Detection of Fusobacterium spp in colorectal tissue samples using reverse transcription polymerase chain reaction with minor groove binder probes: an exploratory research

An unhealthy microbiome is intimately correlated with several disease states, including colorectal cancer, wherein bacteria might be the key to neoplastic initiation and progression. Recent studies revealed an enrichment of Fusobacterium in colorectal tumor tissues relative to surrounding normal mucosa. Given the available evidence, we conducted an exploratory study quantifying the relative expression of Fusobacterium spp in 28 tissue samples from patients treated at Centro Hospitalar de São João belonging to 4 different groups: adenomas, paired normal tissue from patients with adenomas, carcinomas, and paired normal tissue from patients with colorectal carcinomas. To increase reverse transcription polymerase chain reaction quantification sensitivity, minor groove binders fluorescent probes were used, having in mind its implementation into routine clinical practice. Differences of Fusobacterium spp relative abundance between paired neoplastic lesions/normal tissue were examined by Wilcoxon signed-rank test and for all the other 2-group comparisons the Mann-Whitney U test was used. Most of the adenomas studied belonged to clinical specimens showing either tubular or villous low-grade dysplasia and an enrichment of Fusobacterium relative to paired normal tissue was not found (P = .180). In the carcinoma group, 57% of samples displayed a positive status for this bacterium with the highest burden of detectable Fusobacterium belonging to a specimen with positive regional lymph node metastasis. This is the first Portuguese study confirming a trend toward an overabundance of Fusobacterium in colorectal carcinomas compared to adenomas and paired samples of normal-looking mucosa, in keeping with the role of this bacterium in colorectal carcinogenesis. Further studies are needed to elucidate the relevance of Fusobacterium detection for the prevention and treatment of colorectal cancer

Cell-Free DNA (cfDNA) Regulates Metabolic Remodeling in the ES-2 Ovarian Carcinoma Cell Line, Influencing Cell Proliferation, Quiescence, and Chemoresistance in a Cell-of-Origin-Specific Manner

Funding Information: This work benefited from access to CERMAX, ITQB-NOVA, Oeiras, Portugal, with equipment funded by FCT, project AAC 01/SAICT/2016. Funding Information: The institutions are funded by Funda\u00E7\u00E3o para a Ci\u00EAncia e a Tecnologia/Minist\u00E9rio da Ci\u00EAncia, Tecnologia e Ensino Superior (FCT/MCTES, Portugal) through national funds to iNOVA4Health (UIDB/04462/2020 and UIDP/04462/2020), to MOSTMICRO-ITQB (UIDB/04612/2020 and UIDP/04612/2020) and the Associated Laboratory LS4FUTURE (LA/P/0087/2020). No Self s.r.l. grant funded the NOVA Medical School and Instituto Portugu\u00EAs de Oncologia de Lisboa, Francisco Gentil, to support the development of Isabel Lemos PhD project. Isabel Lemos was funded by an FCT individual Ph.D. fellowship (UI/BD/154203/2022). Catarina Freitas-Dias was funded by the No Self project. Ana Hip\u00F3lito was funded by a FCT individual Ph.D. fellowship (SFRH/BD/148441/2019). Luis G. Gon\u00E7alves was financed by a FCT contract according to DL57/2016, (SFRH/BPD/111100/2015). This work benefited from access to CERMAX, ITQB-NOVA, Oeiras, Portugal with equipment funded by FCT, project AAC 01/SAICT/2016. Publisher Copyright: © 2025 by the authors.Background: The cell-free DNA (cfDNA) is an extracellular fragmented DNA found in body fluids in physiological and pathophysiological contexts. In cancer, cfDNA has been pointed out as a marker for disease diagnosis, staging, and prognosis; however, little is known about its biological role. Methods: The role of cfDNA released by ES-2 ovarian cancer cells was investigated, along with the impact of glucose bioavailability and culture duration in the cfDNA-induced phenotype. The effect of cfDNA on ES-2 cell proliferation was evaluated by proliferation curves, and cell migration was assessed through wound healing. We explored the impact of different cfDNA variants on ES-2 cells’ metabolic profile using nuclear magnetic resonance (NMR) spectroscopy and cisplatin resistance through flow cytometry. Moreover, we assessed the protein levels of DNA-sensitive Toll-like receptor 9 (TLR9) by immunofluorescence and its colocalization with lysosome-associated membrane protein 1 (LAMP1). Results: This study demonstrated that despite inducing similar effects, different variants of cfDNA promote different effects on cells derived from the ES-2 cell line. We observed instant reactions of adopting the metabolic profile that brings back the cell functioning of more favorable culture conditions supporting proliferation and resembling the cell of origin of the cfDNA variant, as observed in unselected ES-2 cells. However, as a long-term selective factor, certain cfDNA variants induced quiescence that favors the chemoresistance of a subset of cancer cells. Conclusions: Therefore, different tumoral microenvironments may generate cfDNA variants that will impact cancer cells differently, orchestrating the disease fate.publishersversionpublishe

