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To analyze the expression of glucose-dependent insulinotropic polypeptide receptor (GIPR) in somatotropinomas specimens and compare clinical, biochemical, radiological, therapeutic, molecular, and pathological data among those who overexpressed (GIPR +) and those who did not overexpress (GIPR - ) GIPR.Clinical, biochemical, radiological, molecular, and pathological data were collected. GNAS1 sequencing was performed with the Sanger method. Protein expression of somatostatin receptor subtypes 2 and 5 and CAM 5.2 were analyzed by immunohistochemistry. Quantitative real-time PCR was performed to analyze the mRNA expression of GIPR with the TaqMan® method. Positive expression was considered when the fold change (FC) was above 17.2 (GIPR +).A total of 74 patients (54% female) were included. Eighteen tumors (24%) were GIPR + . Gsp mutation was detected in 30 tumors (40%). GIPR + tumors were more frequently densely granulated adenomas (83% vs 47%, p = 0.028). There was no difference in clinical, biochemical, radiological, therapeutic (surgical cure or response to medical therapy), or other pathological features between GIPR + and GIPR - tumors. Twenty-eight out of 56 (50%) GIPR - tumors harbored a gsp mutation, whereas two out of 18 (11%) GIPR + tumors harbored a gsp mutation (p = 0.005).We described, for the first time, that GIPR + and gsp mutations are not mutually exclusive, but gsp mutations are less common in GIPR + tumors. GIPR + and GIPR - tumors have similar clinical, biochemical, radiological, therapeutic, and pathological features, with the exception of a high frequency of densely granulated adenomas among GIPR + tumors.

Ouabain is a classic Na+K+ATPase ligand and it has been described to have neuroprotective effects on neurons and glial cells at nanomolar concentrations. In the present work, the neuroprotective and immunomodulatory potential of ouabain was evaluated in neonatal rat retinal cells using an optic nerve axotomy model in vitro. After axotomy, cultured retinal cells were treated with ouabain (3 nM) at different periods. The levels of important inflammatory receptors in the retina such as TNFR1/2, TLR4, and CD14 were analyzed. We observed that TNFR1, TLR4, and CD14 were decreased in all tested periods (15 min, 45 min, 24 h, and 48 h). On the other hand, TNFR2 was increased after 24 h, suggesting an anti-inflammatory potential for ouabain. Moreover, we showed that ouabain also decreased Iba-1 (microglial marker) density. Subsequently, analyses of retrograde labeling of retinal ganglion cells (RGC) were performed after 48 h and showed that ouabain-induced RGC survival depends on autophagy. Using an autophagy inhibitor (3-methyladenine), we observed a complete blockage of the ouabain effect. Western blot analyses showed that ouabain increases the levels of autophagy proteins (LC3 and Beclin-1) coupled to p-CREB transcription factor and leads to autophagosome formation. Additionally, we found that the ratio of cleaved/pro-caspase-3 did not change after ouabain treatment; however, p-JNK density was enhanced. Also, ouabain decreased reactive oxygen species production immediately after axotomy. Taken together, our results suggest that ouabain controls neuroinflammation in the retina following optic nerve axotomy and promotes RGC neuroprotection through activation of the autophagy pathway.

Ouabain is a classic Na

RAS genes are among the most frequently mutated genes in cancer, where their mutation frequency varies according to the distinct RAS isoforms and tumour types. Despite occurring more prevalent in malignant tumours, RAS mutations were also observed in few benign tumours. Pituitary adenomas are examples of benign tumours which vary in size and aggressiveness. The present study was performed to investigate, via liquid biopsy and tissue analysis, the presence of K-RAS mutations in a pituitary macroadenoma. Molecular analysis was performed to investigate K-RAS mutations using the droplet digital PCR (ddPCR) method by evaluating both plasma (liquid biopsy) and the solid tumour of a patient diagnosed with a giant clinically non-functioning pituitary tumour. The patient underwent surgical resection due to visual loss, and the histopathological analysis showed a gonadotrophic pituitary macroadenoma. The molecular analysis revealed the presence of mutant K-RAS both in the plasma and in the tumour tissue which, to our knowledge, has not been previously reported in the literature. Our findings highlight the exceptional capacity of the digital PCR in detecting low frequency mutations (below 1%), since we detected, for the first time, K-RAS mutations in pituitary macroadenoma. The potential impact of K-RAS mutations in these tumours should be further investigated.

