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1.
J Am Chem Soc ; 143(20): 7655-7670, 2021 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-33988982

RESUMO

Aptamers, synthetic single-strand oligonucleotides that are similar in function to antibodies, are promising as therapeutics because of their minimal side effects. However, the stability and bioavailability of the aptamers pose a challenge. We developed aptamers converted from RNA aptamer to modified DNA aptamers that target phospho-AXL with improved stability and bioavailability. On the basis of the comparative analysis of a library of 17 converted modified DNA aptamers, we selected aptamer candidates, GLB-G25 and GLB-A04, that exhibited the highest bioavailability, stability, and robust antitumor effect in in vitro experiments. Backbone modifications such as thiophosphate or dithiophosphate and a covalent modification of the 5'-end of the aptamer with polyethylene glycol optimized the pharmacokinetic properties, improved the stability of the aptamers in vivo by reducing nuclease hydrolysis and renal clearance, and achieved high and sustained inhibition of AXL at a very low dose. Treatment with these modified aptamers in ovarian cancer orthotopic mouse models significantly reduced tumor growth and the number of metastases. This effective silencing of the phospho-AXL target thus demonstrated that aptamer specificity and bioavailability can be improved by the chemical modification of existing aptamers for phospho-AXL. These results lay the foundation for the translation of these aptamer candidates and companion biomarkers to the clinic.

2.
Annu Int Conf IEEE Eng Med Biol Soc ; 2020: 4903-4908, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-33019088

RESUMO

Haptic feedback can render real-time force interactions with computer simulated objects. In several telerobotic applications, it is desired that a haptic simulation reflects a physical task space or interaction accurately. This is particularly true when excessive applied force can result in disastrous consequences, as with the case of robot-assisted minimally invasive surgery (RMIS) and tissue damage. Since force cannot be directly measured in RMIS, non-contact methods are desired. A promising direction of non-contact force estimation involves the primary use of vision sensors to estimate deformation. However, the required fidelity of non-contact force rendering of deformable interaction to maintain surgical operator performance is not well established. This work attempts to empirically evaluate the degree to which haptic feedback may deviate from ground truth yet result in acceptable teleoperated performance in a simulated RMIS-based palpation task. A preliminary user-study is conducted to verify the utility of the simulation platform, and the results of this work have implications in haptic feedback for RMIS and inform guidelines for vision-based tool-tissue force estimation. An adaptive thresholding method is used to collect the minimum and maximum tolerable errors in force orientation and magnitude of presented haptic feedback to maintain sufficient performance.


Assuntos
Robótica , Interface Usuário-Computador , Retroalimentação , Retroalimentação Sensorial , Palpação
4.
EBioMedicine ; 40: 290-304, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30655206

RESUMO

BACKGROUND: Inflammatory mediator prostaglandin E2-prostaglandin E2 receptor EP3 (PTGER3) signaling is critical for tumor-associated angiogenesis, tumor growth, and chemoresistance. However, the mechanism underlying these effects in ovarian cancer is not known. METHODS: An association between higher tumoral expression of PTGER3 and shorter patient survival in the ovarian cancer dataset of The Cancer Genome Atlas prompted investigation of the antitumor effects of PTGER3 downmodulation. PTGER3 mRNA and protein levels were higher in cisplatin-resistant ovarian cancer cells than in their cisplatin-sensitive counterparts. FINDINGS: Silencing of PTGER3 via siRNA in cancer cells was associated with decreased cell growth and less invasiveness, as well as cell-cycle arrest and increased apoptosis, mediated through the Ras-MAPK/Erk-ETS1-ELK1/CFTR1 axis. Furthermore, sustained PTGER3 silencing with multistage vector and liposomal 2'-F-phosphorodithioate-siRNA-mediated silencing of PTGER3 combined with cisplatin resulted in robust antitumor effects in cisplatin-resistant ovarian cancer models. INTERPRETATION: These findings identify PTGER3 as a potential therapeutic target in chemoresistant ovarian cancers expressing high levels of this oncogenic protein. FUND: National Institutes of Health/National Cancer Institute, USA.


