Specific berenil–DNA interactions: an approach for separation of plasmid isoforms by pseudo–affinity chromatography

Small molecules, like some antibiotics and anticancer agents that bind DNA with high specificity can represent a relevant alternative as ligands in affinity processes for plasmid DNA (pDNA) purification. In the present study, pDNA binding affinities of berberine, berenil, kanamycin and neomycin were evaluated by a competitive displacement assay with ethidium bromide using a fluorimetric titration technique. The binding between pDNA and ethidium bromide was tested in different buffer conditions varying the type and the salt concentration, and was performed both in absence and in presence of the studied compounds. The results showed that the minor groove binder berenil has the higher pDNA binding constant. Chromatographic experiments using a derivatized column with berenil as ligand, showed a total retention of pDNA using 1.3 M ammonium sulphate in eluent buffer. A selective separation of supercoiled and open circular isoforms was achieved by further decreasing salt concentration to 0.6 M and then to 0 M. These results suggest a promising application of berenil as ligand for specific pDNA supercoiled (sc) isoform purification by pseudo-affinity chromatography.C. Caramelo-Nunes acknowledges a fellowship (SFRH/BD/64918/2009) from the Portuguese Foundation for Science and Technology (FCT)

Digital pathology workflow implementation at ipatimup

The advantages of the digital methodology are well known. In this paper, we provide a detailed description of the process for the digital transformation of the pathology laboratory at IPATIMUP, the major modifications that operate throughout the processing pipeline, and the advantages of its implementation. The model of digital workflow implementation at IPATIMUP demonstrates that careful planning and adoption of simple measures related to time, space, and sample management can be adopted by any pathology laboratory to achieve higher quality and easy digital transformation

A peripheral blood mononuclear cell-based in vitro model: A tool to explore indoleamine 2, 3-dioxygenase-1 (IDO1)

Background: Proinflammatory cytokines powerfully induce the rate-limiting enzyme indoleamine 2, 3-dioxygenase-1 (IDO-1) in dendritic cells (DCs) and monocytes, it converts tryptophan (Trp) into L-kynurenine (KYN), along the kynurenine pathway (KP). This mechanism represents a crucial innate immunity regulator that can modulate T cells. This work explores the role of IDO1 in lymphocyte proliferation within a specific proinflammatory milieu. Methods: Peripheral blood mononuclera cells (PBMCs) were isolated from buffy coats taken from healthy blood donors and exposed to a pro-inflammatory milieu triggered by a double-hit stimulus: lipopolysaccharide (LPS) plus anti-CD3/CD28. The IDO1 mRNA levels in the PBMCs were measured by RT-PCR; the IDO1 activity was analyzed using the KYN/Trp ratio, measured by HPLC-EC; and lymphocyte proliferation was measured by flow cytometry. Trp and epacadostat (EP) were used as an IDO1 substrate and inhibitor, respectively. KYN, which is known to modulate Teffs, was tested as a positive control in lymphocyte proliferation. Results: IDO1 expression and activity in PBMCs increased in an in vitro pro-inflammatory milieu. The lymphoid stimulus increased IDO1 expression and activity, which supports the interaction between the activated lymphocytes and the circulating myeloid IDO1-expressing cells. The addition of Trp decreased lymphocyte proliferation but EP, which abrogated the IDO1 function, had no impact on proliferation. Additionally, incubation with KYN seemed to decrease the lymphocyte proliferation. Conclusion: IDO1 inhibition did not change T lymphocyte proliferation. We present herein an in vitro experimental model suitable to measure IDO1 expression and activity in circulating myeloid cells

Selective ablating urologic cancers with cold atmospheric plasma

info:eu-repo/semantics/publishedVersio

Adenosine Deaminase Two and Immunoglobulin M Accurately Differentiate Adult Sneddon's Syndrome of Unknown Cause

BACKGROUND: The association that exists between livedo reticularis (LR) and stroke is known as Sneddon's syndrome (SnS). The disorder is classified as primary SnS (PSnS), if the cause remains unknown and secondary SnS. The condition is rare and it occurs mainly sporadically. In 2014, 2 independent teams described a new genetic disorder with childhood-onset, which was called deficiency of adenosine deaminase 2 (DADA2), characterized by recurrent fevers and vascular pathologic features that included LR and stroke. All the patients carried recessively inherited mutations in cat eye syndrome chromosome region candidate 1 gene (CECR1), encoding the adenosine deaminase 2 (ADA2) protein. Genetic testing is the standard for the diagnosis of DADA2. However, the diagnostic accuracy of more affordable laboratorial analysis in CECR1-mutated individuals remains to be established. We aim to determine whether plasma ADA2 activity and serum immunoglobulin M (IgM) levels can distinguish (1) DADA2 from other adult patients within the SnS spectrum, and (2) healthy CECR1 heterozygous (HHZ) from healthy controls (HC). METHODS: ADA2 activity in plasma and serum IgM concentrations was measured in adult patients within the SnS spectrum, healthy first-degree relatives and HC. Genetic results were used as the reference standard. The primary outcome measures were sensitivity and specificity derived from receiver operating curve analysis. RESULTS: A total of 73 participants were included in the study: 26 patients with PSnS with no CECR1 mutation (PSnS), 6 bi-allelic (DADA2 patients) and 7 HHZ CECR1 mutations and 34 HC. Plasma ADA2 activity and serum IgM levels were significantly lower in DADA2 patients than in PSnS. With the use of the best indexes, plasma ADA2 activity differentiated PSnS from DADA2 with a sensitivity and specificity of 100.0% and HHZ from HC with a sensitivity of 97.1% and specificity of 85.7%. Serum IgM levels also differentiated PSnS from DADA2 with a sensitivity of 85.2% and specificity of 83.3%. CONCLUSION: Serum IgM levels might be used as a triage tool and plasma ADA2 activity performs perfectly as a diagnostic test for DADA2 in adult patients within the SnS spectrum. ADA2 activity in plasma also reliably distinguishes HHZ from HC.info:eu-repo/semantics/publishedVersio

