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关键词： Early life stress   Cortex   HPA axis   Oxidative stress   Inflammation  

摘要： Maternal stress has been described as an important component in the offspring's cerebral development, altering the susceptibility to diseases in later life. Moreover, the postnatal period is essential for the development and integration of several peripheral and central systems related to the control of homeostasis. Thus, this study aimed to evaluate the effects of prenatal stress on the activation of cortical neurons, by performing experiments both under basal conditions and after KCl-induced depolarization. Female mice were divided in two groups: control and prenatal restraint stress. Cortical neurons from the offspring were obtained at gestational day 18. The effects of prenatal stress and KCl stimulations on cellular mortality, autophagy, gene expression, oxidative stress, and inflammation were evaluated. We found that neurons from PNS mice have decreased necrosis and autophagy after depolarization. Moreover, prenatal stress modulated the HPA axis, as observed by the increased GR and decreased 5HTr1 mRNA expression. The BDNF is an important factor for neuronal function and results demonstrated that KCl-induced depolarization increased the gene expression of BDNF I, BDNF IV, and TR kappa B. Furthermore, prenatal stress and KCl treatment induced significant alterations in oxidative and inflammatory markers. In conclusion, prenatal stress and stimulation with KCl may influence several markers related to neurodevelopment in cortical neurons from neonate mice, supporting the well-known long-term effects of maternal stress.

关键词： Brain tumors   Glioma   Glioblastoma   Store-operated Ca2+ entry   Purinergic signalling  

摘要： Brain tumors comprise a large series of tumor cancer from benign to highly malignant gliomas and metastases from primary tumors outside the brain. Intracellular Ca2+ homeostasis is involved in a large series of cell functions including cell proliferation, migration, and cell death. Store-operated Ca2+ entry (SOCE), the most important Ca2+ entry pathway in non-excitable cells, is involved in cell proliferation and migration and enhanced in tumor cells from breast cancer, colon cancer and cell lines derived from glioblastoma but there are almost no studies in human primary glioblastoma cells or other brain tumors. We have developed a single procedure to obtain primary cells from a large series (n = 49) of human brain tumors including schwannomas, meningiomas, oligodendrogliomas, astrocytomas, glioblastomas and brain metastases from ovary, breast and lung. Cells were characterized by immunofluorescence and subjected to Ca2+ imaging to investigate resting intracellular Ca2+ levels, Ca2+ responses to physiological agonists as well as voltage-operated Ca2+ entry and SOCE. We found significant differences in resting intracellular Ca2+ and Ca2+ responses to plasma membrane depolarization and ATP among the different tumor cells. Only malignant tumor cells, displayed Ca2+ responses to ATP. SOCE is significantly increased in malignant gliomas whereas voltage-gated Ca2+ entry is decreased. In addition, SOCE is significantly larger in high grade gliomas than in low grade gliomas suggesting that SOCE increases with glioma progression. These data may provide new insights on the role of intracellular Ca2+ and purinergic signalling in brain tumors.

关键词： Bovine   Primary cells   Respiratory epithelial cells   Influenza D virus   Nasal turbinate   Soft palate   Trachea  

摘要： Influenza D virus (IDV) is a novel type of influenza virus that infects and causes respiratory illness in bovines. Lack of host-specific in vitro model that can recapitulate morphology and physiology of in vivo airway epithelial cells has impeded the study of IDV infection. Here, we established and characterized bovine primary respiratory epithelial cells from nasal turbinate, soft palate, and trachea of the same calf. All three cell types showed characteristics peculiar of epithelial cells, polarized into apical-basolateral membrane, and formed tight junctions. Furthermore, these cells expressed both alpha-2,3- and alpha-2,6-linked sialic acids with alpha-2,3 linkage being more abundant. IDV strains replicated to high titers in these cells, while influenza A and B viruses exhibited moderate to low titers, with influenza C virus replication not detected. These findings suggest that bovine primary airway epithelial cells can be utilized to model infection biology and pathophysiology of IDV and other respiratory pathogens.