Cell-Free DNA (cfDNA) Regulates Metabolic Remodeling, Sustaining Proliferation, Quiescence, and Migration in MDA-MB-231, a Triple-Negative Breast Carcinoma (TNBC) Cell Line

Funding Information: This work benefited from access to CERMAX, ITQB-NOVA, Oeiras, Portugal, with equipment funded by FCT, project AAC 01/SAICT/2016. Funding Information: The institutions are funded by Funda\u00E7\u00E3o para a Ci\u00EAncia e a Tecnologia/Minist\u00E9rio da Ci\u00EAncia, Tecnologia e Ensino Superior (FCT/MCTES, Portugal) through national funds to iNOVA4Health (UIDB/04462/2020 and UIDP/04462/2020), MOSTMICRO-ITQB (UIDB/04612/2020 and UIDP/04612/2020), and the Associated Laboratory LS4FUTURE (LA/P/0087/2020). No Self s.r.l. grant funded the NOVA Medical School and Instituto Portugu\u00EAs de Oncologia de Lisboa, Francisco Gentil, to support the development of Isabel Lemos\u2019s PhD project. Isabel Lemos was funded by an FCT individual Ph.D. fellowship (UI/BD/154203/2022). Catarina Freitas-Dias was funded by the NoSelf project. Ana Hip\u00F3lito was funded by an FCT individual Ph.D. fellowship (SFRH/BD/148441/2019). Luis G. Gon\u00E7alves was financed by an FCT contract according to DL57/2016 [SFRH/BPD/111100/2015]. Publisher Copyright: © 2025 by the authors.Background: The clinical relevance of circulating cell-free DNA (cfDNA) in oncology has gained significant attention, with its potential as a biomarker for cancer diagnosis and monitoring. However, its precise role in cancer biology and progression remains unclear. cfDNA in cancer patients’ blood has been shown to activate signaling pathways, such as those mediated by toll-like receptors (TLRs), suggesting its involvement in cancer cell adaptation to the tumor microenvironment. Methods: This impact of cfDNA released from MDA-MB-231, a triple-negative breast cancer (TNBC) cell line was assessed, focusing on glucose availability and culture duration. The impact of cfDNA on the proliferation of MDA-MB-231 cells was investigated using proliferation curves, while cellular migration was evaluated through wound healing assays. The metabolic alterations induced by distinct cfDNA variants in MDA-MB-231 cells were investigated through nuclear magnetic resonance (NMR) spectroscopy, and their effect on cisplatin resistance was evaluated using flow cytometry. Furthermore, the expression levels of DNA-sensitive Toll-like receptor 9 (TLR9) were quantified via immunofluorescence, alongside its colocalization with lysosome-associated membrane protein 1 (LAMP1). Results: This study indicates that cfDNA facilitates metabolic adaptation, particularly under metabolic stress, by modulating glucose and glutamine consumption, key pathways in tumor cell metabolism. Exposure to cfDNA induced distinct metabolic shifts, favoring energy production through oxidative phosphorylation. The anti-cancer activity of cfDNA isolated from conditioned media of cells cultured under stressful conditions is influenced by the culture duration, emphasizing the importance of adaptation and se-lection in releasing cfDNA that can drive pro-tumoral processes. Additionally, cfDNA exposure influenced cell proliferation, quiescence, and migration, processes linked to metastasis and treatment resistance. These findings underscore cfDNA as a key mediator of metabolic reprogramming and adaptive responses in cancer cells, contributing to tumor progression and therapy resistance. Furthermore, the activation of TLR9 signaling suggests a mechanistic basis for cfDNA-induced phenotypic changes. Conclusions: Overall, cfDNA serves as a crucial signaling molecule in the tumor microenvironment, orchestrating adaptive processes that enhance cancer cell survival and progression.publishersversionpublishe

