bond between the phenoxymethyl oxygen of VT-1598 and the imidazole ring nitrogen of His374, the CYP51 residue that is highly conserved across fungal pathogens and fungus specific. Comparative structural analysis of A. fumigatus CYP51/voriconazole and Candida albicans CYP51/VT-1161 complexes supports the role of H bonding in fungal CYP51/inhibitor complexes and emphasizes the importance of an optimal distance between this interaction and the inhibitor-heme iron interaction. Cellular experiments using two A. fumigatus strains (strains 32820 and 1022) displayed a direct correlation between the effects of the drugs on CYP51B activity and fungal growth inhibition, indicating the noteworthy anti-A. fumigatus potency of VT-1598 and confirming its promise as a broad-spectrum antifungal agent.


Assuntos
Antifúngicos/farmacologia , Aspergillus fumigatus/efeitos dos fármacos , Aspergillus fumigatus/enzimologia , Drogas em Investigação/farmacologia , Esterol 14-Desmetilase/metabolismo , Aspergillus fumigatus/genética , Candida albicans/efeitos dos fármacos , Candida albicans/enzimologia , Candida albicans/genética , Fluconazol/farmacologia , Cetoconazol/farmacologia , Testes de Sensibilidade Microbiana , Piridinas/farmacologia , Esterol 14-Desmetilase/genética , Tetrazóis/farmacologia , Triazóis/farmacologia , Voriconazol/farmacologia
5.
Antimicrob Agents Chemother ; 60(2): 1058-66, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26643331

RESUMO

A novel antifungal drug candidate, the 1-tetrazole-based agent VT-1161 [(R)-2-(2,4-difluorophenyl)-1,1-difluoro-3-(1H-tetrazol-1-yl)-1-{5-[4-(2,2,2-trifluoroethoxy)phenyl]pyridin-2-yl}propan-2-ol], which is currently in two phase 2b antifungal clinical trials, was found to be a tight-binding ligand (apparent dissociation constant [Kd], 24 nM) and a potent inhibitor of cytochrome P450 sterol 14α-demethylase (CYP51) from the protozoan pathogen Trypanosoma cruzi. Moreover, VT-1161 revealed a high level of antiparasitic activity against amastigotes of the Tulahuen strain of T. cruzi in cellular experiments (50% effective concentration, 2.5 nM) and was active in vivo, causing >99.8% suppression of peak parasitemia in a mouse model of infection with the naturally drug-resistant Y strain of the parasite. The data strongly support the potential utility of VT-1161 in the treatment of Chagas disease. The structural characterization of T. cruzi CYP51 in complex with VT-1161 provides insights into the molecular basis for the compound's inhibitory potency and paves the way for the further rational development of this novel, tetrazole-based inhibitory chemotype both for antiprotozoan chemotherapy and for antifungal chemotherapy.


Assuntos
Inibidores de 14-alfa Desmetilase/farmacologia , Piridinas/farmacologia , Esterol 14-Desmetilase/química , Tetrazóis/farmacologia , Tripanossomicidas/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Inibidores de 14-alfa Desmetilase/química , Animais , Doença de Chagas/tratamento farmacológico , Cristalografia por Raios X , Modelos Animais de Doenças , Feminino , Heme/química , Camundongos , Modelos Moleculares , Conformação Proteica , Piridinas/química , Esterol 14-Desmetilase/metabolismo , Tetrazóis/química , Trypanosoma cruzi/enzimologia
6.
J Infect Dis ; 212(9): 1439-48, 2015 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-25883390

RESUMO

Sterol 14α-demethylases (CYP51) are the enzymes essential for sterol biosynthesis. They serve as clinical targets for antifungal azoles and are considered as targets for treatment of human Trypanosomatidae infections. Recently, we have shown that VNI, a potent and selective inhibitor of trypanosomal CYP51 that we identified and structurally characterized in complex with the enzyme, can cure the acute and chronic forms of Chagas disease. The purpose of this work was to apply the CYP51 structure/function for further development of the VNI scaffold. As anticipated, VFV (R)-N-(1-(3,4'-difluorobiphenyl-4-yl)-2-(1H-imidazol-1-yl)ethyl)-4-(5-phenyl-1,3,4-oxadiazol-2-yl)benzamide, the derivative designed to fill the deepest portion of the CYP51 substrate-binding cavity, reveals a broader antiprotozoan spectrum of action. It has stronger antiparasitic activity in cellular experiments, cures the experimental Chagas disease with 100% efficacy, and suppresses visceral leishmaniasis by 89% (vs 60% for VNI). Oral bioavailability, low off-target activity, favorable pharmacokinetics and tissue distribution characterize VFV as a promising new drug candidate.