Assuntos
Transformação Celular Neoplásica/genética , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias Ovarianas/etiologia , Neoplasias Ovarianas/metabolismo , Receptores de Prostaglandina E Subtipo EP3/genética , Transdução de Sinais/efeitos dos fármacos , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Biomarcadores , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Transformação Celular Neoplásica/metabolismo , Feminino , Técnicas de Silenciamento de Genes , Inativação Gênica , Humanos , Imuno-Histoquímica , Modelos Biológicos , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/patologia , Proteína Proto-Oncogênica c-ets-1/metabolismo , Receptores de Prostaglandina E Subtipo EP3/metabolismo
5.
EBioMedicine ; 38: 100-112, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30487062

RESUMO

BACKGROUND: Circulating miRNAs are known to play important roles in intercellular communication. However, the effects of exosomal miRNAs on cells are not fully understood. METHODS: To investigate the role of exosomal miR-1246 in ovarian cancer (OC) microenvironment, we performed RPPA as well as many other in vitro functional assays in ovarian cancer cells (sensitive; HeyA8, Skov3ip1, A2780 and chemoresistant; HeyA8-MDR, Skov3-TR, A2780-CP20). Therapeutic effect of miR-1246 inhibitor treatment was tested in OC animal model. We showed the effect of OC exosomal miR-1246 uptake on macrophages by co-culture experiments. FINDINGS: Substantial expression of oncogenic miR-1246 OC exosomes was found. We showed that Cav1 gene, which is the direct target of miR-1246, is involved in the process of exosomal transfer. A significantly worse overall prognosis were found for OC patients with high miR-1246 and low Cav1 expression based on TCGA data. miR-1246 expression were significantly higher in paclitaxel-resistant OC exosomes than in their sensitive counterparts. Overexpression of Cav1 and anti-miR-1246 treatment significantly sensitized OC cells to paclitaxel. We showed that Cav1 and multi drug resistance (MDR) gene is involved in the process of exosomal transfer. Our proteomic approach also revealed that miR-1246 inhibits Cav1 and acts through PDGFß receptor at the recipient cells to inhibit cell proliferation. miR-1246 inhibitor treatment in combination with chemotherapy led to reduced tumor burden in vivo. Finally, we demonstrated that when OC cells are co-cultured with macrophages, they are capable of transferring their oncogenic miR-1246 to M2-type macrophages, but not M0-type macrophages. INTERPRETATION: Our results suggest that cancer exosomes may contribute to oncogenesis by manipulating neighboring infiltrating immune cells. This study provide a new mechanistic therapeutic approach to overcome chemoresistance and tumor progression through exosomal miR-1246 in OC patients.


Assuntos
Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Caveolina 1/genética , Resistencia a Medicamentos Antineoplásicos/genética , Exossomos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , MicroRNAs/genética , Neoplasias Ovarianas/genética , Animais , Apoptose/efeitos dos fármacos , Caveolina 1/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Modelos Animais de Doenças , Exossomos/metabolismo , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , MicroRNAs/metabolismo , Modelos Biológicos , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/mortalidade , Interferência de RNA , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Transdução de Sinais , Microambiente Tumoral
6.
Mol Ther Nucleic Acids ; 9: 251-262, 2017 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-29246304

RESUMO

Despite substantial improvements in the treatment strategies, ovarian cancer is still the most lethal gynecological malignancy. Identification of drug treatable therapeutic targets and their safe and effective targeting is critical to improve patient survival in ovarian cancer. AXL receptor tyrosine kinase (RTK) has been proposed to be an important therapeutic target for metastatic and advanced-stage human ovarian cancer. We found that AXL-RTK expression is associated with significantly shorter patient survival based on the The Cancer Genome Atlas patient database. To target AXL-RTK, we developed a chemically modified serum nuclease-stable AXL aptamer (AXL-APTAMER), and we evaluated its in vitro and in vivo antitumor activity using in vitro assays as well as two intraperitoneal animal models. AXL-aptamer treatment inhibited the phosphorylation and the activity of AXL, impaired the migration and invasion ability of ovarian cancer cells, and led to the inhibition of tumor growth and number of intraperitoneal metastatic nodules, which was associated with the inhibition of AXL activity and angiogenesis in tumors. When combined with paclitaxel, in vivo systemic (intravenous [i.v.]) administration of AXL-aptamer treatment markedly enhanced the antitumor efficacy of paclitaxel in mice. Taken together, our data indicate that AXL-aptamers successfully target in vivo AXL-RTK and inhibit its AXL activity and tumor growth and progression, representing a promising strategy for the treatment of ovarian cancer.

