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Introducción. El consumo de alimentos transgénicos constituye un riesgo potencial para la salud. Sin embargo, en el Perú se carece de información actualizada y confiable sobre la presencia de transgénicos en los alimentos y sobre los datos pertinentes en su etiquetado; de igual manera sobre los alimentos que consumen los animales de abasto, cuyos productos van a ser ingeridos por el humano. Objetivo. Determinar la transgenicidad, mediante la detección del promotor 35S, en productos alimenticios industrializados de maíz para consumo humano y animal, que se comercializan en Lima y verificar sí en el etiquetado se menciona si contiene o no secuencias transgénicas. Métodos. Se analizaron 30 muestras de alimentos para consumo humano y 10 para consumo de animales de abasto; y se revisó el etiquetado. Para la extracción del ADN se utilizó el kit Dneasy Mericon Food, para la detección del P35S el método Real Time-PCR empleando el kit Mericon Screen 35S y para determinar la concentración de copias el kit Mericon Quant Mon 810. Resultados. Se detectó el P35S en el 66,66% de las muestras para consumo humano, y en el 90,00% de las muestras para consumo animal. En el etiquetado del 100% de las muestras para consumo humano y animal no se menciona si contiene o no componentes transgénicos. Conclusiones. La detección de contenido transgénico en la mayoría de los alimentos industrializados de maíz para humanos y animales evidencian la necesidad de su mención en el etiquetado y de la implementación de una política exigente en bioseguridad alimentaria.
Introduction. Consumption of transgenic foods constitutes a potential health risk. However, in Peru there is a lack of updated and reliable information on the presence of transgenics in food and on the relevant data on their labeling; in the same way about the food consumed by animals for supply, whose products are going to be ingested by humans. Objetive. To determine the transgenicity, through the detection of the 35S promoter, in industrialized corn food products for human and animal consumption, which are marketed in Lima and to verify if the labeling mentions whether or not it contains transgenic sequences. Methods. 30 food samples for human consumption and 10 for consumption by animals for production were analyzed; and the labeling was revised. The Dneasy Mericon Food kit was used for DNA extraction, the Real Time-PCR method for P35S detection using the Mericon Screen 35S kit, and the Mericon Quant Mon 810 kit to determine the copy concentration. Results. P35S was detected in 66,66% of the samples for human consumption, and in 90.00% of the samples for animal consumption. The labeling of 100% of the samples for human and animal consumption does not mention whether or not it contains transgenic components. Conclusions. The detection of transgenic content in the majority of industrialized corn foods for humans and animals demonstrates the need to mention them on the label and the implementation of a demanding policy on food biosafety.
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Objective:To investigate the effect of KRT5 knockdown in keratinocytes on melanin content in co-cultured melanocytes, and to explain mechanisms underlying formation of hyperpigmented lesions in reticulate pigmented anomaly of the flexures (Dowling-Degos disease, DDD) .Methods:HaCaT cells with heterozygous mutations in the KRT5 gene were obtained by using clustered regularly interspaced short palindromic repeats (CRISPR) -CRISPR-associated protein 9 (Cas9) technology (experimental group) , and HaCaT cells transfected with non-targeting single guide RNA:Cas9 protein complex served as control group, both of which were in vitro co-cultured with primary human melanocyte cells (HEMn) separately. Immunofluorescence study was conducted to determine the expression of cytokeratin and melanosomes in co-cultured cells; melanin content was detected in melanocytes in different co-culture groups, which were obtained by differential trypsinization. Immunohistochemical study was performed to determine the expression of melanocyte-specific premelanosome protein 17 (Pmel17) in skin lesions in a patient with DDD carrying a KRT5 mutation and normal skin tissues in a healthy control. Results:Sanger sequencing showed a heterozygous mutation (c.1delA) at the initiation codon of exon 1 of the KRT5 gene in HaCaT cells in the experimental group, but no mutation in the KRT5 gene in the control group. Western blot analysis showed that the KRT5 protein expression was significantly lower in the experimental group (0.60 ± 0.05) than in the control group (1.00 ± 0.00, t = 32.38, P = 0.001) . Compared with the co-culture system in the control group, the number of Pmel17-labeled melanosomes markedly increased with the melanin content elevated by 52.5% ( t = -3.48, P = 0.025) in the HEMn cells co-cultured with HaCaT cells in the experimental group. Immunohistochemical study showed that the Pmel17 expression increased in the skin lesions in the DDD patient with KRT5 mutation compared with the normal skin tissues in the healthy control. Conclusion:The effect of HaCaT cells with CRISPR-Cas9-induced KRT5 mutation on the co-cultured HEMn melanocytes was verified by the successfully established in vitro co-culture system, which provides a primary cell model for further studies on interaction mechanisms between keratinocytes and melanocytes, and on pathogenesis of skin pigmentation abnormalities.