Assuntos
Antiprotozoários/farmacologia , Benzamidas/farmacologia , Doença de Chagas/tratamento farmacológico , Inibidores das Enzimas do Citocromo P-450/farmacologia , Sistema Enzimático do Citocromo P-450/química , Leishmaniose Visceral/tratamento farmacológico , Oxidiazóis/farmacologia , Animais , Antiprotozoários/farmacocinética , Benzamidas/farmacocinética , Biotransformação , Inibidores das Enzimas do Citocromo P-450/farmacocinética , Modelos Animais de Doenças , Feminino , Humanos , Imidazóis/farmacologia , Concentração Inibidora 50 , Camundongos , Camundongos Endogâmicos BALB C , Microssomos Hepáticos/efeitos dos fármacos , Estrutura Molecular , Oxidiazóis/farmacocinética , Ratos , Relação Estrutura-Atividade , Distribuição Tecidual , Trypanosoma cruzi/efeitos dos fármacos
8.
Infect Immun ; 81(11): 4139-48, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23980110

RESUMO

Human defensins play a fundamental role in the initiation of innate immune responses to some microbial pathogens. Here we show that colonic epithelial model HCT116 cells respond to Trypanosoma cruzi infection by secreting defensin α-1, which reduces infection. We also report the early effects of defensin α-1 on invasive trypomastigotes that involve damage of the flagellar structure to inhibit parasite motility and reduce cellular infection. Short exposure of defensin α-1 to trypomastigotes shows that defensin α-1 binds to the flagellum, resulting in flagellar membrane and axoneme alterations, followed by breaking of the flagellar membrane connected to the trypanosome body, leading to detachment and release of the parasite flagellum. In addition, defensin α-1 induces a significant reduction in parasite motility in a peptide concentration-dependent manner, which is abrogated by anti-defensin α-1 IgG. Preincubation of trypomastigotes with a concentration of defensin α-1 that inhibits 50% trypanosome motility significantly reduced cellular infection by 80%. Thus, human defensin α-1 is an innate immune molecule that is secreted by HCT116 cells in response to T. cruzi infection, inhibits T. cruzi motility, and plays an important role in reducing cellular infection. This is the first report showing a novel cellular innate immune response to a human parasite by secretion of defensin α-1, which neutralizes the motility of a human parasite to reduce cellular infection. The mode of activity of human defensin α-1 against T. cruzi and its function may provide insights for the development of new antiparasitic strategies.


Assuntos
Células Epiteliais/imunologia , Células Epiteliais/parasitologia , Flagelos/imunologia , Locomoção , Trypanosoma cruzi/imunologia , alfa-Defensinas/metabolismo , Membrana Celular/ultraestrutura , Flagelos/fisiologia , Flagelos/ultraestrutura , Células HCT116 , Humanos , Trypanosoma cruzi/fisiologia , Trypanosoma cruzi/ultraestrutura
9.
Invest New Drugs ; 31(3): 535-44, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23054211

RESUMO

Targeting tumor vasculature represents a rational strategy for controlling cancer. (Z)-(+/-)-2-(1-benzylindol-3-ylmethylene)-1-azabicyclo[2.2.2]octan-3-ol (denoted VJ115) is a novel chemical entity that inhibits the enzyme ENOX1, a NADH oxidase. Genetic and small molecule inhibition of ENOX1 inhibits endothelial cell tubule formation and tumor-mediated neo-angiogenesis. Inhibition of ENOX1 radiosensitizes tumor vasculature, a consequence of enhanced apoptosis. However, the molecular mechanisms underlying these observations are not well understood. Herein, we mechanistically link ENOX1-mediated regulation of cellular NADH concentrations with proteomics profiling of endothelial cell protein expression following exposure to VJ115. Pathway Studios network analysis of potential effector molecules identified by the proteomics profiling indicated that a VJ115 exposure capable of altering intracellular NADH concentrations impacted proteins involved in cytoskeletal reorganization. The analysis was validated using RT-PCR and immunoblotting of selected proteins. RNAi knockdown of ENOX1 was shown to suppress expression of stathmin and lamin A/C, proteins identified by the proteomics analysis to be suppressed upon VJ115 exposure. These data support the hypothesis that VJ115 inhibition of ENOX1 can impact expression of proteins involved in cytoskeletal reorganization and support a hypothesis in which ENOX1 activity links elevated cellular NADH concentrations with cytoskeletal reorganization and angiogenesis.


