Glycosylation of cancer extracellular vesicles: Capture strategies, functional roles and potential clinical applications

Glycans are major constituents of extracellular vesicles (EVs). Alterations in the glycosylation pathway are a common feature of cancer cells, which gives rise to de novo or increased synthesis of particular glycans. Therefore, glycans and glycoproteins have been widely used in the clinic as both stratification and prognosis cancer biomarkers. Interestingly, several of the known tumor-associated glycans have already been identified in cancer EVs, highlighting EV glycosylation as a potential source of circulating cancer biomarkers. These particles are crucial vehicles of cell–cell communication, being able to transfer molecular information and to modulate the recipient cell behavior. The presence of particular glycoconjugates has been described to be important for EV protein sorting, uptake and organ-tropism. Furthermore, specific EV glycans or glycoproteins have been described to be able to distinguish tumor EVs from benign EVs. In this review, the application of EV glycosylation in the development of novel EV detection and capture methodologies is discussed. In addition, we highlight the potential of EV glycosylation in the clinical setting for both cancer biomarker discovery and EV therapeutic delivery strategies.This work was funded by FEDER funds through the Operational Programme for Competitiveness Factors COMPETE 2020 (POCI-01-0145-FEDER-016585; POCI-01-0145-FEDER-007274) and national funds through the Foundation for Science and Technology (FCT), under the projects:PTDC/BBB-EBI/0567/2014 to C.A.R and UID/BIM/04293/2013; and the project NORTE-01-0145-FEDER-000029, supported by Norte Portugal Regional Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF)

Modification of T lymphocytes with lentiviral vectors for expression of anti-CD19 chimeric antigen receptor (CAR)

The use of immunotherapy with modified T lymphocytes with chimeric antigen receptor (CAR) has been proven effective in the treatment of leukemias and lymphomas resistant to chemotherapy. CAR possess an extracellular domain derived from variable regions of antibodies and costimulation intracellular domains of T lymphocytes. CD19 protein has been shown to be an ideal target because it is expressed on most B-cell tumors as well as normal B cells, but not in other types of cells. Recent clinical studies involving anti-CD19 CAR T-cells have shown excellent responses in a variety of B-cell tumors, even in patients with relapse after high-dose chemotherapy. This study aimed to produce CD4+ lymphocyte lineage Jurkat (ATCC® TIB-152 ™) modified with a second generation anti-CD19 CAR with 4-1BB as intracellular costimulation domain. Lentiviral vectors were produced in HEK293T (ATCC® CRL-3216 ™) transiently transfected with plasmids containing the coding sequence of the CAR, viral envelope VSV-G, and viral capsid. The viral titer was calculated by real time PCR after transduction of HEK293T cells, resulting in 1.65 x 105 IU/mL. The literature indicates an MOI (multiplicity of infection) from 5 to 10 IU/cell for transduction of lymphocytes. A new batch of virus was produced, and the supernatant was ultracentrifuged at 19200 rpm (Beckman Coulter, SW28 rotor) in order to concentrate the viral particles. The viral titer of the concentrated batch was 1.26 x 108 IU/mL. This new titer is compatible with the necessary to infect 107 cells, amount of pre-expansion cells necessary to obtain the number of cells suitable for infusion into patients (2.5 x 109 to 5 x 109 cells). Then, the infection of Jurkat was performed in a 6-well plate with RPMI 1640 supplemented with 10% fetal bovine serum (FBS), 2 µg/mL Polybrene®, and centrifugation at 1000 rpm for 20 minutes at room temperature. After 16 hours of incubation (37°C, 5% CO2 and 85% humidity), the medium was exchanged for fresh RPMI 1640 10% FBS. After additional 48 hours of incubation under the same conditions, the cells were collected and was their DNA was extracted. We obtained by real-time PCR that the number of integrated viral copies per genome was 35.3 ± 4.5 (mean ± standard deviation) for transduction with MOI of 5 IU/cell. While for MOI of 10 IU/cell, it was obtained 42.6 ± 0.1 copies per genome. It was observed that there was not a significant increase in viral copies when the MOI increased from 5 to 10. This may occur because cell’s surface receptors have been saturated by the large number of viruses. The lentiviral vector used by us has been shown to transduce T lymphocyte satisfactorily. The next steps of the study are the transduction of T lymphocytes from healthy donors and verification of the CAR receptor effectiveness to bind to CD19 of cell B lymphocyte lineages. Grant #2016/08374-5, São Paulo Research Foundation (FAPESP)

