RESUMO
Cutaneous drug-induced reactions are immune-mediated responses that can lead to life-threatening diseases such as drug reaction with eosinophilia and systemic symptoms (DRESS), Stevens-Johnson syndrome, and toxic epidermal necrolysis, collectively known as severe cutaneous adverse reactions (SCARs). Unfortunately, they cannot be predicted during drug development, and, at present, a prognostic biomarker is not available nor are validated in vitro assays for diagnosis. Thus, by using proteomic and microarray miRNA analysis, the cargo of extracellular vesicles obtained from SCARs patients was analyzed and correlated with the severity of the reaction. Confirmatory assays using Western blot and qRT-PCR were performed to validate findings, and bioinformatic tools were used to establish the correlation between protein and miRNAs expression between groups. The proteomic analysis showed an increase in the amount of pro-inflammatory proteins, von Willebrand factor, and C-reactive protein and a decrease in anti-inflammatory and protective proteins in the SCARs group compared with the control group. Additionally, histone protein H2A was enriched in DRESS patients. APO1 and SERPINA4 proteins, highly increased in the control group but absent in the SCARs group, are the target of several overexpressed miRNAs, suggesting that the regulation of these proteins might involve gene silencing and protein repressing mechanisms in the severe patients. According with previous reports showing its presence in plasma and T-cells, microRNA miR-18 was upregulated in extracellular vesicles obtained from the most severe patients. Determination of the unique cargo associated with different disease conditions will help to understand the pathophysiology of these complex reactions and might help to develop novel biomarkers for life-threatening iatrogenic cutaneous disease.
Assuntos
Toxidermias/genética , Vesículas Extracelulares/genética , MicroRNAs/genética , Toxidermias/diagnóstico , Vesículas Extracelulares/química , Vesículas Extracelulares/patologia , Humanos , Proteoma/análise , Proteoma/genética , Proteômica , TranscriptomaRESUMO
INTRODUCTION: The peripheral ossifying fibroma (POF) is a benign reactive lesion that exclusively arises from gingiva. The lesion may gain considerably large sizes and present peculiar clinical and radiographic features that would then allow it to be called a giant POF; in that case, its otherwise simple surgical extraction could create a challenge. Thus, we elect here, for the very first time, a plausible alternative for treating giant POF: piezosurgery followed by placement of platelet-rich fibrin (PRF). CASE PRESENTATION: A 31-year-old black male presented a large asymptomatic nodule on the lower gingiva; the lesion had caused vestibular displacement of teeth and had been present for 18 years. Following the diagnostic hypothesis of a giant POF, an excisional biopsy was performed under local anesthesia using piezosurgery (microvibration of 36,000 times/sec was used in a bone cortical working mode), which confirmed the diagnosis. The surgical procedure was facilitated with the use of piezosurgery followed by placement of PRF, being the trans- and postoperative periods occurred with no complications. One year after the treatment, the patient shows no signs of disease recurrence and remains under follow-up. CONCLUSIONS: Giant POF is a rare gingival reactive lesion that can reach large dimensions, causing teeth displacement, functional, and esthetic impairments. The lesion can be successfully managed with piezosurgery and PRF, as illustrated herein, avoiding extensive bone loss and damage to the surrounding soft tissues.