Assuntos
Inibidores da Angiogênese/farmacologia , Proteínas do Citoesqueleto/metabolismo , Indóis/farmacologia , NADH NADPH Oxirredutases/antagonistas & inibidores , Quinuclidinas/farmacologia , Células Cultivadas , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , NAD/metabolismo , Proteômica
10.
Res Sq ; 2023 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-36747834

RESUMO

COVID-19 is a viral infection that resulted in a global pandemic. In the United States, COVID-19 caused incommensurate deaths, especially among members of minority groups. Previous literature shows comorbidities such as hypertension (HTN), diabetes mellitus (DM) and obesity (OBS) have been implicated in the severity of COVID-19 cases regardless of racial or ethnic group classification. However, minority populations, particularly people of African descent experienced higher mortality as they carry a disproportionately heavier burden in comorbidities cases. In this study we first confirm current literature on COVID-19 incidence and its correlation with the prevalence of comorbidities in various racial and ethnic populations, using anonymous and aggregated data from the Nashville General Hospital at Meharry, an Institute for the Study of Minority Health. We also evaluated the prevalence of comorbidities in minority patients and computed the correlation between the COVID-19 incidence and a permuted prevalence of comorbidities. A total of 959 patients were reviewed and our study indicates COVID-19 patients classified as Non-Hispanic Blacks (NHB) were approximately 3 times more likely to have an HTN or DM or both HTN and DM diagnosis. The chances double to be approximately six times higher when an OBS diagnosis is included singularly or in conjunction with either HTN or DM or both HTN and DM.

11.
Artigo em Inglês | MEDLINE | ID: mdl-37138667

RESUMO

Background: Coronavirus Disease 2019, COVID-19, a viral infection, responsible for the latest pandemic has been shown to particularly affect the older population. Older adults, those aged 65 years and older, and individuals with serious underlying medical conditions are at a higher risk for severe illness from COVID-19 with a greater likelihood for hospitalization, admittance to the intensive care unit (ICU), and mortality. In this article, we describe the incidence and mortality rate found in Long Term Care facilities (LTCFs) and delineate any variations observed across varying types of LTCFs in the state of Tennessee (TN). Methods: Using aggregated data from the Tennessee (TN) Department of Health on COVID-19 Cases and Deaths from June 2020 to November 2021, we compare and contrast the incidence and fatality of COVID-19 among Long Term Care Facilities (LTCFs) in TN and describe the trends observed in these settings. Results: Our study indicates that there were major variations in COVID-19 prevalence rates in Nursing Homes (NHs) - 49% versus Assisted Care Living Facilities (ACLFs) in TN -16%. Although COVID-19 prevalence rates differed for NH and ACLFs, 12% of infected residents died in NHs while 13% of infected residents died in ACLFs. (Odds Ratio [OR]: 1.08 95% Confidence Interval [CI]: 0.93 -1.3, z-score: 1.37, p value: 0.085). Cases were more prevalent in five counties namely Davidson, Shelby, Hamilton, Knox, and Rutherford, majority of which were Metropolitan. Conclusion: As new variants continue to appear, counties with higher prevalence of COVID-19 should take continued effort to protect both resident and staff members especially in NHs settings and Metropolitan cities, where prevalence rate of the illness is higher.

12.
Cancers (Basel) ; 14(17)2022 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-36077843

RESUMO

Claudin-4 is part of the Claudin family of transmembrane tight junction (TJ) proteins found in almost all tissues and, together with adherens junctions and desmosomes, forms epithelial and endothelial junctional complexes. Although the distribution of Claudin-4 occurs in many cell types, the level of expression is cell-specific. Claudin proteins regulate cell proliferation and differentiation by binding cell-signaling ligands, and its expression is upregulated in several cancers. As a result, alterations in Claudin expression patterns or distribution are vital in the pathology of cancer. Profiling the genetic expression of Claudin-4 showed that Claudin-4 is also a receptor for the clostridium perfringens enterotoxin (CPE) and that Claudin-4 has a high sequence similarity with CPE's high-affinity receptor. CPE is cytolytic due to its ability to form pores in cellular membranes, and CPE treatment in breast cancer cells have shown promising results due to the high expression of Claudin-4. The C-terminal fragment of CPE (c-CPE) provides a less toxic alternative for drug delivery into breast cancer cells, particularly metastatic tumors in the brain, especially as Claudin-4 expression in the central nervous system (CNS) is low. Therefore, c-CPE provides a unique avenue for the treatment of breast-brain metastatic tumors.