7.
Cell Discov ; 3: 17029, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28904816

RESUMO

The regulation of microRNA (miRNA) biogenesis, function and degradation involves a range of mechanisms, including interactions with RNA-binding proteins. The potential contribution of regulatory miRNAs to the expression of these RNA interactor proteins that could control other miRNAs expression is still unclear. Here we demonstrate a regulatory circuit involving oncogenic and tumor-suppressor miRNAs and an RNA-binding protein in a chemotherapy-resistant ovarian cancer model. We identified and characterized miR-15a-5p and miR-25-3p as negative regulators of hnRNPA1 expression, which is required for the processing of miR-18a-3p, an inhibitor of the K-RAS oncogene. The inhibition of miR-25-3p and miR-15a-5p decreased the proliferation, motility, invasiveness and angiogenic potential and increased apoptosis when combined with docetaxel. Alteration of this regulatory circuit causes poor overall survival outcome in ovarian cancer patients. These results highlight miR-15a-5p and miR-25-3p as key regulators of miR-18a-3p expression and its downstream target K-RAS, through direct modulation of hnRNPA1 expression. Our results demonstrate the therapeutic potential of inhibiting miR-25-3p and miR-15a-5p and the use of miR-18a-3p/KRAS ratio as a prominent outcome prognostic factor.

8.
Oncotarget ; 8(12): 20145-20164, 2017 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-28423620

RESUMO

Exosomes have emerged as important mediators of diverse biological functions including tumor suppression, tumor progression, invasion, immune escape and cell-to-cell communication, through the release of molecules such as mRNAs, miRNAs, and proteins. Here, we identified differentially expressed exosomal miRNAs between normal epithelial ovarian cell line and both resistant and sensitive ovarian cancer (OC) cell lines. We found miR-940 as abundant in exosomes from SKOV3-IP1, HeyA8, and HeyA8-MDR cells. The high expression of miR-940 is associated with better survival in patients with ovarian serous cystadenocarcinoma. Ectopic expression of miR-940 inhibited proliferation, colony formation, invasion, and migration and triggered G0/G1 cell cycle arrest and apoptosis in OC cells. Overexpression of miR-940 also inhibited tumor cell growth in vivo. We showed that proto-oncogene tyrosine-protein kinase (SRC) is directly targeted by miR-940 and that miR-940 inhibited SRC expression at mRNA and protein levels. Following this inhibition, the expression of proteins downstream of SRC, such as FAK, paxillin and Akt was also reduced. Collectively, our results suggest that OC cells secrete the tumor-suppressive miR-940 into the extracellular environment via exosomes, to maintain their invasiveness and tumorigenic phenotype.


Assuntos
Biomarcadores Tumorais/metabolismo , Exossomos/genética , Regulação Neoplásica da Expressão Gênica , MicroRNAs/genética , Neoplasias Ovarianas/patologia , Quinases da Família src/metabolismo , Animais , Apoptose , Biomarcadores Tumorais/genética , Proliferação de Células , Feminino , Genes Supressores de Tumor , Humanos , Camundongos , Camundongos Nus , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Transdução de Sinais , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto , Quinases da Família src/genética
9.
Mol Cancer Ther ; 16(6): 1114-1123, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28265009