Made available in DSpace on 2022-04-29T08:29:59Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-07-01 Context: Artificial intelligence (AI), in particular machine learning (ML), may be used to deeply analyze biomarkers of response to first-generation somatostatin receptor ligands (fg-SRLs) in the treatment of acromegaly. Objective: To develop a prediction model of therapeutic response of acromegaly to fg-SRL. Methods: Patients with acromegaly not cured by primary surgical treatment and who had adjuvant therapy with fg-SRL for at least 6 months after surgery were included. Patients were considered controlled if they presented growth hormone (GH)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of COVID-19, a pandemic associated with substantial morbidity and mortality. Despite of this, no vaccine or approved drug is available to eradicate the virus. In this manuscript, we present an alternative study area that may contribute to development of diagnostic biomarkers and therapeutic targets for COVID-19. We analyzed sixty SARS-CoV-2 genomes to identify regions that could work as virus-encoded miRNA seed sponges and potentially bind to human miRNA seed sites and prevent interaction with their native targets thereby relieving native miRNA suppression. MicroRNAs (miRNAs) are evolutionally conserved single-stranded RNAs that regulate gene expression at the posttranscriptional level by disrupting translation. MiRNAs are key players in variety of biological processes that regulate differentiation, development and activation of immune cells in both innate and adaptive immunity. We find 34 miRNAs for positive-sense viral RNA and 45 miRNAs for negative-sense that can strongly bind to certain key SARS-CoV-2 genes. The disruption and dysfunction of miRNAs may perturb the immune response and stimulate the release of inflammatory cytokines altering the cellular response to viral infection. Previous studies demonstrate that miRNAs have the potential to be used as diagnostic and therapeutic biomarkers. Therefore, its discovery and validation are essential for improving the diagnosis of infection and clinical monitoring in COVID-19.

Mammarenaviruses are a diverse genus of emerging viruses that include several causative agents of severe viral hemorrhagic fevers with high mortality in humans. Although these viruses share many similarities, important differences with regard to pathogenicity, type of immune response, and molecular mechanisms during virus infection are different between and within New World and Old World viral infections. Viruses rely exclusively on the host cellular machinery to translate their genome, and therefore to replicate and propagate. miRNAs are the crucial factor in diverse biological processes such as antiviral defense, oncogenesis, and cell development. The viral infection can exert a profound impact on the cellular miRNA expression profile, and numerous RNA viruses have been reported to interact directly with cellular miRNAs and/or to use these miRNAs to augment their replication potential. Our present study indicates that mammarenavirus infection induces metabolic reprogramming of host cells, probably manipulating cellular microRNAs. A number of metabolic pathways, including valine, leucine, and isoleucine biosynthesis, d-Glutamine and d-glutamate metabolism, thiamine metabolism, and pools of several amino acids were impacted by the predicted miRNAs that would no longer regulate these pathways. A deeper understanding of mechanisms by which mammarenaviruses handle these signaling pathways is critical for understanding the virus/host interactions and potential diagnostic and therapeutic targets, through the inhibition of specific pathologic metabolic pathways.

Knowledge of viral load is essential for formulating strategies for antiviral treatment, vaccination, and epidemiological control of COVID-19. Moreover, patients identification with high viral load could also be useful to understand risk factors such as age, comorbidities, severity of symptoms and hypoxia to decide the need for hospitalization. Several studies are evaluating the importance of analyzing viral load in different types of samples, clinical outcomes and viral transmission pathways. However, in a great number of emerging studies cycle threshold (Ct) values by itself is often used as a viral load indicator, which may be a mistake. In this study, we compared tracheal aspirate with nasopharyngeal samples obtained from critically ill COVID-19 patients and demonstrate how the raw Ct could lead to misinterpretation of results. Further, we analyzed nasopharyngeal swabs positive samples and propose a method to reduce evaluation error that could occur from using raw Ct. Based on these findings, we show the impact that normalization of Ct values has on interpretation of viral load data from different biological samples from patients with COVID-19, transmission and lastly in relations with clinical outcomes.ImportanceIn a pandemic, prevention of disease transmission is key. Reliable data for profiles of viral load are needed and important to guide antiviral treatment, infection control and vaccination. The differential expression of SARS-CoV-2 viral RNA among patient groups is a current topic of interest and viral load has been associated with a diversity of outcomes. However, in a great number of emerging studies cycle threshold (Ct) values by itself is often used as a viral load indicator, which may be a mistake. In this study, we compared tracheal aspirate with nasopharyngeal samples obtained from critically ill COVID-19 patients and demonstrate how the raw Ct could lead to misinterpretation of results. Based on these findings, we show the impact that normalization of Ct values has on interpretation of viral load data from different biological samples from patients with COVID-19, transmission and lastly in relations with clinical outcomes.

Knowledge of viral load is essential to formulate strategies for antiviral treatment, vaccination, and epidemiological control of COVID-19. Moreover, identification of patients with high viral loads can also be useful to understand risk factors such as age, comorbidities, severity of symptoms and hypoxia, to decide on the need for hospitalization. Several ongoing studies are analyzing viral load in different types of samples and evaluating its relationship with clinical outcomes and viral transmission pathways. However, in a great number of emerging studies, cycle threshold (Ct) values alone are often used as viral load indicators, which may be a mistake. In this study, we compared tracheal aspirate with nasopharyngeal swab samples obtained from critically ill COVID-19 patients and here we report how the raw Ct can lead to misinterpretation of results. Furthermore, based on analysis of nasopharyngeal swab samples we propose a method to reduce evaluation errors that could occur from using raw Ct data. Based on these findings, we show the impact that normalization of Ct values has on interpretation of SARS-CoV-2 viral load from different biological samples.