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β-thalassemia is a single-gene genetic disease caused by β globin gene mutations leading to the fact that red blood cells are unable to form normal adult hemoglobin, and then patients develop hemolytic anemia. Current treatment regimens mainly include allogenetic hematologic stem cell transplantation, symptomatic regular blood transfusions and the use of iron removers to reduce iron load. Some severe patients have quite poor prognoses and deadly consequences if not treated timely. Genetically modified autohematopoietic stem cells can provide a new treatment option for patients with β thalassemia, which may achieve a long-term and stable increase in hemoglobin level through a single dose, making one-time cure β-thalassemia possible. This paper reviews the key elements of clinical trial design for β-thalassemia gene therapy from the aspects of efficacy evaluation endpoints, clinical trial design, enrollment population, and subject monitoring in order to provide a reference for pharma-therapeutic research and development enterprises.
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Objective To construct F344 rat bone marrow mesenchymal stem cell line (MSC) modified with human hepatocyte growth factor (hHGF) gene.Methods Recombinant virus containing hHGF was obtained by transfecting the packaging cell line 293 FT with lentiviral vector pLV/EF1α-hHGF-IRES-eGFP.MSCs derived from F344 rat bone marrow were then tranfected with packed lentiviral vector.Purified MSCs expressing hHGF was obtained by screening culture with G418.MSCs and MSCs transfected with empty vector were used as control.The expression of hHGF protein was detected by Western blot (eGFP-MSCs).The hHGF-transfected MSCs were cultured in osteoblast-inducing culture medium and osteoblast phenotype was assayed by alizarin Red staining.The cells were also cultured in adipogenesis medium and stained with Oil Red O for identification.Results The expression of hHGF protein was significantly up-regulated in the hHGF-MSCs as compared with MSCs and eGFP-MSCs.hHGF-MSCs readily differentiated into mineralizing cells or adipocytes when incubated in differentiation medium.Conclusion A F344 rat MSC line that stably expresses HGF is successfully established.
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Objective To investigate the effect of human hepatocyte growth factor (hHGF) genetic modification on the ameliorating effects of mesenchymal stem cells (MSCs) implantation on pulmonary microvascular rarefaction in a rat model of pulmonary hypertension (PH).Methods MSCs were obtained from F344 rats and transduced with lentiviral vector modified with human HGF (hHGF-MSCs) or empty vector (EGFP-MSCs).Sixty-six 7 week old male F344 rats weighing 180-250 g were used in this study.PH was induced by left pneumonectomy and subcutaneous monocrotaline (MCT) 60 mg/kg injected at 2 weeks after operation.The animals with PH were randomly divided into 3 groups:control group (group C),EGFP-MSCs group (group E) and HGF-MSCs group (group H).Groups H and E received hHGF-MSCs or EGFP-MSCs 5 × 105 in DMEM 1 ml iv at 3 weeks after subcutaneous MCT injection,while group C received plain DMEM 1 ml.Mean pulmonary arterial pressure (mPAP) was measured and right ventricular hypertrophy and angiogenesis in the lung were assessed and the content of rat HGF (rHGF) and hHGF protein in lung tissue and pulmonary capillary density (by immuno-histochemistry) was measured at 2 weeks after MSCs implantation.The survival rates within 45 days after MCT administration were compared among the 3 groups.Results No hHGF was detected in groups C and E.Both hHGF-MSCs and EGFP-MSCs significantly reduced MPAP and right ventricular hypertrophy and increased pulmonary capillary density and survival rates in groups H and E as compared with group C and the efficacy of hHGF-MSCs was significantly greater than that of EGFP-MSCs.Barium angiography revealed that distal pulmonary vasculature was significantly increased in group H as compared with groups E and C.The survival of the rats receiving hHGF-MSCs was significantly longer in group H than that in groups E and C.Conclusion hHGF genetic modification can improve the ameliorating effects of MSCs implantation on PH-related microvascular rarefaction.
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ObjectiveTo construct human embryonic kidney cells (HEK293) modified with human preproenkephalin (hPPE) gene.MethodshPPE gene fragments were obtained from recombinant plasmid pcDNA3.1( + )/hPPE by using restriction endonuelease Hind Ⅲ and Not Ⅰ.Homologous recombination of lentivirus and hPPE gene was produced by using recombinant DNA technology.HEK293 cells were then transfected with the recombinant lentivirus vectors.The expression of hPPE gene in HEK293 cells was detected by Western blot.ResultsThe results of DNA sequencing indicated that the positive clone of recombinant lentivirus was completely consistent with sequencing of hPPE in Genebank.The titer of the concentrated virus was 2.07 × 108 TU/ml.GFP fluorescence was not seen in HEK293 cells transfected with the lentiviral vector under fluorescence microscope.A strong fluorescence was seen in HEK293 cells transfected with Ubc-GFP-L.V.empty viral vector.Positive expression of hPPE was demonstrated in HEK293 cells transfected with lentiviral vector by Western blot.Conclusion HEK293 cells modified with hPPE gene were successfully constructed and the target gene hPPE was stably expressed in HEK293 cells.