RESUMO

To address the need for efficient and biocompatible delivery systems for systemic siRNA delivery, we developed 1,2-Dioleoyl-sn-Glycero-3-Phosphatidylcholine (DOPC) nanoliposomal EphA2-targeted therapeutic (EPHARNA). Here, we performed safety studies of EPHARNA in murine and primate models. Single dosing of EPHARNA was tested at 5 concentrations in mice (N = 15 per group) and groups were sacrificed on days 1, 14, and 28 for evaluation of clinical pathology and organ toxicity. Multiple dosing of EPHARNA was tested in mice and Rhesus macaques twice weekly at two dose levels in each model. Possible effects on hematologic parameters, serum chemistry, coagulation, and organ toxicity were assessed. Following single-dose EPHARNA administration to mice, no gross pathologic or dose-related microscopic findings were observed in either the acute (24 hours) or recovery (14 and 28 days) phases. The no-observed-adverse-effect level (NOAEL) for EPHARNA is considered >225 µg/kg when administered as a single injection intravenously in CD-1 mice. With twice weekly injection, EPHARNA appeared to stimulate a mild to moderate inflammatory response in a dose-related fashion. There appeared to be a mild hemolytic reaction in the female mice. In Rhesus macaques, minimal to moderate infiltration of mononuclear cells was found in some organs including the gastrointestinal tract, heart, and kidney. No differences attributed to EPHARNA were observed. These results demonstrate that EPHARNA is well tolerated at all doses tested. These data, combined with previously published in vivo validation studies, have led to an ongoing first-in-human phase I clinical trial (NCT01591356). Mol Cancer Ther; 16(6); 1114-23. ©2017 AACR.


Assuntos
Lipossomos , Nanopartículas , Fosfatidilcolinas , Interferência de RNA , RNA Interferente Pequeno/genética , Receptor EphA2/genética , Animais , Biomarcadores , Avaliação Pré-Clínica de Medicamentos , Feminino , Humanos , Macaca mulatta , Masculino , Camundongos , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/efeitos adversos , RNA Interferente Pequeno/farmacocinética , Distribuição Tecidual
10.
Cancer Res ; 76(24): 7194-7207, 2016 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-27742688

RESUMO

Cancer cells actively promote their tumorigenic behavior by reprogramming gene expression. Loading intraluminal vesicles with specific miRNAs and releasing them into the tumor microenvironment as exosomes is one mechanism of reprogramming whose regulation remains to be elucidated. Here, we report that miR-6126 is ubiquitously released in high abundance from both chemosensitive and chemoresistant ovarian cancer cells via exosomes. Overexpression of miR-6126 was confirmed in healthy ovarian tissue compared with ovarian cancer patient samples and correlated with better overall survival in patients with high-grade serous ovarian cancer. miR-6126 acted as a tumor suppressor by directly targeting integrin-ß1, a key regulator of cancer cell metastasis. miR-6126 mimic treatment of cancer cells resulted in increased miR-6126 and decreased integrin-ß1 mRNA levels in the exosome. Functional analysis showed that treatment of endothelial cells with miR-6126 mimic significantly reduced tube formation as well as invasion and migration capacities of ovarian cancer cells in vitro Administration of miR-6126 mimic in an orthotopic mouse model of ovarian cancer elicited a relative reduction in tumor growth, proliferating cells, and microvessel density. miR-6126 inhibition promoted oncogenic behavior by leading ovarian cancer cells to release more exosomes. Our findings provide new insights into the role of exosomal miRNA-mediated tumor progression and suggest a new therapeutic approach to disrupt oncogenic phenotypes in tumors. Cancer Res; 76(24); 7194-207. ©2016 AACR.


Assuntos
Exossomos/metabolismo , MicroRNAs/metabolismo , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Animais , Western Blotting , Feminino , Genes Supressores de Tumor , Xenoenxertos , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Camundongos , Camundongos Nus , Reação em Cadeia da Polimerase , Transcriptoma , Transfecção
11.
ISRN Dermatol ; 2013: 739054, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24223310

RESUMO

Uncontrolled blood sugar is a major cause of vascular complications and delayed wound healing in diabetes mellitus. During wound healing process, normally, apoptosis is responsible for events such as removal of inflammatory cells and evolution of granulation tissue into scar which occur during the late phase of wound healing. Early apoptosis can lead to abnormal wound healing by removing granulation tissue including fibroblast, endothelial cell, and small vessels. To determine the role of apoptosis in association with hyperglycemia in diabetic wound healing, apoptosis-related intracellular marker such as expression of Bcl-2 protein by immunohistochemistry and normal histology has been studied. Histological findings show higher level of apoptosis and diminished granulation tissue formation in diabetic rats wounds along with minimal expression of Bcl-2 in diabetic rats wounds when compared with nondiabetic rats wounds. It can be concluded from this study that elevated blood sugar level may be associated with increased apoptosis and the least expression of Bcl-2 protein which might cause deregulation of the wound healing processes in streptozotocin-induced diabetic rats.