13.
PLoS Negl Trop Dis ; 16(1): e0010074, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34986160

RESUMO

The protozoan parasite, Trypanosoma cruzi, causes severe morbidity and mortality in afflicted individuals. Approximately 30% of T. cruzi infected individuals present with cardiac pathology. The invasive forms of the parasite are carried in the vascular system to infect other cells of the body. During transportation, the molecular mechanisms by which the parasite signals and interact with host endothelial cells (EC) especially heart endothelium is currently unknown. The parasite increases host thrombospondin-1 (TSP1) expression and activates the Wnt/ß-catenin and hippo signaling pathways during the early phase of infection. The links between TSP1 and activation of the signaling pathways and their impact on parasite infectivity during the early phase of infection remain unknown. To elucidate the significance of TSP1 function in YAP/ß-catenin colocalization and how they impact parasite infectivity during the early phase of infection, we challenged mouse heart endothelial cells (MHEC) from wild type (WT) and TSP1 knockout mice with T. cruzi and evaluated Wnt signaling, YAP/ß-catenin crosstalk, and how they affect parasite infection. We found that in the absence of TSP1, the parasite induced the expression of Wnt-5a to a maximum at 2 h (1.73±0.13), P< 0.001 and enhanced the level of phosphorylated glycogen synthase kinase 3ß at the same time point (2.99±0.24), P<0.001. In WT MHEC, the levels of Wnt-5a were toned down and the level of p-GSK-3ß was lowest at 2 h (0.47±0.06), P< 0.01 compared to uninfected control. This was accompanied by a continuous significant increase in the nuclear colocalization of ß-catenin/YAP in TSP1 KO MHEC with a maximum Pearson correlation coefficient of (0.67±0.02), P< 0.05 at 6 h. In WT MHEC, the nuclear colocalization of ß-catenin/YAP remained steady and showed a reduction at 6 h (0.29±0.007), P< 0.05. These results indicate that TSP1 plays an important role in regulating ß-catenin/YAP colocalization during the early phase of T. cruzi infection. Importantly, dysregulation of this crosstalk by pre-incubation of WT MHEC with a ß-catenin inhibitor, endo-IWR 1, dramatically reduced the level of infection of WT MHEC. Parasite infectivity of inhibitor treated WT MHEC was similar to the level of infection of TSP1 KO MHEC. These results indicate that the ß-catenin pathway induced by the parasite and regulated by TSP1 during the early phase of T. cruzi infection is an important potential therapeutic target, which can be explored for the prophylactic prevention of T. cruzi infection.


Assuntos
Doença de Chagas/patologia , Via de Sinalização Hippo/fisiologia , Trombospondina 1/metabolismo , Via de Sinalização Wnt/fisiologia , Proteínas de Sinalização YAP/metabolismo , beta Catenina/metabolismo , Transporte Ativo do Núcleo Celular/fisiologia , Animais , Células Endoteliais/parasitologia , Endotélio/citologia , Endotélio/parasitologia , Glicogênio Sintase Quinase 3 beta/metabolismo , Coração/parasitologia , Camundongos , Camundongos Knockout , Ratos , Trombospondina 1/genética , Trypanosoma cruzi/metabolismo , Proteína Wnt-5a/metabolismo , beta Catenina/antagonistas & inibidores
14.
Microorganisms ; 9(9)2021 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-34576860

RESUMO

Acute and chronic upper respiratory illnesses such as asthma, and allergic rhinitis (AR) have been linked to the presence of microorganisms in the nose. Microorganisms can exist in symbiotic or commensal relationships with the human body. However, in certain cases, opportunistic pathogens can take over, leading to altered states (dysbiosis) and causing disease. Thus, the microflora present in a host can be useful to reflect health status. The human body contains 10 trillion to 100 trillion microorganisms. Of these populations, certain pathogens have been identified to promote or undermine wellbeing. Therefore, knowledge of the microbiome is potentially helpful as a diagnostic tool for many diseases. Variations have been recognized in the types of microbes that inhabit various populations based on geography, diet, and lifestyle choices and various microbiota have been shown to modulate immune responses in allergic disease. Interestingly, the diseases affected by these changes are prevalent in certain racial or ethnic populations. These prevalent microbiome variations in these groups suggest that the presence of these microorganisms may be significantly associated with health disparities. We review current research in the search for correlations between ethnic diversity, microbiome communities in the nasal cavity and health outcomes in neurological and respiratory functions.