The role of UDP-Glc:glycoprotein glucosyltransferase 1 in the maturation of an obligate substrate prosaposin

A natural substrate for UGT1 is confirmed, revealing how the enzyme functions in the calnexin chaperone system as a quality control step in protein folding

Influence of Aldehyde Dehydrogenase Inhibition on Stemness of Endometrial Cancer Stem Cells

Publisher Copyright: © 2024 by the authors.Endometrial cancer is one of the most common gynaecological malignancies. Although often diagnosed at an early stage, there is a subset of patients with recurrent and metastatic disease for whom current treatments are not effective. Cancer stem cells (CSCs) play a pivotal role in triggering tumorigenesis, disease progression, recurrence, and metastasis, as high aldehyde dehydrogenase (ALDH) activity is associated with invasiveness and chemotherapy resistance. Therefore, this study aimed to evaluate the effects of ALDH inhibition in endometrial CSCs. ECC-1 and RL95-2 cells were submitted to a sphere-forming protocol to obtain endometrial CSCs. ALDH inhibition was evaluated through ALDH activity and expression, sphere-forming capacity, self-renewal, projection area, and CD133, CD44, CD24, and P53 expression. A mass spectrometry-based proteomic study was performed to determine the proteomic profile of endometrial cancer cells upon N,N-diethylaminobenzaldehyde (DEAB). DEAB reduced ALDH activity and expression, along with a significant decrease in sphere-forming capacity and projection area, with increased CD133 expression. Additionally, DEAB modulated P53 expression. Endometrial cancer cells display a distinct proteomic profile upon DEAB, sharing 75 up-regulated and 30 down-regulated proteins. In conclusion, DEAB inhibits ALDH activity and expression, influencing endometrial CSC phenotype. Furthermore, ALDH18A1, SdhA, and UBAP2L should be explored as novel molecular targets for endometrial cancer.publishersversionpublishe

Cold atmospheric plasma, a novel approach against bladder cancer, with higher sensitivity for the high-grade cell line

Antitumor therapies based on Cold Atmospheric Plasma (CAP) are an emerging medical field. In this work, we evaluated CAP effects on bladder cancer. Two bladder cancer cell lines were used, HT-1376 (stage III) and TCCSUP (stage IV). Cell proliferation assays were performed evaluating metabolic activity (MTT assay) and protein content (SRB assay). Cell viability, cell cycle, and mitochondrial membrane potential (Δψm) were assessed using flow cytometry. Reactive oxygen and nitrogen species (RONS) and reduced glutathione (GSH) were evaluated by fluorescence. The assays were carried out with different CAP exposure times. For both cell lines, we obtained a significant reduction in metabolic activity and protein content. There was a decrease in cell viability, as well as a cell cycle arrest in S phase. The Δψm was significantly reduced. There was an increase in superoxide and nitric oxide and a decrease in peroxide contents, while GSH content did not change. These results were dependent on the exposure time, with small differences for both cell lines, but overall, they were more pronounced in the TCCSUP cell line. CAP showed to have a promising antitumor effect on bladder cancer, with higher sensitivity for the high-grade cell line.info:eu-repo/semantics/publishedVersio

Copy number variants prioritization after array-CGH analysis - a cohort of 1000 patients

Array-based comparative genomic hybridization has been assumed to be the first genetic test offered to detect genomic imbalances in patients with unexplained intellectual disability with or without dysmorphisms, multiple congenital anomalies, learning difficulties and autism spectrum disorders. Our study contributes to the genotype/phenotype correlation with the delineation of laboratory criteria which help to classify the different copy number variants (CNVs) detected. We clustered our findings into five classes ranging from an imbalance detected in a microdeletion/duplication syndrome region (class I) to imbalances that had previously been reported in normal subjects in the Database of Genomic Variants (DGV) and thus considered common variants (class IV).info:eu-repo/semantics/publishedVersio