关键词： anaemia   red blood cells   transfusion   meningioma  

摘要： Simple Summary The transfusion of red blood cells (RBC) in patients undergoing major elective cranial surgery is associated with increased morbidity and mortality. This study sought to identify the clinical outcome of RBC transfusions in skull base and non-skull base meningioma patients including the identification of risk factors for RBC transfusion. Data underline that preoperative anaemia rate was significantly higher in transfused patients (17.7%) compared to patients without RBC transfusion (6.2%). We could further show that RBC transfusion was associated with increased postoperative complications and increased hospital length of stay. After multivariate analyses, risk factors for RBC transfusion were preoperative American Society of Anaesthesiologists (ASA) physical status score, tumor size, surgical time, and intraoperative blood loss. We concluded that blood loss due to large tumors or localization near large vessels are the main triggers for RBC transfusion in meningioma patients paired with a potential preselection that masks the effect of preoperative anaemia in multivariate analysis. So far, this has not been investigated in a large cohort (n = 423) of skull base and non-skull base meningioma patients. Transfusion of red blood cells (RBC) in patients undergoing major elective cranial surgery is associated with increased morbidity, mortality and prolonged hospital length of stay (LOS). This retrospective single center study aims to identify the clinical outcome of RBC transfusions on skull base and non-skull base meningioma patients including the identification of risk factors for RBC transfusion. Between October 2009 and October 2016, 423 patients underwent primary meningioma resection. Of these, 68 (16.1%) received RBC transfusion and 355 (83.9%) did not receive RBC units. Preoperative anaemia rate was significantly higher in transfused patients (17.7%) compared to patients without RBC transfusion (6.2%;p = 0.0015). In transfused patients, postoperative

关键词： Persistence   GLP-1 Receptor Agonist   Basal Insulin   Type 2 Diabetes   Loose Combination  

摘要： Aims: The purpose of this study is to assess the persistence of Canadians with Type 2 diabetes mellitus (T2D) on loose-dose combination treatment (i.e., administered by separate devices) with a glucagon-like peptide 1 receptor agonist (GLP-1 RA) and basal insulin over 12 months. Methods: This study is a retrospective cohort study of T2D adults using a Canadian longitudinal prescription database over a 5-year period. Cohort 1 (n = 12,411) is a primary cohort including only individuals inexperienced with the combination therapy at index. Cohort 2 (n =13,498) is an exploratory cohort and includes everyone regardless of previous experience on the loose-dose combination therapy. The primary endpoint is the proportion of individuals persistent and average days persistent to the loose-dose combination therapy at 12 months in Canada. Results: In Cohort 1, overall persistence was 47% in the 12-month period post-index. Persistence is similar when including all inexperienced and subsequent loose-dose combination experiences in Cohort 2 (45%). Conclusions: Canadian T2D adults taking a loose-dose combination therapy of a GLP-1 RA and basal insulin had overall low persistence and lower than reports from previous studies of GLP-1 RA or basal insulin alone. Improving persistence to combination therapy with GLP-1 RA plus basal insulin is an important issue to explore in clinical practice. (c) 2021 Elsevier B.V. All rights reserved.

关键词： carcinoma   fluorodeoxyglucose F18 positron‐   emission tomography   neoplasms   second primary   squamous cell   unknown primary  

摘要： Background To assess the effect of 18-fluorodeoxyglucose positron emission tomography (FDG-PET) in the pretherapeutic staging of N classification, detection rate of distant metastases, and second primaries. Methods Retrospective study on patients with head and neck carcinoma. We compared pretherapeutic N classification by ultrasound, computed tomography (CT)/magnetic resonance imaging (MRI), and FDG-PET-CT/MRI. Results A change in the N classification due to FDG-PET-CT/MRI was observed in 116 patients (39.5%) compared to N classification by ultrasound and fine-needle aspiration cytology. Patients with advanced nodal classification (>N2a) were more likely to be reclassified. Distant metastases were detected in 19 patients and a total of 36 second primaries were diagnosed by FDG-PET-CT/MRI. Detection of distant metastases was more likely in regional advanced disease (>N2a). Smokers (>10 py) had a significantly higher risk of second primary. Conclusion FDG-PET-CT/MRI leads to a significant change in pretherapeutic N classification. The cumulative incidence of distant metastases and second primaries was 18.7%.

关键词： colorectal cancer   saffron   safranal   crocin   HCT116   MLH1   MSH3   DNA damage and repair   apoptosis  

