Identification of Streptococcus pneumoniae by a real-time PCR assay targeting SP2020.

Real-time PCR targeting lytA (the major autolysin gene) and piaB (permease gene of the pia ABC transporter) are currently used as the gold-standard culture-independent assays for Streptococcus pneumoniae identification. We evaluated the performance of a new real-time PCR assay - targeting SP2020 (putative transcriptional regulator gene) - and compared its performance with the assays previously described. A collection of 150 pneumococci, 433 non-pneumococci and 240 polymicrobial samples (obtained from nasopharynx, oropharynx, and saliva; 80 from each site) was tested. SP2020 and lytA-CDC assays had the best performance (sensitivity of 100% for each compared to 95.3% for piaB). The specificity for lytA and piaB was 99.5% and for SP2020 was 99.8%. Misidentifications occurred for the three genes: lytA, piaB and SP2020 were found in non-pneumococcal strains; piaB was absent in some pneumococci including a serotype 6B strain. Combining lytA and SP2020 assays resulted in no misidentifications. Most polymicrobial samples (88.8%) yielded concordant results for the three molecular targets. The remaining samples seemed to contain non-typeable pneumococci (0.8%), and non-pneumococci positive for lytA (1.7%) or SP2020 (8.7%). We propose that combined detection of both lytA-CDC and SP2020 is a powerful strategy for the identification of pneumococcus either in pure cultures or in polymicrobial samples

Lymphatic vessel density and VEGF-C expression are significantly different among benign and malignant thyroid lesions

Thyroid cancer is the most frequent endocrine neoplasia worldwide. The route for metastasis and loco-regional invasion preferentially occurs by lymphatic vessels. For this reason, the assessment of lymphatic vessel density (LVD) is supposed to represent both a prognostic parameter and also a potential therapeutic target. In order to evaluate the value of LVD in benign and malignant thyroid lesions, we analyzed 110 thyroidectomy specimens using D2-40, a specific marker for lymphatic vessels and vascular endothelial growth factor C (VEGF-C), the most potent molecule of lymphatic proliferation. LVD was significantly different between papillary and follicular carcinomas in total (p = 0.045) and peritumoral area (p = 0.042). Follicular adenoma and follicular carcinoma showed an important difference of intra- (p = 0.019) and peritumoral (p = 0.033) LVD. VEGF-C was more markedly expressed in malignancies than in benign lesions (p = 0.0001). Almost all cancers with high positive VEGF-C expression also exhibited increased peritumoral LVD (p = 0.049) when compared with the benign lesions. Indeed, the high peritumoral LVD of malignant thyroid lesions is an important finding for surgery planning and supports the practice of total thyroidectomy in malignant thyroid neoplasm's since the lymphatic peritumoral vessels definitely are an escape path for tumor cells

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

Telomerase promoter mutations in cancer: an emerging molecular biomarker?

João Vinagre, Vasco Pinto and Ricardo Celestino contributed equally to the manuscript.Cell immortalization has been considered for a long time as a classic hallmark of cancer cells. Besides telomerase reactivation, such immortalization could be due to telomere maintenance through the “alternative mechanism of telomere lengthening” (ALT) but the mechanisms underlying both forms of reactivation remained elusive. Mutations in the coding region of telomerase gene are very rare in the cancer setting, despite being associated with some degenerative diseases. Recently, mutations in telomerase (TERT) gene promoter were found in sporadic and familial melanoma and subsequently in several cancer models, notably in gliomas, thyroid cancer and bladder cancer. The importance of these findings has been reinforced by the association of TERT mutations in some cancer types with tumour aggressiveness and patient survival. In the first part of this review, we summarize the data on the biology of telomeres and telomerase, available methodological approaches and non-neoplastic diseases associated with telomere dysfunction. In the second part, we review the information on telomerase expression and genetic alterations in the most relevant types of cancer (skin, thyroid, bladder and central nervous system) on record, and discuss the value of telomerase as a new biomarker with impact on the prognosis and survival of the patients and as a putative therapeutic target

A Sox2–Sox9 signalling axis maintains human breast luminal progenitor and breast cancer stem cells

Increased cancer stem cell content during development of resistance to tamoxifen in breast cancer is driven by multiple signals, including Sox2-dependent activation of Wnt signalling. Here, we show that Sox2 increases and estrogen reduces the expression of the transcription factor Sox9. Gain and loss of function assays indicate that Sox9 is implicated in the maintenance of human breast luminal progenitor cells. CRISPR/Cas knockout of Sox9 reduces growth of tamoxifen-resistant breast tumours in vivo. Mechanistically, Sox9 acts downstream of Sox2 to control luminal progenitor cell content and is required for expression of the cancer stem cell marker ALDH1A3 and Wnt signalling activity. Sox9 is elevated in breast cancer patients after endocrine therapy failure. This new regulatory axis highlights the relevance of SOX family transcription factors as potential therapeutic targets in breast cancer

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

Visceral dissemination of mucocutaneous leishmaniasis in a kidney transplant recipient

Intracellular protozoan of the genus Leishmania, endemic in the Mediterranean basin, are the cause of cutaneous (CL), mucocutaneous (MCL), and visceral leishmaniasis (VL). A 75-year-old woman was admitted nine years after a second kidney transplant (KT), due to persistent pancytopenia and fever. She presented edema and erythema of the nose in the last two years and an exophytic nodular lesion located on the left arm, with areas of peripheral necrosis and central ulceration in the last 18 months. A bone marrow biopsy revealed features compatible with Leishmania amastigotes, and polymerase chain reaction test (PCR) for Leishmania infantum was positive. Moreover, biopsy and PCR for L. infantum of the cutaneous lesion on the patient’s left arm and nose and PCR from peripheral blood were positive. Thus, a diagnosis of CL, MCL, and VL was made, and liposomal amphotericin B was initiated, but the patient had an unfavorable outcome and died. This is the first report of a KT recipient presenting with the entire spectrum of leishmaniasis. In Portugal, this infection is rare—so a high degree of clinical suspicion is required for its diagnosis, especially in endemic regions, as visceral leishmaniasis is a potentially life-threatening infection

Theoretical investigation of the phase behaviour of model ternary mixtures containing n

International audienceWe have used the hetero-SAFT-VR approach to investigate the phase equilibria of a number of binary and ternary mixtures of n -alkanes, perfluoro-n -alkanes, and perfluoroalkylalkane diblock surfactants. We focussed our work on the understanding of the microscopic conditions that control the phase behaviour of these mixtures, with a particular emphasis of the effect on the liquid-liquid separation and the stabilisation of n -alkane + perfluoro-n -alkane mixtures when a diblock surfactant is added. We selected the n -heptane + perfluoromethane binary mixture, and studied the changes on the phase behaviour when a symmetric (same number of alkyl and perfluoroalkyl chemical groups) or an asymmetric (different number of alkyl and perfluoroalkyl chemical groups) diblock surfactants is added to the binary mixture. We have obtained the phase diagrams of a wide range of binary and ternary mixtures at different thermodynamic conditions. We have found a variety of interesting behaviours as we modify the alkyl or/and the perfluoroalkyl chain-length of the diblock surfactants: the usual changes in the vapour-liquid phase separation, changes in the type of phase diagrams (typically from type I to type V phase behaviour according to the Scott and Konynenburg classification), azeotropy, and Bancroft points. We noted that the main effect of adding a symmetric or an asymmetric surfactant to the n -heptane + perfluoromethane mixture is to stabilise the system, i.e., to decrease the two-phase (liquid-liquid) immiscibility region of the ternary diagram as the surfactant concentration is increased