15.
J Med Chem ; 64(23): 17511-17522, 2021 12 09.
Artigo em Inglês | MEDLINE | ID: mdl-34842434

RESUMO

Naegleria fowleri is the protozoan pathogen that causes primary amoebic meningoencephalitis (PAM), with the death rate exceeding 97%. The amoeba makes sterols and can be targeted by sterol biosynthesis inhibitors. Here, we characterized N. fowleri sterol 14-demethylase, including catalytic properties and inhibition by clinical antifungal drugs and experimental substituted azoles with favorable pharmacokinetics and low toxicity. None of them inhibited the enzyme stoichiometrically. The highest potencies were displayed by posaconazole (IC50 = 0.69 µM) and two of our compounds (IC50 = 1.3 and 0.35 µM). Because both these compounds penetrate the brain with concentrations reaching minimal inhibitory concentration (MIC) values in an N. fowleri cellular assay, we report them as potential drug candidates for PAM. The 2.1 Å crystal structure, in complex with the strongest inhibitor, provides an explanation connecting the enzyme weaker substrate specificity with lower sensitivity to inhibition. It also provides insight into the enzyme/ligand molecular recognition process and suggests directions for the design of more potent inhibitors.


Assuntos
Inibidores de 14-alfa Desmetilase/farmacologia , Naegleria fowleri/enzimologia , Esterol 14-Desmetilase/metabolismo , Ligantes , Esterol 14-Desmetilase/efeitos dos fármacos , Especificidade por Substrato
16.
PLoS One ; 16(3): e0246393, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33690604

RESUMO

Evidence link bacterial enterotoxins to apparent crypt-cell like cells (CCLCs), and Alpha Defensin 5 (DEFA5) expansion in the colonic mucosa of Crohn's colitis disease (CC) patients. These areas of ectopic ileal metaplasia, positive for Paneth cell (PC) markers are consistent with diagnosis of CC. Retrospectively, we: 1. Identified 21 patients with indeterminate colitis (IC) between 2000-2007 and were reevaluation their final clinical diagnosis in 2014 after a followed-up for mean 8.7±3.7 (range, 4-14) years. Their initial biopsies were analyzed by DEFA5 bioassay. 2. Differentiated ulcer-associated cell lineage (UACL) analysis by immunohistochemistry (IHC) of the CC patients, stained for Mucin 6 (MUC6) and DEFA5. 3. Treated human immortalized colonic epithelial cells (NCM460) and colonoids with pure DEFA5 on the secretion of signatures after 24hr. The control colonoids were not treated. 4. Treated colonoids with/without enterotoxins for 14 days and the spent medium were collected and determined by quantitative expression of DEFA5, CCLCs and other biologic signatures. The experiments were repeated twice. Three statistical methods were used: (i) Univariate analysis; (ii) LASSO; and (iii) Elastic net. DEFA5 bioassay discriminated CC and ulcerative colitis (UC) in a cohort of IC patients with accuracy. A fit logistic model with group CC and UC as the outcome and the DEFA5 as independent variable differentiator with a positive predictive value of 96 percent. IHC staining of CC for MUC6 and DEFA5 stained in different locations indicating that DEFA5 is not co-expressed in UACL and is therefore NOT the genesis of CC, rather a secretagogue for specific signature(s) that underlie the distinct crypt pathobiology of CC. Notably, we observed expansion of signatures after DEFA5 treatment on NCM460 and colonoids cells expressed at different times, intervals, and intensity. These factors are key stem cell niche regulators leading to DEFA5 secreting CCLCs differentiation 'the colonic ectopy ileal metaplasia formation' conspicuously of pathogenic importance in CC.