摘要： Background: Colorectal cancer (CRC) is one of the most common cancers worldwide. One of its subtypes is associated with defective mismatch repair (dMMR) genes. Saffron has many potentially protective roles against colon malignancy. However, these roles in the context of dMMR tumors have not been explored. In this study, we aimed to investigate the effects of saffron and its constituents in CRC cell lines with dMMR. Methods: Saffron crude extracts and specific compounds (safranal and crocin) were used in the human colorectal cancer cell lines HCT116, HCT116+3 (inserted MLH1), HCT116+5 (inserted MSH3), and HCT116+3+5 (inserted MLH1 and MSH3). CDC25b, p-H2AX, TPDP1, and GAPDH were analyzed by Western blot. Proliferation and cytotoxicity were analyzed by MTT. The scratch wound assay was also performed. Results: Saffron crude extracts restricted (up to 70%) the proliferation in colon cells with deficient MMR (HCT116) compared to proficient MMR. The wound healing assay indicates that deficient MMR cells are doing better (up to 90%) than proficient MMR cells when treated with saffron. CDC25b and TDP1 downregulated (up to 20-fold) in proficient MMR cells compared to deficient MMR cells, while p.H2AX was significantly upregulated in both cell types, particularly at >10 mg/mL saffron in a concentration-dependent manner. The reduction in cellular proliferation was accompanied with upregulation of caspase 3 and 7. The major active saffron compounds, safranal and crocin reproduced most of the saffron crude extracts' effects. Conclusions: Saffron's anti-proliferative effect is significant in cells with deficient MMR. This novel effect may have therapeutic implications and benefits for MSI CRC patients who are generally not recommended for the 5-fluorouracil-based treatment.

关键词： Extracellular vesicles   Keratinocytes   Langerhans cells   Cytokine   LC3b-II   Toll-like receptor   Autophagy  

摘要： Cells can communicate through the extracellular vesicles (EVs) they secrete. Pathogen associated molecular patterns (PAMPs), alter the biophysical and communicative properties of EVs released from cells, but the functional consequences of these changes are unknown. Characterization of keratinocyte-derived EVs after poly (I:C) treatment (poly(I:C)-EVs) showed slight differences in levels of EV markers TSG101 and Alix, a loss of CD63 and were positive for autophagosome marker LC3b-II and the cytokine IL36? compared to EVs from unstimulated keratinocytes (control-EVs). Flagellin treatment (flagellin-EVs) led to an EV marker profile like control-EVs but lacked LC3b-II. Flagellin-EVs also lacked IL-36? despite nearly identical intracellular levels. While poly(I:C) treatment led to the clear emergence of a 200 nm diameter EV sub-population, these were not found in flagellin-EVs. EV associated IL-36? colocalized with LC3b-II in density gradient analysis, equilibrating to 1.10 g/ mL, indicating a common EV species. Poly(I:C), but not flagellin, induced intracellular vesicles positive for IL36?, LC3b-II, Alix and TSG101, consistent with fusion of autophagosomes and multivesicular bodies. Simultaneous rapamycin and flagellin treatment induced similar intracellular vesicles but was insufficient for the release of IL-36?+/LC3b-II+ EVs. Finally, a qRT-PCR array screen showed eight cytokine/chemokine transcripts were altered (p < 0.05) in monocyte-derived Langerhans cells (LCs) when stimulated with poly(I:C)-EVs while three were altered when LCs were stimulated with flagellin-EVs compared to control-EVs. After independent confirmation, poly(I:C)-EVs upregulated BMP6 (p = 0.035) and flagellin-EVs upregulated CXCL8 (p = 0.005), VEGFA (p = 0.018) and PTGS2 (p = 0.020) compared to control-EVs. We conclude that exogenous signals derived from pathogens can alter keratinocyte-mediated modulation of the local immune responses by inducing changes in the types of EVs secreted and responses

关键词： fibroblasts   non‐   viral transfection   viral vectors  

摘要： Cultured fibroblast cells, especially dermal cells, are used for various types of scientific research, particularly within the medical field. Desirable features of the cells include their ease of isolation, rapid cellular growth, and high degree of robustness. Currently, fibroblasts are mainly used to obtain pluripotent cells via a reprogramming process. Dermal fibroblasts, are particularly useful for gene therapies used for promoting wound healing or minimizing skin aging. In recent years, fibroblast transfection efficiencies have significantly improved. In order to introduce molecules (most often DNA or RNA) into cells, viral-based systems (transduction) or non-viral methods (transfection) that include physical/mechanical processes or lipid reagents may be used. In this article, we describe critical points that should be considered when selecting a method for transfecting fibroblasts. The most effective methods used for the transfection of fibroblasts include both viral-based and non-viral nucleofection systems. These methods result in a high level of transgene expression and are superior in terms of transfection efficacy and viability.

关键词： ubiquitination   proteasomal degradation   auxin   degron   inducible degradation   optogenetic approach  

摘要： One of the most informative methods to study the roles of individual proteins in cell functions is based on changing their intracellular concentrations. Genetic knockouts or knockdowns are most commonly used for the purpose. However, acting directly at the level of an expressed protein is more informative or convenient to perform in some cases. This action should ideally be controlled in time and reversible. The review analyzes the current data on systems developed to achieve controlled degradation of proteins via their ubiquitination with subsequent proteasome-mediated degradation or other mechanisms.