Characterization of the striatal extracellular matrix in a mouse model of Parkinson’s disease

Parkinson’s disease’s etiology is unknown, although evidence suggests the involvement of oxidative modifications of intracellular components in disease pathobiology. Despite the known involvement of the extracellular matrix in physiology and disease, the influence of oxidative stress on the matrix has been neglected. The chemical modifications that might accumulate in matrix components due to their long half-live and the low amount of extracellular antioxidants could also contribute to the disease and explain ineffective cellular therapies. The enriched striatal extracellular matrix from a mouse model of Parkinson’s disease was characterized by Raman spectroscopy. We found a matrix fingerprint of increased oxalate content and oxidative modifications. To uncover the effects of these changes on brain cells, we morphologically characterized the primary microglia used to repopulate this matrix and further quantified the effects on cellular mechanical stress by an intracellular fluorescence resonance energy transfer (FRET)-mechanosensor using the U-2 OS cell line. Our data suggest changes in microglia survival and morphology, and a decrease in cytoskeletal tension in response to the modified matrix from both hemispheres of 6-hydroxydopamine (6-OHDA)-lesioned animals. Collectively, these data suggest that the extracellular matrix is modified, and underscore the need for its thorough investigation, which may reveal new ways to improve therapies or may even reveal new therapies.This research was funded by FEDER (Fundo Europeu de Desenvolvimento Regional) funds through the COMPETE 2020 Operational Program for Competitiveness and Internationalization (POCI), Portugal 2020, and Portuguese funds through FCT (ID/BIM/04293/2020), UnIC (UID/IC/00051/2019), iBiMED (UID/BIM/04501/2020 and POCI-01-0145-FEDER-007628), and LAQV/REQUIMTE (UIDB/50006/2020) research units as well as RV’s Fellowship Grant (IF/00286/2015). Ana Freitas acknowledges FCT for her PhD scholarship (SFRH/BD/111423/2015), Miguel Aroso is hired through the Scientific Employment Stimulus from FCT (CEECIND/03415/2017), and M.L. has an FCT RJEC Id 3762 contract.The authors thank Eduardo D Martín Montiel for his support, fruitful discussions, suggestions, and technical and scientific help. The authors also thank Sofia Lamas and all the i3S Animal facility personnel for their support with the animals throughout the study. Raman spectroscopy, together with wide field and confocal microscopy, were performed at the i3S Scientific Platform Bioimaging, member of the PPBI (Plataforma Portuguesa de Bioimagem, POCI-01-0145-FEDER-022122)

Unusual presentation of peritonitis with persistent clear aspirate: a case report

<p>Abstract</p> <p>Introduction</p> <p>Peritonitis is the most frequent complication of peritoneal dialysis. Diagnosis of peritonitis includes symptoms and signs of peritonitis with a cloudy aspirate of more than 100 WBC/ml, as well as positive cultures. Although sterile peritonitis has been reported in the literature, to the best of our knowledge this is the first report of an unusual presentation of peritonitis without any white blood cells in the peritoneal aspirate despite multiple positive peritoneal cultures.</p> <p>Case presentation</p> <p>An 82-year-old Caucasian man who had been on continuous cycling peritoneal dialysis for 12 years was admitted to our hospital with general malaise, loss of appetite, weight loss and somnolence. He did not describe abdominal pain or fever. Even though his peritoneal fluid was consistently negative for leukocytes and clear, he had peritonitis with different organisms consecutively.</p> <p>Conclusions</p> <p>Our case report shows that any patient on peritoneal dialysis presenting with evidence of infection (fever, peripheral leukocytosis) without an obvious cause should have aspirate cultures done even if the aspirate is clear and abdominal pain is absent. Our case report may change the initial work-up and management of these patients. We believe this report is of interest to general medicine and emergency room physicians as well as nephrologists.</p

Prevalence of diabetes mellitus and impaired glucose tolerance in a rural community of Angola