Assuntos
Colite Ulcerativa/metabolismo , Colo/citologia , Doença de Crohn/metabolismo , Enterotoxinas/farmacologia , Organoides/citologia , alfa-Defensinas/metabolismo , Idoso , Linhagem da Célula , Células Cultivadas , Colite Ulcerativa/microbiologia , Colite Ulcerativa/patologia , Colo/efeitos dos fármacos , Colo/metabolismo , Doença de Crohn/microbiologia , Doença de Crohn/patologia , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Feminino , Humanos , Modelos Logísticos , Masculino , Mucina-6/metabolismo , Técnicas de Cultura de Órgãos , Organoides/efeitos dos fármacos , Organoides/metabolismo , Proteômica , Estudos Retrospectivos
17.
Toxicol Appl Pharmacol ; 244(1): 21-6, 2010 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-19560482

RESUMO

Nrf2 (NF-E2-related factor 2) is a master transcription factor containing a powerful acidic transcriptional activation domain. Nrf2-dependent gene expression impacts cancer chemoprevention strategies, inflammatory responses, and progression of neurodegenerative diseases. Under basal conditions, association of Nrf2 with the CUL3 adaptor protein Keap1 results in the rapid Nrf2 ubiquitylation and proteasome-dependent degradation. Inhibition of Keap1 function blocks ubiquitylation of Nrf2, allowing newly synthesized Nrf2 to translocate into the nucleus, bind to ARE sites and direct target gene expression. Site-directed mutagenesis experiments coupled with proteomic analysis support a model in which Keap1 contains at least 2 distinct cysteine motifs. The first is located at Cys 151 in the BTB domain. The second is located in the intervening domain and centers around Cys 273 and 288. Adduction or oxidation at Cys151 has been shown to produce a conformational change in Keap1 that results in dissociation of Keap1 from CUL3, thereby inhibiting Nrf2 ubiquitylation. Thus, adduction captures specific chemical information and translates it into biochemical information via changes in structural conformation.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Culina/metabolismo , Cisteína/metabolismo , Proteínas do Citoesqueleto/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Adaptadoras de Transdução de Sinal/química , Motivos de Aminoácidos , Animais , Proteínas do Citoesqueleto/química , Regulação da Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Proteína 1 Associada a ECH Semelhante a Kelch , Oxirredução , Complexo de Endopeptidases do Proteassoma/metabolismo , Estrutura Terciária de Proteína , Relação Estrutura-Atividade , Compostos de Sulfidrila/metabolismo , Ativação Transcricional , Ubiquitinação
18.
FASEB J ; 23(9): 2986-95, 2009 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19395476

RESUMO

There is a need for novel strategies that target tumor vasculature, specifically those that synergize with cytotoxic therapy, in order to overcome resistance that can develop with current therapeutics. A chemistry-driven drug discovery screen was employed to identify novel compounds that inhibit endothelial cell tubule formation. Cell-based phenotypic screening revealed that noncytotoxic concentrations of (Z)-(+/-)-2-(1-benzenesulfonylindol-3-ylmethylene)-1-azabicyclo[2. 2.2]octan-3-ol (analog I) and (Z)-(+/-)-2-(1-benzylindol-3-ylmethylene)-1-azabicyclo[2.2.2]octan-3-ol (analog II) inhibited endothelial cell migration and the ability to form capillary-like structures in Matrigel by > or =70%. The ability to undergo neoangiogenesis, as measured in a window-chamber model, was also inhibited by 70%. Screening of biochemical pathways revealed that analog II inhibited the enzyme ENOX1 (EC(50) = 10 microM). Retroviral-mediated shRNA suppression of endothelial ENOX1 expression inhibited cell migration and tubule formation, recapitulating the effects observed with the small-molecule analogs. Genetic or chemical suppression of ENOX1 significantly increased radiation-mediated Caspase3-activated apoptosis, coincident with suppression of p70S6K1 phosphorylation. Administration of analog II prior to fractionated X-irradiation significantly diminished the number and density of tumor microvessels, as well as delayed syngeneic and xenograft tumor growth compared to results obtained with radiation alone. Analysis of necropsies suggests that the analog was well tolerated. These results suggest that targeting ENOX1 activity represents a novel therapeutic strategy for enhancing the radiation response of tumors.


