RESUMEN
Archaeological pottery are the most numerous objects found during excavations and reflect the culinary practices of the past. However, their functionality for cooking/storing specific foods or drinks cannot be deduced solely from comparing their shapes and sizes. Analysis of protein residues bound to ceramics can reveal the protein/animal type through their amino acid sequence, thus enabling direct identification of food types. Therefore, the aim of our experimental study was to test sixteen aptamers for the analysis of proteinaceous organic residues found within the porous structure of pottery. Traditionally prepared archaeological ceramic replicas were cooked for 5 days in various food/protein suspensions, were UV aged, buried for a year, excavated, and extensively cleaned. Their shards were analysed using immunofluorescence microscopy with aptamers. Results show that eight aptamers (Clone1 and Kirby for egg residuals; seqU5 and BLG14 for milk residuals; HA for blood residuals; Gli4 for gluten residuals; Par1 for fish residuals; and D1 for collagen residuals) produced a successful/specific immunofluorescence microscopy result when they were hybridised to shards containing target protein residuals. Interestingly, on whole egg control samples, when the egg lysozyme-targeting aptamer Kirby was used, fluorescence intensity was 3.1 times greater compared to that observed with anti-ovalbumin antibodies.
Asunto(s)
Aptámeros de Nucleótidos , Cerámica , Cerámica/química , Aptámeros de Nucleótidos/química , Animales , Arqueología , Proteínas/química , Proteínas/análisis , Microscopía FluorescenteRESUMEN
As the most abundant leukocyte in circulation, the neutrophil plays a far-reaching role in maintaining homeostasis. Within the context of disease, however, neutrophils can potentiate various pathophysiological mechanisms with disastrous consequences for patients. The role of the neutrophil in disease is complex with mechanisms like NETosis driving the progression of several pathologies. NETosis involves neutrophils extruding protein-decorated DNA webs called neutrophil extracellular traps (NETs), which facilitate the progression of inflammatory, non-infectious, and neoplastic pathologies. The need to visualize NETs has thus never been greater. Current approaches for visualizing NETs are limited in specificity and sensitivity, involving non-specific fluorescent DNA dyes or co-stains of neutrophil and DNA markers. Improved methodologies are needed to robustly distinguish NETs from other cell-free DNA. Excitingly, a novel NET-specific posttranslational modification involving cleavage on the N-terminus of histone H3 has recently been identified. Here, we demonstrate that this single marker is superior to the conventional use of the co-stain of the neutrophil marker, myeloperoxidase, and, the DNA marker, histone H3 citrullination in visualizing neutrophil NETosis. This is due to this single marker's unparalleled ability to identify, not only more NETs but also their formation at earlier stages of NETosis. Moreover, we additionally propose a stepwise mechanism of neutrophil NETosis in which a histone H3 cleavage event precedes histone H3 citrullination. Taken together, these results demonstrate a novel method for visualizing NETs, allowing for continued exploration of their multifaceted roles in immunity and disease.
Asunto(s)
Trampas Extracelulares , Humanos , Trampas Extracelulares/metabolismo , Histonas/metabolismo , Neutrófilos/metabolismo , Citrulinación , ADN/metabolismoRESUMEN
In legumes, the seed storage proteins accumulate within specialized organelles called protein storage vacuoles (PSVs). In several plant species, PSVs are differentiated into subdomains that accumulate different kinds of proteins. Even though the existence of subdomains is common in cereals and legumes, it has not been reported in soybean PSVs. The two most abundant seed proteins of soybean, 7S and 11S globulins, have different temporal accumulation patterns and exhibit considerable solubility differences that could result in differential accretion of these proteins within the PSVs. Here, we employed confocal fluorescent microscopy to examine the presence or absence of subdomains within the soybean PSVs. Eosin-stained sections of FAA-fixed paraffin embedded soybean seeds, when viewed by confocal fluorescence microscopy, revealed the presence of intricate subdomains within the PSVs. However, fluorescence immunolabeling studies demonstrated that the 7S and 11S globulins were evenly distributed within the PSVs and failed to corroborate the existence of subdomains within the PSVs. Similarly, confocal scanning microscopy examination of free-hand, vibratome and cryostat sections also failed to demonstrate the existence of subdomains within PSVs. The subdomains, which were prominently seen in PSVs of FAA-fixed soybean seeds, were not observed when the seeds were fixed either in glutaraldehyde/paraformaldehyde or glutaraldehyde. Our studies demonstrate that the apparent subdomains observed in FAA-fixed seeds may be a fixation artifact.