Background: Somatic mutations in the ubiquitin-specific peptidase 8 (USP8) gene have been described in Cushing’s disease (CD). These mutations increase proopiomelanocortin transcription resulting in ACTH production and seem to correlate with somatostatin receptor type 5 (SST5) expression. Aims: Screen USP8 in patients with corticotropinomas and correlate USP8 mutational status with SST5 expression in CD. Methods: Tumor DNA was extracted and then exon 14 amplified by PCR. SST5 was assessed by immunohistochemistry (clone UMB4) and quantified multiplying the percentage of positive cells (0,0%; 80%, 4) and intensity (mild, 1; moderate, 2; intense, 3), giving a score (IRS) from 0-12 with ≥ 6 considered high. Results: Among 59 patients, 38 had CD and 21 silent corticotropinomas. In CD, 13 (34.2%) patients had pathogenic mutations (6 had p.Ser719del; 5 had p.Pro720Arg and 2 had p.Pro720Gln). In the mutated CD group, all were women and had median age of 34.5 years (20-46). Median ACTH was 64.7pg/mL [(34.8-330.0), normal

Protein kinase C (PKC) is a family of serine/threonine kinases related to several phenomena as cell proliferation, differentiation and survival. Our previous data demonstrated that treatment of axotomized neonatal rat retinal cell cultures for 48 h with phorbol 12-myristate 13-acetate (PMA), a PKC activator, increases retinal ganglion cells (RGCs) survival. Moreover, this treatment decreases M1 receptors (M1R) and modulates BDNF levels. The aim of this work was to assess the possible involvement of neurotrophins BDNF and NGF in the modulation of M1R levels induced by PKC activation, and its involvement on RGCs survival. Our results show that PMA (50 ng/mL) treatment, via PKC delta activation, modulates NGF, BDNF and M1R levels. BDNF and NGF mediate the decrease of M1R levels induced by PMA treatment. M1R activation is essential to PMA neuroprotective effect on RGCs as telenzepine (M1R selective antagonist) abolished it. Based on our results we suggest that PKC delta activation modulates neurotrophins levels by a signaling pathway that involves M1R activation and ultimately leading to an increase in RGCs survival in vitro.

AimNumerous studies have demonstrated that exercise acutely prevents the reduction in flow-mediated dilation induced by mental stress in subjects with metabolic syndrome (MetS). However, it is unknown whether a similar effect occurs in endothelial progenitors cells (EPCs). This study investigated whether exercise protects from the deleterious effect of mental stress on cultured EPCs in healthy subjects and those with MetS.Main methodsTen healthy subjects (aged 31±2) and ten subjects with MetS (aged 36±2) were enrolled. Subjects underwent a mental stress test, followed immediately by either 40min of leg cycling or rest across two randomized sessions: mental stress+non-exercise control (MS) and mental stress+exercise (MS+EXE). The Stroop Color-Word Test was used to elicit mental stress. Blood samples were drawn at baseline and following sessions to isolate mononuclear cells. These cells were cultured in fibronectin-coated plates for seven days, and EPCs were identified by immunofluorescence (acLDL+/ UEA-I Lectin+).Key findingsAll subjects presented similar increases in mean blood pressure and heart rate during the mental stress test (P0.05). The EPC response to MS and MS+EXE was increased in healthy subjects, whereas it was decreased in subjects with MetS (P

Increased levels of adhesion molecules or metalloproteinases (MMPs) may indicate endothelial dysfunction. Exercise mobilizes circulating angiogenic cells (CACs) from bone marrow in healthy subjects, improving vascular function. However, it is unclear whether this mechanism is preserved in the early stages of metabolic syndrome (early MetS). We aimed to evaluate the acute effects of exercise on adhesion molecules, angiogenic factors, MMPs, and CACs in early MetS. Fifteen subjects with early MetS and nine healthy controls underwent an exercise session and a nonexercise session, randomly. Adhesion molecules, angiogenic factors, CACs, and MMPs were evaluated before and after exercise or nonexercise sessions. At baseline, levels of sE-selectin, sICAM-1, and MMP-9 were higher in early MetS than in controls (P≤0.03). After exercise, sE-selectin, sICAM-1, and MMP-9 levels were still higher in early MetS (P<0.05). Subjects with early MetS presented less CACs (P=0.02) and higher MMP-9 activity (P≤0.04), while healthy controls presented higher MMP-2 activity after exercise. There was no difference between moments in nonexercise session (P>0.05). In conclusion, subjects with early MetS already presented impaired endothelial function at rest along with a decrease in CACs and an increase in MMP-9 activity in response to exercise.