Assuntos
Endotélio Vascular/citologia , Neovascularização Patológica/tratamento farmacológico , Proteína Dissulfeto Redutase (Glutationa)/antagonistas & inibidores , Quinuclidinas/farmacologia , Fatores de Transcrição/antagonistas & inibidores , Movimento Celular/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Células Cultivadas , Avaliação Pré-Clínica de Medicamentos , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/fisiologia , Humanos , Indóis , Proteínas de Membrana/antagonistas & inibidores , Neoplasias/irrigação sanguínea , Neoplasias/terapia , Neovascularização Patológica/radioterapia , Quinuclidinas/uso terapêutico
19.
Expert Opin Drug Discov ; 14(11): 1161-1174, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31411084

RESUMO

Introduction: Chagas disease affects 8-10 million people worldwide, mainly in Latin America. The current therapy for Chagas disease is limited to nifurtimox and benznidazole, which are effective in treating only the acute phase of the disease but with severe side effects. Therefore, there is an unmet need for new drugs and for the exploration of innovative approaches which may lead to the discovery of new effective and safe drugs for its treatment. Areas covered: The authors report and discuss recent approaches including structure-based design that have led to the discovery of new promising small molecule candidates for Chagas disease which affect prime targets that intervene in the sterol pathway of T. cruzi. Other trypanosome targets, phenotypic screening, the use of artificial intelligence and the challenges with Chagas disease drug discovery are also discussed. Expert opinion: The application of recent scientific innovations to the field of Chagas disease have led to the discovery of new promising drug candidates for Chagas disease. Phenotypic screening brought new hits and opportunities for drug discovery. Artificial intelligence also has the potential to accelerate drug discovery in Chagas disease and further research into this is warranted.


Assuntos
Doença de Chagas/tratamento farmacológico , Descoberta de Drogas/métodos , Tripanossomicidas/farmacologia , Animais , Inteligência Artificial , Doença de Chagas/parasitologia , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Tripanossomicidas/efeitos adversos
20.
ACS Infect Dis ; 5(3): 365-371, 2019 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-30625275

RESUMO

Up to now, no vaccines are available for Chagas disease, and the current therapy is largely unsatisfactory. Novel imidazole-based scaffolds of protozoan sterol 14α-demethylase (CYP51) inhibitors have demonstrated potent antiparasitic activity with no acute toxicity. Presently our aim was to investigate the effectiveness of the experimental 14α-demethylase inhibitor VFV in the mouse models of Trypanosoma cruzi infection using a naturally drug-resistant Colombiana strain, under monotherapy and in association with the reference drug, benznidazole (Bz). The treatment with VFV resulted in complete parasitemia suppression and 100% animal survival when administered orally (given in 10% DMSO plus 5% Arabic gum) at 25 mg/kg (bid) for 60 days. However, as parasite relapse was found using VFV alone under this treatment scheme, the coadministration of VFV with Bz was assayed giving simultaneously (for 60 days, bid) by oral route, under two different drug vehicles (10% DMSO plus 5% Gum Arabic with or without 3% Tween 80). All tested mice groups resulted in >99.9% of parasitemia decrease and 100% animal survival. qPCR analysis performed on cyclophosphamide immunosuppressed mice revealed that, although presenting lack of cure, VFV given as monotherapy was 14-fold more active than Bz, and the coadministration of Bz plus VFV (given simultaneously, using 10% DMSO plus 5% Gum Arabic as vehicle) resulted in 106-fold lower blood parasitism as compared to the monotherapy of Bz. Another interesting finding was the parasitological cure in 70% of the animals treated with Bz and VFV when the coadministration was given using the VFV suspension in 10% DMSO + Arabic gum + Tween 80 (a formulation that we have found to provide a better pharmacokinetics), even after immunosuppression using cyclophosphamide cycles, supporting the promising aspect of the drug coadministration in improving the efficacy of therapeutic arsenal against T. cruzi.


Assuntos
Inibidores de 14-alfa Desmetilase/administração & dosagem , Doença de Chagas/tratamento farmacológico , Nitroimidazóis/administração & dosagem , Proteínas de Protozoários/antagonistas & inibidores , Tripanossomicidas/administração & dosagem , Trypanosoma cruzi/efeitos dos fármacos , Trypanosoma cruzi/enzimologia , Inibidores de 14-alfa Desmetilase/química , Animais , Doença de Chagas/parasitologia , Modelos Animais de Doenças , Sinergismo Farmacológico , Quimioterapia Combinada , Humanos , Masculino , Camundongos , Nitroimidazóis/química , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Esterol 14-Desmetilase/química , Esterol 14-Desmetilase/metabolismo , Tripanossomicidas/química , Trypanosoma cruzi/química
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