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OBJECTIVE: The aim of this work was to investigate the occurrence of Roundup Ready soybean in enteral nutrition formulas sold in Brazil. METHODS: A duplex Polymerase Chain Reaction based on the amplification of the lectin gene and the construction of the recombinant deoxyribonucleic acid of transgenic glyphosate-tolerant soybean (35S promoter and chloroplast transit peptide gene) was performed in order to analyze the deoxyribonucleic acid obtained from nine soy protein isolate-containing formulas. RESULTS: Despite the highly processed nature of the food matrices, amplifiable deoxyribonucleic acid templates were obtained from all tested samples, as judged by the amplification of the lectin gene sequence. However, amplicons relative to the presence of Roundup Ready soybean were restricted to one of the nine enteral nutrition formulas analyzed as well as to the soybean reference powder, as expected. Quantitative analysis of the genetically modified formula by real-time Polymerase Chain Reaction showed a content of approximately 0.3 percent (w/w) of recombinant deoxyribonucleic acid from the Roundup Ready soybean. CONCLUSION: The results show that one of the formulas contained genetically modified soy, pointing to the need of regulating the use of transgenic substances and of specific labeling in this product category.
OBJETIVO: Investigar a ocorrência de soja transgênica em fórmulas de suporte nutricional comercializadas no Brasil. MÉTODOS: Foi desenvolvido o método da reação em cadeia da polimerase duplex, com base na amplificação do gene na lectina, e na construção do ácido desoxirribonucléico recombinante da soja transgênica tolerante a glifosato (promotor 35S e gene de peptídeo de trânsito de cloroplasto), a fim de avaliar o ácido desoxirribonucléico extraído a partir das nove fórmulas contendo isolado protéico de soja. RESULTADOS: Apesar do alto grau de processamento aos quais os produtos avaliados foram submetidos, foi possível extrair ácido desoxirribonucléico amplificável a partir de todas as amostras, demonstrado pela amplificação do gene endógeno (lectina). Adicionalmente, o fragmento relativo à modificação genética da soja transgênica foi detectado em uma das nove amostras avaliadas, bem como na amostra relativa ao material de referência contendo 1,0 por cento de organismo geneticamente modificado. As análises quantitativas realizadas a partir da reação em cadeia da polimerase em tempo real revelaram a presença de aproximadamente 0,3 por cento de ácido desoxirribonucléico recombinante derivado de organismo geneticamente modificado na amostra de fórmula que apresentou resultado positivo. CONCLUSÃO: Os resultados demonstram que uma das fórmulas analisadas apresentava ingredientes derivados de soja geneticamente modificada, apontando para a necessidade de regulamentar a utilização de transgênicos, e de rotulagem específica nessa categoria de produtos.
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Soybeans , Enteral Nutrition , Organisms, Genetically Modified , Polymerase Chain Reaction/methodsABSTRACT
Objective To investigate the analgesic effect of intrathecal(IT)human bone marrow mesenchymal stem cells(hMSC)genetically modified with human proenkephalin gene(PENK)in a rat model of neuropathic pain.Methods Forty male SD rats weighing 160-180 g in which IT catheters were successfully implanted without complication were randomly divided into 4 gorups(n = 10 each): group A normal control;group B neuropathic pain(NP);group C NP + hMSC-pBABE and group D NP + hMSC-PENK.Neuropathic pain was induced with chronic constrictive injury(CCI).Four loose ligatures were placed on the main stem of sciatic nerve with 4-0 chronic catgut.IT normal saline 10 μl,hMSC-pBABE cell suspension 10 μl(2 × 108-3 × 108/μl)and hMSCPENK cell suspension 10 μl(2 × 108-3 × 108/μl)were injected in group B,C and D respectively on the 3rd day after operation.Paw-withdrawal latency(PWL)to noxious thermal stimulation was measured before(baseline)and at 3,5,7,9 and 14 d after operation.The animals were killed on the 14th day after last PWL measurement.RNA was extracted from the spinal cord for determination of proenkephalin mRNA expression.Results PWL was significantly decreased after operation as compared with the baseline values before operation in group B,C and D.PWL was significantly longer at 7,9,14 d after operation in group D than in group B and C but there was no significant difference in PWL after operation between group B and C.PENK mRNA expression was significantly lower in group B and C than in group A,but was significantly higher in group D than in group B and C.There was no significant difference in PENK mRNA expression between group B and C.Conclusion Intratheccal human bone marrow mesenchymal stem cells genetically modified with human proenkephalin gene can relieve neuropathic pain in rats.
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Objective To construct h n bone marrow mesenchymal stem cell line genetically modified with human proenkephalin gene. Methods The packaging cell line Phoenix-293T was transfected with the recombinant pBABE-PENK vector to aquire virus. The recombinant virus was then collected and used to infect hMSCs. Stable expression of proenkephalin gene and leucine enkephalin protein and the concentration of leucine enkephalin protein were detected by RT-PCR, immunofluorescence and ELISA respectively. Results The expression of proenkephalin gene and leucine enkephalin protein were significantly up-regulated in the hMSC-PENK cells, and the concentration of leucine enkephalin protein was also increased in the culture medium. Conclusion A human mesenchymal stem cell line that expresses proenkephalin gene and secrets enkephalin was successfully established.