Asunto(s)
Globulinas , Glycine max , Antígenos de Plantas/metabolismo , Cotiledón/metabolismo , Globulinas/metabolismo , Glutaral/metabolismo , Microscopía Confocal , Microscopía Fluorescente , Proteínas de Almacenamiento de Semillas/metabolismo , Semillas/metabolismo , Proteínas de Soja/metabolismo , Glycine max/metabolismo , Vacuolas/metabolismoRESUMEN
Neuroendocrine neoplasms (NENs) are rare neoplasms with heterogeneous clinical behavior. Alteration in human microbiota was reported in association with carcinogenesis in different solid tumors. However, few studies addressed the role of microbiota in NEN. We here aimed at evaluating the presence of bacterial infiltration in neuroendocrine tumoral tissue. To assess the presence of bacteria, 20 specimens from pancreatic NEN (pan-NEN) and 20 from intestinal NEN (I-NEN) were evaluated through Fluorescent In situ Hybridization and confocal microscopy. Demographic data, pre-operative investigations, operative findings, pathological diagnosis, follow-up, and survival data were evaluated. Among I-NEN, bacteria were detected in 15/20 (75%) specimens, with high variability in microbial distribution. In eight patients, a high infiltration of microorganisms was observed. Among pan-NEN, 18/20 (90%) showed microorganisms' infiltration, with a homogeneous microbial distribution. Bacterial localization in pan-NEN was observed in the proximity of blood vessels. A higher bacterial infiltration in the tumoral specimen as compared with non-tumoral tissue was reported in 10/20 pan-NEN (50%). No significant differences were observed in mean bacterial count according to age, sex, ki67%, site, tumor stage. Mean bacterial count did not result to be a predictor of disease-specific survival. This preliminary study demonstrates the presence of a significant microbiota in the NEN microenvironment. Further research is needed to investigate the potential etiological or clinical role of microbiota in NEN.
Asunto(s)
Microbiota , Tumores Neuroendocrinos , Humanos , Hibridación Fluorescente in Situ , Tumores Neuroendocrinos/patología , Proyectos Piloto , Microambiente TumoralRESUMEN
Significant increases in male infertility and the still unresolved questions on the compatibility and interpretation of current methods in infertility diagnostics call for new protocols. Morphology, genome damage, RNA content and quantity are currently in practice as the major parameters in evaluation of sperm quality. However, results of various methods are not always in mutual concordance. In this study, in vivo acridine orange (AO) staining, which is presently in application in the estimation of genome damage in reticulocytes, was adjusted for spermatozoa staining. Ten men suffering from oligoasthenoteratozoospermia (OAT) and 10 healthy fertile men were analysed using in vivo AO staining. Microscopic analysis was performed by fluorescent and confocal fluorescent microscopy. Our results show that this method preserves spermatozoa membranes, which enables new insight into spermatozoa genome damage, RNA content in residual cytoplasm, damage of neck area with mitochondrion and tail pathology. The introduced method explains the difference between results of sperm DNA fragmentation assay and the globally used AO staining and opens new options for the development of automated systems. In conclusion, the results of our study offer (a) an innovative approach to the analysis of spermatozoa pathology, (b) enable localization and quantification of RNA in residual cytoplasm, (c) a significant contribution to research of aetiology of infertility in men, (d) open new perspectives for the automatization of sperm quality estimation and (e) improve the personalized approach in the selection of in vitro fertilization protocols.