Rab27a and Rab27b control different steps of the exosome secretion pathway

Exosomes are secreted membrane vesicles that share structural and biochemical characteristics with intraluminal vesicles of multivesicular endosomes (MVEs). Exosomes could be involved in intercellular communication and in the pathogenesis of infectious and degenerative diseases. The molecular mechanisms of exosome biogenesis and secretion are, however, poorly understood. Using an RNA interference (RNAi) screen, we identified five Rab GTPases that promote exosome secretion in HeLa cells. Among these, Rab27a and Rab27b were found to function in MVE docking at the plasma membrane. The size of MVEs was strongly increased by Rab27a silencing, whereas MVEs were redistributed towards the perinuclear region upon Rab27b silencing. Thus, the two Rab27 isoforms have different roles in the exosomal pathway. In addition, silencing two known Rab27 effectors, Slp4 (also known as SYTL4, synaptotagmin-like 4) and Slac2b (also known as EXPH5, exophilin 5), inhibited exosome secretion and phenocopied silencing of Rab27a and Rab27b, respectively. Our results therefore strengthen the link between MVEs and exosomes, and introduce ways of manipulating exosome secretion in vivo

Does urbanization explain differences in interactions between an insect herbivore and its natural enemies and mutualists?

Urbanization can alter the composition of arthropod communities. However, little is known about how urbanization affects ecological interactions. Using experimental colonies of the black bean aphid Aphis fabae Scopoli reared on Vicia faba L, we asked if patterns of predator-prey, host-parasitoid and ant-aphid mutualisms varied along an urbanization gradient across a large town in southern England. We recorded the presence of naturally occurring predators, parasitoid wasps and mutualistic ants together with aphid abundance. We examined how biotic (green areas and plant richness) and abiotic features (impervious surfaces and distance to town center) affected (1) aphid colony size, (2) the likelihood of finding predators, mutualistic ants and aphid mummies (indicating the presence of parasitoids), and (3) how the interplay among these factors affected patterns of parasitoid attack, predator abundance, mutualistic interactions and aphid abundance. The best model to predict aphid abundance was the number of mutualistic ants attending the colonies. Aphid predators responded negatively to both the proportion of impervious surfaces and to the number of mutualistic ants farming the colonies, and positively to aphid population size, whereas parasitized aphids were found in colonies with higher numbers of aphids and ants. The number of mutualistic ants attending was positively associated with aphid colony size and negatively with the number of aphid predators. Our findings suggest that for insect-natural enemy interactions, urbanization may affect some groups, while not influencing others, and that local effects (mutualists, host plant presence) will also be key determinants of how urban ecological communities are formed

Cholesteryl Hemiazelate Present in Cardiovascular Disease Patients Causes Lysosome Dysfunction in Murine Fibroblasts

Funding Information: This work was financially supported by the FCT (Foundation for Science and Technology of the Portuguese Ministry of Science and Higher Education), Refs. 2022.01305.PTDC and 2022.03249.PTDC. The Coimbra Chemistry Centre (CQC) is supported by the FCT through projects UIDB/00313/2020 and UIDP/00313/2020. E.L. is the holder of a PhD fellowship from the FCT (2021.06265.BD). A.R.A.M. was funded by the FCT Stimulus of Scientific Employment Individual Support Call 2017 (CEECIND/01006/2017). R.P. was funded by the NHLBI Division of Intramural Research (ZIA HL006151-10). Publisher Copyright: © 2023 by the authors.There is growing evidence supporting the role of fibroblasts in all stages of atherosclerosis, from the initial phase to fibrous cap and plaque formation. In the arterial wall, as with macrophages and vascular smooth muscle cells, fibroblasts are exposed to a myriad of LDL lipids, including the lipid species formed during the oxidation of their polyunsaturated fatty acids of cholesteryl esters (PUFA-CEs). Recently, our group identified the final oxidation products of the PUFA-CEs, cholesteryl hemiesters (ChE), in tissues from cardiovascular disease patients. Cholesteryl hemiazelate (ChA), the most prevalent lipid of this family, is sufficient to impact lysosome function in macrophages and vascular smooth muscle cells, with consequences for their homeostasis. Here, we show that the lysosomal compartment of ChA-treated fibroblasts also becomes dysfunctional. Indeed, fibroblasts exposed to ChA exhibited a perinuclear accumulation of enlarged lysosomes full of neutral lipids. However, this outcome did not trigger de novo lysosome biogenesis, and only the lysosomal transcription factor E3 (TFE3) was slightly transcriptionally upregulated. As a consequence, autophagy was inhibited, probably via mTORC1 activation, culminating in fibroblasts’ apoptosis. Our findings suggest that the impairment of lysosome function and autophagy and the induction of apoptosis in fibroblasts may represent an additional mechanism by which ChA can contribute to the progression of atherosclerosis.publishersversionpublishe

