In the past few years, several researchers have focused their attention on nucleic acid mimics due to the increasing necessity of developing a more robust recognition of DNA or RNA sequences. ...Fluorescence in situ hybridization (FISH) is an example of a method where the use of these novel nucleic acid monomers might be crucial to the success of the analysis. To achieve the expected accuracy in detection, FISH probes should have high binding affinity towards their complementary strands and discriminate effectively the noncomplementary strands. In this study, we investigate the effect of different chemical modifications in fluorescent probes on their ability to successfully detect the complementary target and discriminate the mismatched base pairs by FISH. To our knowledge, this paper presents the first study where this analysis is performed with different types of FISH probes directly in biological targets, Helicobacter pylori and Helicobacter acinonychis. This is also the first study where unlocked nucleic acids (UNA) were used as chemistry modification in oligonucleotides for FISH methodologies. The effectiveness in detecting the specific target and in mismatch discrimination appears to be improved using locked nucleic acids (LNA)/2′-O-methyl RNA (2′OMe) or peptide nucleic acid (PNA) in comparison to LNA/DNA, LNA/UNA, or DNA probes. Further, the use of LNA modifications together with 2′OMe monomers allowed the use of shorter fluorescent probes and increased the range of hybridization temperatures at which FISH would work.
The thermodynamics and kinetics of DNA hybridization, i.e. the process of self-assembly of one, two or more complementary nucleic acid strands, has been studied for many years. The appearance of the ...nearest-neighbor model led to several theoretical and experimental papers on DNA thermodynamics that provide reasonably accurate thermodynamic information on nucleic acid duplexes and allow estimation of the melting temperature. Because there are no thermodynamic models specifically developed to predict the hybridization temperature of a probe used in a fluorescence in situ hybridization (FISH) procedure, the melting temperature is used as a reference, together with corrections for certain compounds that are used during FISH. However, the quantitative relation between melting and experimental FISH temperatures is poorly described. In this review, various models used to predict the melting temperature for rRNA targets, for DNA oligonucleotides and for nucleic acid mimics (chemically modified oligonucleotides), will be addressed in detail, together with a critical assessment of how this information should be used in FISH.
Tumor-associated lymph vessels and lymph node involvement are critical staging criteria in several cancers. In skin squamous cell carcinoma, lymph vessels play a role in cancer development and ...metastatic spread. However, their relationship with the cancer stem cell niche at early tumor stages remains unclear. To address this gap, we studied the lymph vessel localization at the cancer stem cell niche and observed an association from benign skin lesions to malignant stages of skin squamous cell carcinoma. By co-culturing lymphatic endothelial cells with cancer cell lines representing the initiation and promotion stages, and conducting RNA profiling, we observed a reciprocal induction of cell adhesion, immunity regulation, and vessel remodeling genes, suggesting dynamic interactions between lymphatic and cancer cells. Additionally, imaging analyses of the cultured cells revealed the establishment of heterotypic contacts between cancer cells and lymph endothelial cells, potentially contributing to the observed distribution and maintenance at the cancer stem cell niche, inducing downstream cellular responses. Our data provide evidence for an association of lymph vessels from the early stages of skin squamous cell carcinoma development, opening new avenues for better comprehending their involvement in cancer progression.
•Five image analysis methods to quantify fluorescence intensity were proposed.•FISHji3, FISHji4 and FISHji5 showed high accuracy.•Automatic FISHji methods allowed a fast and easy quantification of ...fluorescence.
Fluorescence in situ hybridization (FISH) is based on the use of fluorescent staining dyes, however, the signal intensity of the images obtained by microscopy is seldom quantified with accuracy by the researcher. The development of innovative digital image processing programs and tools has been trying to overcome this problem, however, the determination of fluorescent intensity in microscopy images still has issues due to the lack of precision in the results and the complexity of existing software.
This work presents FISHji, a set of new ImageJ methods for automated quantification of fluorescence in images obtained by epifluorescence microscopy. To validate the methods, results obtained by FISHji were compared with results obtained by flow cytometry. The mean correlation between FISHji and flow cytometry was high and significant, showing that the imaging methods are able to accurately assess the signal intensity of fluorescence images. FISHji are available for non-commercial use at http://paginas.fe.up.pt/∼nazevedo/.
Augmentation and reshaping of body volume, particularly in the gluteal area, presents a significant challenge in aesthetic surgery. Hyaluronic acid (HA) fillers have emerged as an effective and safe ...tool for such indications, but literature examining nonsurgical gluteal reshaping with HA remains limited. This study aims to evaluate the long-term safety of using recommended volumes of HA body fillers for nonsurgical gluteal augmentation.