12.
ScientificWorldJournal ; 2013: 363652, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23509434

RESUMO

To aid the clinical diagnosis of typhoid fever in India, where most hospitals and primary health centres have no facilities for culture, we report on the development of a novel and rapid immunodiagnostic kit for the direct detection of Salmonella Typhi--specific IgG antibodies against S. Typhi flagellar H antigen. The disease often does not show a specific clinical picture, and can be confused with other febrile illness such as malaria, dengue fever and Staphylococcus aureus. To overcome the problem of cross reactivity specific epitope of the flagellar H antigen was immobilised on the testing kit strip eliminating chances of cross reactivity and false positive results thereby increasing the specificity of the test. Since the immunodiagnostic kit, uses the flagellar H antigen from bacteria present in our country, the antibodies present in the serum of patients of our country will have maximum binding affinity, enhancing the sensitivity of our test kit. The immunodiagnostic kit on analysis gave a positive result with clinically diagnosed typhoid positive patient serum and negative results were obtained with the sera of clinically diagnosed malaria, abscess of Staphylococcus aureus and Visceral leishmaniasis (Kala-azar) patients.


Assuntos
Antígenos de Bactérias/imunologia , Kit de Reagentes para Diagnóstico , Salmonella typhi/imunologia , Febre Tifoide/diagnóstico , Western Blotting , Reações Cruzadas , Eletroforese em Gel de Poliacrilamida , Epitopos/análise , Epitopos/imunologia , Flagelina/imunologia , Humanos , Soros Imunes , Testes Imunológicos/métodos , Índia , Leishmaniose Visceral/diagnóstico , Leishmaniose Visceral/imunologia , Malária/diagnóstico , Malária/imunologia , Staphylococcus aureus/imunologia , Febre Tifoide/imunologia , Febre Tifoide/microbiologia
13.
ISRN Biochem ; 2013: 182601, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-25937956

RESUMO

Helicobacter pylori (H. pylori) infection is associated with increased oxidative stress and serum prolidase activity (SPA) in many diseases. We aimed to observe SPA and oxidative stress in nonulcer dyspepsia (NUD) infected with and without H. pylori among eastern Indians. 106 patients with H. pylori positive NUD, 82 patients with H. pylori negative NUD, and 50 healthy individuals were selected. SPA, total antioxidant capacity (TAOC), and total oxidant status (TOS) were measured with the use of spectrophotometer and an automated measurement method. SPA, TOS, and oxidative stress index (OSI) were significantly higher in patients with H. pylori positive than H. pylori negative NUD and healthy individuals (all P < 0.0001), whereas TAOC was significantly lower (P < 0.0001). Nonsignificant, increased SPA (P value = 0.6083) and decreased TAOC (P value = 0.1186) were observed in patients with H. pylori negative NUD than healthy individuals, while increased TOS and OSI were significant (P < 0.0001). Weak, nonsignificant correlations were observed between serum prolidase activity and TAOC, TOS, and OSI in H. pylori positive cases. Thus, increased SPA along with increased oxidative stress was observed, which seem to be closely associated with H. pylori infection. SPA and oxidative stress seem to be used as biomarkers for H. pylori infection in NUD.

14.
Cancer Biol Ther ; 8(11): 1027-34, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19395869

RESUMO

EphA2 gene silencing has been shown to result in antitumor efficacy. Here we considered whether silencing additional targets downstream of EphA2 would further enhance the therapeutic effect. EphA2 targeted siRNA was tested in combination with either FAK or Src targeted siRNA using DOPC nanoliposomes in orthotopic models of ovarian carcinoma. The effects of therapy were determined by changes in tumor weight, proliferation (Ki-67), and microvessel density (CD31). In our initial in vivo study, EphA2 plus FAK silencing resulted in the greatest reduction in tumor growth (by 73%, p < 0.005) as compared to control siRNA alone. In the SKOV3ip1 and HeyA8 ovarian cancer models, EphA2 siRNA-DOPC treatment resulted in a 50-67% decrease in tumor growth (p < 0.02, for both), and FAK siRNA-DOPC resulted in a 61-62% decrease in tumor growth (p < 0.009, p < 0.05, respectively). EphA2 plus FAK siRNA-DOPC treatment resulted in a significant reduction (SKOV3ip1: 76%, p < 0.007, HeyA8: 90%, p < 0.003) in tumor growth compared to control siRNA-DOPC. Combination treatment with EphA2 + FAK siRNA-DOPC resulted in significant decreases in tumor cell proliferation (p < 0.001) and microvessel density compared to control siRNA-DOPC (80%; p < 0.001), or the monotherapy groups (p values <0.001). These data suggest that the antitumor efficacy of in vivo EphA2 targeting is enhanced in combination with FAK silencing. Dual targeting of EphA2 and FAK may have therapeutic implications for ovarian cancer management.