Assuntos
Fibroma Ossificante , Doenças da Gengiva , Piezocirurgia , Fibrina Rica em Plaquetas , Adulto , Fibroma Ossificante/terapia , Doenças da Gengiva/terapia , Humanos , Masculino , Recidiva Local de NeoplasiaRESUMO
Synthetic biology and genetic engineering in algae offer an unprecedented opportunity to develop species with traits that can help solve the problems associated with food and energy supply in the 21st century. In the green alga Chlamydomonas reinhardtii, foreign genes can be expressed from the chloroplast genome for molecular farming and metabolic engineering to obtain commodities and high-value molecules. To introduce these genes, selectable markers, which rely mostly on the use of antibiotics, are needed. This has risen social concern associated with the potential risk of horizontal gene transfer across life kingdoms, which has led to a quest for antibiotic-free selectable markers. Phosphorus (P) is a scarce nutrient element that most organisms can only assimilate in its most oxidized form as phosphate (Pi); however, some organisms are able to oxidize phosphite (Phi) to Pi prior to incorporation into the central metabolism of P. As an alternative to the use of the two positive selectable makers already available for chloroplast transformation in C. reinhardtii, the aadA and the aphA-6 genes, that require the use of antibiotics, we investigated if a phosphite-based selection method could be used for the direct recovery of chloroplast transformed lines in this alga. Here we show that following bombardment with a vector carrying the ptxD gene from Pseudomonas stutzeri WM88, only cells that integrate and express the gene proliferate and form colonies using Phi as the sole P source. Our results demonstrate that a selectable marker based on the assimilation of Phi can be used for chloroplasts transformation in a biotechnologically relevant organism. The portable selectable marker we have developed is, in more than 18 years, the latest addition to the markers available for selection of chloroplast transformed cells in C. reinhardtii. The ptxD gene will contribute to the repertoire of tools available for synthetic biology and genetic engineering in the chloroplast of C. reinhardtii.
Assuntos
Proteínas de Bactérias/genética , Chlamydomonas reinhardtii/genética , Cloroplastos/genética , NADH NADPH Oxirredutases/genética , Fosfitos/metabolismo , Fósforo/metabolismo , Proteínas de Algas/genética , Proteínas de Algas/metabolismo , Proteínas de Bactérias/metabolismo , Chlamydomonas reinhardtii/metabolismo , Cloroplastos/metabolismo , Engenharia Genética/métodos , Marcadores Genéticos , Vetores Genéticos/química , Vetores Genéticos/metabolismo , NADH NADPH Oxirredutases/metabolismo , Fosfitos/farmacologia , Pseudomonas stutzeri/química , Pseudomonas stutzeri/genética , Seleção Genética , Transformação GenéticaRESUMO
The clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9 (CRISPR/Cas9) technology is a versatile and useful tool to perform genome editing in different organisms ranging from bacteria and yeast to plants and mammalian cells. For a couple of years, it was believed that the system was inefficient and toxic in the alga Chlamydomonas reinhardtii. However, recently the system has been successfully implemented in this model organism, albeit relying mostly on the electroporation of ribonucleoproteins (RNPs) into cell wall deficient strains. This requires a constant source of RNPs and limits the application of the technology to strains that are not necessarily the most relevant from a biotechnological point of view. Here, we show that transient expression of the Streptococcus pyogenes Cas9 gene and sgRNAs, targeted to the single-copy nuclear apt9 gene, encoding an adenine phosphoribosyl transferase (APT), results in efficient disruption at the expected locus. Introduction of indels to the apt9 locus results in cell insensitivity to the otherwise toxic compound 2-fluoroadenine (2-FA). We have used agitation with glass beads and particle bombardment to introduce the plasmids carrying the coding sequences for Cas9 and the sgRNAs in a cell-walled strain of C. reinhardtii (CC-125). Using sgRNAs targeting exons 1 and 3 of apt9, we obtained disruption efficiencies of 3 and 30% on preselected 2-FA resistant colonies, respectively. Our results show that transient expression of Cas9 and a sgRNA can be used for editing of the nuclear genome inexpensively and at high efficiency. Targeting of the APT gene could potentially be used as a pre-selection marker for multiplexed editing or disruption of genes of interest.