A retrospective, observational study was carried out across multiple centers in Italy and the United Arab Emirates. The study involved participants between 22 and 53 years of age who underwent gluteal augmentation using HA body filler (HYAcorp MLF1/2) between 2017 and 2021, with up to 4 years and 7 months of follow-up. Participants and investigators independently evaluated the procedure's effectiveness by comparing pre- and posttreatment photographs. The Global Aesthetic Improvement Scale was used to assess posttreatment satisfaction by both participants and investigators. All adverse effects (AEs) were recorded.
The study included a diverse group of 91 participants. No serious adverse events were reported, with the majority of AE occurring shortly after treatment and resolving in 1 week. AEs were more frequently observed in participants with previous treatments using different substances in the treatment area.
The real-world application of HA body filler (HYAcorp MLF1/2) for gluteal augmentation in the participants of this study showed the treatment's effectiveness, with no severe adverse events reported among the participants. High levels of satisfaction were reported among both participants and investigators.
This short insight covers some of the recent topics relevant to the field of cadherin-catenin adhesion in mediating connections between different cell types, so-called heterotypic or heterocellular ...connections, in both homeostasis and cancer. These scientific discoveries are increasing our understanding of how multiple cells residing in complex tissues can be instructed by cadherin adhesion receptors to regulate tissue architecture and function and how these cadherin-mediated heterocellular connections spur tumor growth and the acquisition of malignant characteristics in tumor cells. Overall, the findings that have emerged over the past few years are elucidating the complexity of the functional roles of the cadherin-catenin complexes. Future exciting research lies ahead in order to understand the physical basis of these heterotypic interactions and their influence on the behavior of heterogeneous cellular populations as well as their roles in mediating phenotypic and genetic changes as cells evolve through complex environments during morphogenesis and cancer.
In this chapter, we describe a fluorescence in vivo hybridization (FIVH) protocol, using nucleic acid probes, for the detection of the bacterium Helicobacter pylori in the gastric mucosa of an ...infected C57BL/6 mouse model. This protocol should be easily extended to other microorganisms not only as a way to identify in vivo important microorganisms and their patterns of distribution within specific or at different anatomic sites, but also to better understand interaction mechanisms involving the microbiome and the human body.
Helicobacter pylori infects more than 50% of the worldwide population. It is mostly found deep in the gastric mucus lining of the stomach, being a major cause of peptic ulcers and gastric ...adenocarcinoma. To face the increasing resistance of H. pylori to antibiotics, antimicrobial nucleic acid mimics are a promising alternative. In particular, locked nucleic acids (LNA)/2’-OMethyl RNA (2'OMe) have shown to specifically target H. pylori, as evidenced by in situ hybridization. The success of in vivo hybridization depends on the ability of these nucleic acids to penetrate the major physical barriers—the highly viscoelastic gastric mucus and the bacterial cell envelope. We found that LNA/2'OMe is capable of diffusing rapidly through native, undiluted, gastric mucus isolated from porcine stomachs, without degradation. Moreover, although LNA/2'OMe hybridization was still successful without permeabilization and fixation of the bacteria, which is normally part of in vitro studies, the ability of LNA/2'OMe to efficiently hybridize with H. pylori was hampered by the presence of mucus. Future research should focus on developing nanocarriers that shield LNA/2'OMe from components in the gastric mucus, while remaining capable of diffusing through the mucus and delivering these nucleic acid mimics directly into the bacteria.
Different cancer stem cell (CSC) populations can be found in many types of cancer, including squamous cell carcinoma (SSC). Diverse reports showed that CSC play a crucial role in the relapse of ...different types of cancer. CSC sustains tumor growth due to their capacity to self-renew and their potential to initiate secondary tumors with metastatic cancer features. Therefore, the development of methods for the isolation of CSC is a key step to explore the mechanisms underlying CSC maintenance. In this chapter, we provide a method for isolating CSC from cutaneous SSC using immunofluorescence labeling to allow the specific purification of CSC by fluorescence-activated cell sorting (FACS). This method is based on the use of CSC membrane markers, allowing as well the isolation CSC from different mouse strains.
The understanding of the human microbiome and its influence upon human life has long been a subject of study. Hence, methods that allow the direct detection and visualization of microorganisms and ...microbial consortia (e.g. biofilms) within the human body would be invaluable. In here, we assessed the possibility of developing a variant of fluorescence in situ hybridization (FISH), named fluorescence in vivo hybridization (FIVH), for the detection of Helicobacter pylori. Using oligonucleotide variations comprising locked nucleic acids (LNA) and 2'-O-methyl RNAs (2'OMe) with two types of backbone linkages (phosphate or phosphorothioate), we were able to successfully identify two probes that hybridize at 37 °C with high specificity and sensitivity for H. pylori, both in pure cultures and in gastric biopsies. Furthermore, the use of this type of probes implied that toxic compounds typically used in FISH were either found to be unnecessary or could be replaced by a non-toxic substitute. We show here for the first time that the use of advanced LNA probes in FIVH conditions provides an accurate, simple and fast method for H. pylori detection and location, which could be used in the future for potential in vivo applications either for this microorganism or for others.
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