Assuntos
Proteína-Tirosina Quinases de Adesão Focal/genética , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/terapia , Receptor EphA2/genética , Inibidores da Angiogênese/genética , Animais , Apoptose/genética , Processos de Crescimento Celular/genética , Modelos Animais de Doenças , Feminino , Proteína-Tirosina Quinases de Adesão Focal/deficiência , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Humanos , Imuno-Histoquímica , Antígeno Ki-67/metabolismo , Camundongos , Camundongos Nus , Neoplasias Ovarianas/enzimologia , Neoplasias Ovarianas/patologia , RNA Interferente Pequeno/genética , Receptor EphA2/deficiência
15.
BMC Genomics ; 10: 85, 2009 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-19232123

RESUMO

BACKGROUND: The variations within an individual's HLA (Human Leukocyte Antigen) genes have been linked to many immunological events, e.g. susceptibility to disease, response to vaccines, and the success of blood, tissue, and organ transplants. Although the microarray format has the potential to achieve high-resolution typing, this has yet to be attained due to inefficiencies of current probe design strategies. RESULTS: We present a novel three-step approach for the design of high-throughput microarray assays for HLA typing. This approach first selects sequences containing the SNPs present in all alleles of the locus of interest and next calculates the number of base changes necessary to convert a candidate probe sequences to the closest subsequence within the set of sequences that are likely to be present in the sample including the remainder of the human genome in order to identify those candidate probes which are "ultraspecific" for the allele of interest. Due to the high specificity of these sequences, it is possible that preliminary steps such as PCR amplification are no longer necessary. Lastly, the minimum number of these ultraspecific probes is selected such that the highest resolution typing can be achieved for the minimal cost of production. As an example, an array was designed and in silico results were obtained for typing of the HLA-B locus. CONCLUSION: The assay presented here provides a higher resolution than has previously been developed and includes more alleles than previously considered. Based upon the in silico and preliminary experimental results, we believe that the proposed approach can be readily applied to any highly polymorphic gene system.


Assuntos
Sondas de DNA/genética , Teste de Histocompatibilidade/métodos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Alelos , Genoma Humano , Antígenos HLA-B/genética , Humanos , Polimorfismo de Nucleotídeo Único , Sensibilidade e Especificidade , Análise de Sequência de DNA
16.
World J Surg ; 31(4): 644-53, 2007 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-17334862

RESUMO

BACKGROUND: Sirolimus (rapamycin), a strong immunosuppressive agent, is administered to renal transplant patients to prevent rejection. The rapamycin signaling pathway [mammalian target of rapamycin (mTOR)] has been implicated in transcriptional regulation. METHODS: We used high-density oligonucleotide human microarrays to evaluate the effects of sirolimus treatment on gene expression in renal transplant patients. With this technique, we assessed selected genes in the rapamycin signaling, immunosuppression, insulin signaling, and triglyceride metabolism pathways. RESULTS: Filtered data from both treated and untreated patients showed variability within each group. Significant fold changes were observed in genes from the immunosuppression and insulin signaling pathways but not the rapamycin signaling pathway. The triglyceride metabolism pathway revealed a significant reduction of message levels in lipoprotein and triglyceride synthesis genes. CONCLUSIONS: These results show that using oligonucleotide microarrays to analyze the effects of sirolimus treatment in patients with renal transplant is an effective way to evaluate gene message levels in multiple pathways.