Assuntos
Adenina Fosforribosiltransferase/genética , Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas/genética , Chlamydomonas reinhardtii/genética , Genes Reporter/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Eletroporação/métodos , Edição de Genes/métodos , Plasmídeos/genética , RNA Guia de Cinetoplastídeos/genética , Ribonucleoproteínas/genéticaRESUMO
microRNAs are noncoding RNAs of 20-24 nucleotides (nt) in length that act as repressors of genes and are important in key developmental processes in the entire life cycle of plants. To determine the function of a microRNA, the first step is to resolve its expression pattern; this can be achieved by in situ hybridization, RNA blot assays, or quantitative PCR. However, the study of the expression of a MIR gene is straightforward with the use of reporter proteins such as ß-D-glucuronidase (GUS), GFP, or mCherry. To do this, it is necessary to clone the promoter region of the MIR gene and place it upstream of the reporter gene; in this way the activity of the promoter will be a direct reflection of the expression of the MIR gene. Here, we indicate step by step how to make transcriptional fusion constructs from the cloning of a promoter region of a MIR gene fused to the classical reporter proteins GUS and mCherry, the latter with codon optimization for better expression in Arabidopsis thaliana. This method is particularly useful to dissect the promoter region of a MIR gene and to find its expression pattern in a tissue and developmental specific manner.
Assuntos
Arabidopsis/genética , Regulação da Expressão Gênica de Plantas/genética , MicroRNAs/genética , RNA de Plantas/genética , Proteínas Recombinantes de Fusão/genética , Clonagem Molecular , Genes de Plantas/genética , Genes Reporter/genética , Glucuronidase/genética , Regiões Promotoras Genéticas/genética , Transcrição Gênica/genéticaRESUMO
KEY MESSAGE: Two intercistronic regions were identified as functional intercistronic expression elements (IEE) for the simultaneous expression of aphA-6 and gfp in a synthetic operon in the chloroplast of C. reinhardtii. Chlamydomonas reinhardtii, a biflagellate photosynthetic microalga, has been widely used in basic and applied science. Already three decades ago, Chlamydomonas had its chloroplast genome transformed and to this day constitutes the only alga routinely used in transplastomic technology. Despite the fact that over a 100 foreign genes have been expressed from the chloroplast genome, little has been done to address the challenge of expressing multiple genes in the form of operons, a development that is needed and crucial to push forward metabolic engineering and synthetic biology in this organism. Here, we studied five intercistronic regions and investigated if they can be used as intercistronic expression elements (IEE) in synthetic operons to drive the expression of foreign genes in the chloroplast of C. reinhardtii. The intercistronic regions were those from the psbB-psbT, psbN-psbH, psaC-petL, petL-trnN and tscA-chlN chloroplast operons, and the foreign genes were the aminoglycoside 3'-phosphotransferase (aphA-6), which confers resistance to kanamycin, and the green fluorescent protein gene (gfp). While all the intercistronic regions yielded lines that were resistant to kanamycin, only two (obtained with intercistronic regions from psbN-psbH and tscA-chlN) were identified as functional IEEs, yielding lines in which the second cistron (gfp) was translated and generated GFP. The IEEs we have identified could be useful for the stacking of genes for metabolic engineering or synthetic biology circuits in the chloroplast of C. reinhardtii.
Assuntos
Chlamydomonas reinhardtii/genética , Cloroplastos/metabolismo , DNA Intergênico/genética , Genes de Plantas/genética , Óperon/genética , Plantas Geneticamente Modificadas/genética , Cloroplastos/genética , Regulação da Expressão Gênica de Plantas/genética , Engenharia Genética/métodos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Canamicina Quinase/genética , Canamicina Quinase/metabolismo , Engenharia Metabólica/métodos , Plantas Geneticamente Modificadas/metabolismoRESUMO
Light-up aptamers are practical tools to image RNA localization in vivo. A now classical light-up aptamer system is the combination of the 3,5-difluoro-4-hydroxybenzylidene (DFHBI) fluorogen and the RNA aptamer Spinach, which has been successfully used in bacterial and mammalian cells. However, light-up aptamers have not been used in algae. Here, we show that a simple vector, carrying Spinach, transcriptionally fused to the aphA-6 gene, can be effectively used to generate a functional light-up aptamer in the chloroplast of Chlamydomonas reinhardtii. After incubation with DFHBI, lines expressing the aphA-6/Spinach mRNA were observed with laser confocal microscopy to evaluate the functionality of the light-up aptamer in the chloroplast of C. reinhardtii. Clear and strong fluorescence was localized to the chloroplast, in the form of discrete spots. There was no background fluorescence in the strain lacking Spinach. Light-up aptamers could be further engineered to image RNA or to develop genetically encoded biosensors in algae.