Coarctation of the aorta and mild to moderate developmental delay in a child with a de novo deletion of chromosome 15(q21.1q22.2)

BACKGROUND: Deletion of 15q21q22 is a rare chromosomal anomaly. To date, there have been nine reports describing ten individuals with different segmental losses involving 15q21 and 15q22. Many of these individuals have common features of growth retardation, hypotonia and moderate to severe mental retardation. Congenital heart disease has been described in three individuals with interstitial deletion involving this region of chromosome 15. CASE PRESENTATION: We report a child with coarctation of the aorta, partial agenesis of corpus callosum and mild to moderate developmental delay, with a de novo deletion of 15q21.1q22.2, detected by the array Comparative Genomic Hybridization (CGH). We utilized chromosome 15-specific microarray-based CGH to define the chromosomal breakpoints in this patient. CONCLUSION: This is the first description of mapping of an interstitial deletion involving the chromosome 15q21q22 segment using the chromosome 15-specific array-CGH. The report also expands the spectrum of clinical phenotype associated with 15q21q22 deletion

A Cysteine Protease Is Critical for Babesia spp. Transmission in Haemaphysalis Ticks

Vector ticks possess a unique system that enables them to digest large amounts of host blood and to transmit various animal and human pathogens, suggesting the existence of evolutionally acquired proteolytic mechanisms. We report here the molecular and reverse genetic characterization of a multifunctional cysteine protease, longipain, from the babesial parasite vector tick Haemaphysalis longicornis. Longipain shares structural similarity with papain-family cysteine proteases obtained from invertebrates and vertebrates. Endogenous longipain was mainly expressed in the midgut epithelium and was specifically localized at lysosomal vacuoles and possibly released into the lumen. Its expression was up-regulated by host blood feeding. Enzymatic functional assays using in vitro and in vivo substrates revealed that longipain hydrolysis occurs over a broad range of pH and temperature. Haemoparasiticidal assays showed that longipain dose-dependently killed tick-borne Babesia parasites, and its babesiacidal effect occurred via specific adherence to the parasite membranes. Disruption of endogenous longipain by RNA interference revealed that longipain is involved in the digestion of the host blood meal. In addition, the knockdown ticks contained an increased number of parasites, suggesting that longipain exerts a killing effect against the midgut-stage Babesia parasites in ticks. Our results suggest that longipain is essential for tick survival, and may have a role in controlling the transmission of tick-transmittable Babesia parasites

Fatores associados à pneumonia em crianças Yanomami internadas por condições sensíveis à atenção primária na região norte do Brasil

Resumo Em países em desenvolvimento, a pneumonia é a principal causa de adoecimento e morte na infância, principalmente em grupos vulneráveis. O objetivo deste estudo foi analisar os fatores associados à pneumonia em crianças Yanomami internadas por condições sensíveis à atenção primária (ICSAP). As internações foram divididas em dois grupos: i) pneumonias e ii) demais causas, de acordo com a lista brasileira de ICSAP. Foram estimadas taxas ajustadas de ICSAP e utilizou-se a regressão logística não condicional para analisar fatores associados. Mais de 90% dos registros foram considerados ICSAP. A taxa padronizada de ICSAP foi 18,6/1.000. As chances de internação por pneumonia foram 2,7 (IC: 1,3-5,4) vezes maiores em crianças de 0,1 a 5,9 meses; 1,9 (IC: 1,1-3,3) vezes maiores nas crianças que tiveram a duração da hospitalização variando de 8 a 14 dias; e 3,0 (IC: 1,2-7,5) vezes maiores nas crianças com diagnóstico secundário de desnutrição. O excesso de internações evitáveis é um forte indício da baixa qualidade das ações em terras indígenas e do limitado acesso à atenção primária, contrariando o modelo assistencial proposto pelo subsistema de saúde indígena no Brasil que, em tese, deveria privilegiar tecnologias assistenciais ancoradas na atenção primária