Assuntos
Expressão Gênica/efeitos dos fármacos , Imunossupressores/uso terapêutico , Transplante de Rim , Sirolimo/uso terapêutico , Feminino , Rejeição de Enxerto/prevenção & controle , Humanos , Insulina/metabolismo , Interleucina-2/genética , Masculino , Análise de Sequência com Séries de Oligonucleotídeos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Transcrição Genética , Resultado do Tratamento , Triglicerídeos/metabolismo
17.
Curr Oncol Rep ; 8(6): 484-91, 2006 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-17040626

RESUMO

The completion of the human genome project, along with the ancillary technologies derived from this effort, provides the ability to comprehensively analyze patient tumors as well as the individual patient's own genetic make-up at the DNA, RNA, and protein level. As a result, novel molecular screening techniques have the potential to push the boundaries of detection to even smaller tumors and also to allow accurate risk assessment, cancer prevention, and treatment planning in individual women. This review focuses on advances over the past 2 years in the use of molecular signatures and circulating tumor cells for early breast cancer detection and for prediction of response to therapy.


Assuntos
Neoplasias da Mama/diagnóstico , Neoplasias da Mama/terapia , Carcinoma/diagnóstico , Carcinoma/terapia , Técnicas de Diagnóstico Molecular/métodos , Biomarcadores Tumorais/análise , Biomarcadores Tumorais/sangue , Neoplasias da Mama/prevenção & controle , Vacinas Anticâncer/uso terapêutico , Carcinoma/prevenção & controle , DNA/análise , Diagnóstico Precoce , Feminino , Predisposição Genética para Doença , Humanos , Células Neoplásicas Circulantes/patologia , Planejamento de Assistência ao Paciente , Prognóstico , Proteínas/análise , RNA/análise , Sensibilidade e Especificidade
19.
FEBS J ; 273(2): 398-408, 2006 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-16403026

RESUMO

Reliable detection and identification of pathogens in complex biological samples, in the presence of contaminating DNA from a variety of sources, is an important and challenging diagnostic problem for the development of field tests. The problem is compounded by the difficulty of finding a single, unique genomic sequence that is present simultaneously in all genomes of a species of closely related pathogens and absent in the genomes of the host or the organisms that contribute to the sample background. Here we describe 'host-blind probe design'- a novel strategy of designing probes based on highly frequent genomic signatures found in the pathogen genomes of interest but absent from the host genome. Upon hybridization, an array of such informative probes will produce a unique pattern that is a genetic fingerprint for each pathogen strain. This multiprobe approach was applied to 83 dengue virus genome sequences, available in public databases, to design and perform in silico microarray experiments. The resulting patterns allow one to unequivocally distinguish the four major serotypes, and within each serotype to identify the most similar strain among those that have been completely sequenced. In an environment where dengue is indigenous, this would allow investigators to determine if a particular isolate belongs to an ongoing outbreak or is a previously circulating version. Using our probe set, the probability that misdiagnosis at the serotype level would occur is approximately 1 : 10(150).


Assuntos
Primers do DNA , Sondas de DNA , Vírus da Dengue/genética , Genoma Viral , Vírus da Dengue/classificação , Humanos , Reação em Cadeia da Polimerase
20.
Proteome Sci ; 3: 9, 2005 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-16281978

RESUMO

This study presents the development and application of protein lysate microarray (LMA) technology for verification of presence and quantification of human tissue samples for protein biomarkers. Sub-picogram range sensitivity has been achieved on LMA using a non-enzymatic protein detection methodology. Results from a set of quality control experiments are presented and demonstrate the high sensitivity and reproducibility of the LMA methodology. The optimized LMA methodology has been applied for verification of the presence and quantification of disease markers for atherosclerosis. LMA were used to measure lipoprotein [a] and apolipoprotein B100 in 52 carotid endarterectomy samples. The data generated by LMA were validated by ELISA using the same protein lysates. The correlations of protein amounts estimated by LMA and ELISA were highly significant, with r2 > or = 0.98 (p < or = 0.001) for lipoprotein [a] and with r2 > or = 0.94 (p < or = 0.001) for apolipoprotein B100. This is the first report to compare data generated using proteins microarrays with ELISA, a standard technology for the verification of the presence of protein biomarkers. The sensitivity, reproducibility, and high-throughput quality of LMA technology make it a potentially powerful technology for profiling disease specific protein markers in clinical samples.

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