Assuntos
Aptâmeros de Nucleotídeos/genética , Chlamydomonas reinhardtii/genética , Cloroplastos/genética , Compostos de Benzil , Fluorescência , Corantes Fluorescentes , Imidazolinas , Canamicina Quinase/genética , RNA Mensageiro/genética , RNA de Plantas/genéticaRESUMO
AIM: The objective of this prospective study was to investigate outcomes of a lip repositioning technique for the treatment of excessive gingival display. MATERIALS AND METHODS: Thirteen consecutively treated patients with excessive gingival display were treated with a modified lip repositioning technique. Treatment consisted of the removal of two strips of mucosa, bilaterally to the maxillary labial frenum and coronal repositioning of the new mucosal margin. The clinical dimensions of gingival display, upper lip and vermillion length were measured at baseline, 3 and 6 months post-operatively. Subjects completed surveys to evaluate satisfaction with outcomes. RESULTS: The baseline gingival display of 5.8 ± 2.1 mm significantly decreased to 1.4 ± 1.0 mm at 3 months (p < 0.0001) and was maintained until 6 months (1.3 ± 1.6 mm). The reduction in gingival display strongly correlated to the combined change in upper lip and vermillion length (r(2) = 0.60, p = 0.0018). Subjects were satisfied with their smile after surgery and would likely choose to undergo the procedure again (92%). The worst part of undergoing the procedure was the discomfort or the inability to move the lip during the early healing (69%). CONCLUSION: Treatment of excessive gingival display by means of a modified lip repositioning technique results in high level of patient satisfaction and predictable outcomes that are stable in the short term.
Assuntos
Gengiva/patologia , Lábio/cirurgia , Sorriso , Adulto , Estética Dentária , Feminino , Seguimentos , Humanos , Freio Labial/cirurgia , Lábio/patologia , Masculino , Pessoa de Meia-Idade , Mucosa Bucal/cirurgia , Dor Pós-Operatória/etiologia , Satisfação do Paciente , Complicações Pós-Operatórias , Estudos Prospectivos , Resultado do Tratamento , Cicatrização/fisiologia , Adulto JovemRESUMO
Cervical carcinoma (CC) is one of the most common cancers among women worldwide and the first cause of death among the Mexican female population. Human papillomavirus (HPV) infection is the most important etiologic factor for CC. Of the oncogenic types, HPV16 and HPV18 are found in 60-70% of invasive CCs worldwide. HPV18 appears to be associated with a more aggressive form of cervical neoplasia than HPV16 infection. At present, there are no studies on differentially expressed cellular genes between transformed cells harboring HPV16 and HPV18 sequences. Based on previous complementary DNA microarray data from our group, 13 genes were found to be differentially overexpressed between HPV16- and HPV18-transformed cells. These genes were as follows: E6BP, UBE4A, C20orf14, ATF7, ABCC8, SLC6A12, WASF3, SUV39H1, SPAG8, CCNC, E2FFE, BIRC5, and DEDD. Differential expression of six selected genes was confirmed by real-time reverse transcription-polymerase chain reaction (RT-PCR). All real-time RT-PCRs confirmed differential expression between HPV18 and HPV(-) samples. The present work identifies genes from signaling pathways triggered by HPV transformation that could be differentially deregulated between HPV16(+) and HPV18(+) samples.