<p>Abstract</p> <p>Background</p> <p>To determine the prevalence of diabetes mellitus (DM) and impaired glucose tolerance (IGT) in a rural community (Bengo) of Angola.</p> <p>Methods</p> <p>A random sample of 421 subjects aged 30 to 69 years (30% men and 70% women) was selected from three villages of Bengo province. This cross-sectional home survey was conducted using a sampling design of stage conglomerates. First, clinical and anthropometric data were obtained and fasting capillary glucose level was determined. Subjects who screened positive (fasting capillary glucose ≥ 100 mg/dl and < 200 mg/dl) and each sixth consecutive subject who screened negative (fasting capillary glucose < 100 mg/dl) were submitted to the second phase of survey, consisting of the 75-g oral glucose tolerance test. Data was analyzed by the use of SAS statistical software.</p> <p>Results</p> <p>The prevalence rates of diabetes mellitus and IGT were 2.8% and 8.1%, respectively. The age group with the highest prevalence of diabetes was 60 to 69 years (42%). Impaired glucose tolerance prevalence was 38% in the 40 to 49 year age group and it increased with age, considering that the 50 to 59 and 60 to 69 year age groups as a whole represent 50% of all subjects with impaired glucose tolerance. The prevalence of diabetes mellitus did not differ significantly between men (3.2%) and women (2.7%) (p = 0.47). On the other hand, the prevalence of impaired glucose tolerance among women showed almost twice that found in men (9.1% vs. 5.6%, respectively). Overweight was present in 66.7% of the individuals with diabetes mellitus and 26.5% of individuals with impaired glucose tolerance showed overweight or obesity.</p> <p>Conclusions</p> <p>Although the prevalence of diabetes mellitus was low, the prevalence of impaired glucose tolerance is considered to be within an intermediary range, suggesting a future increase in the frequency of diabetes in this population.</p

COVID-SGIS: A Smart Tool for Dynamic Monitoring and Temporal Forecasting of Covid-19

Background: The global burden of the new coronavirus SARS-CoV-2 is increasing at an unprecedented rate. The current spread of Covid-19 in Brazil is problematic causing a huge public health burden to its population and national health-care service. To evaluate strategies for alleviating such problems, it is necessary to forecast the number of cases and deaths in order to aid the stakeholders in the process of making decisions against the disease. We propose a novel system for real-time forecast of the cumulative cases of Covid-19 in Brazil. / Methods: We developed the novel COVID-SGIS application for the real-time surveillance, forecast and spatial visualization of Covid-19 for Brazil. This system captures routinely reported Covid-19 information from 27 federative units from the Brazil.io database. It utilizes all Covid-19 confirmed case data that have been notified through the National Notification System, from March to May 2020. Time series ARIMA models were integrated for the forecast of cumulative number of Covid-19 cases and deaths. These include 6-days forecasts as graphical outputs for each federative unit in Brazil, separately, with its corresponding 95% CI for statistical significance. In addition, a worst and best scenarios are presented. / Results: The following federative units (out of 27) were flagged by our ARIMA models showing statistically significant increasing temporal patterns of Covid-19 cases during the specified day-to-day period: Bahia, Maranhão, Piauí, Rio Grande do Norte, Amapá, Rondônia, where their day-to-day forecasts were within the 95% CI limits. Equally, the same findings were observed for Espírito Santo, Minas Gerais, Paraná, and Santa Catarina. The overall percentage error between the forecasted values and the actual values varied between 2.56 and 6.50%. For the days when the forecasts fell outside the forecast interval, the percentage errors in relation to the worst case scenario were below 5%. / Conclusion: The proposed method for dynamic forecasting may be used to guide social policies and plan direct interventions in a cost-effective, concise, and robust manner. This novel tools can play an important role for guiding the course of action against the Covid-19 pandemic for Brazil and country neighbors in South America

The yeast P5 type ATPase, Spf1, regulates manganese transport into the endoplasmic reticulum

The endoplasmic reticulum (ER) is a large, multifunctional and essential organelle. Despite intense research, the function of more than a third of ER proteins remains unknown even in the well-studied model organism Saccharomyces cerevisiae. One such protein is Spf1, which is a highly conserved, ER localized, putative P-type ATPase. Deletion of SPF1 causes a wide variety of phenotypes including severe ER stress suggesting that this protein is essential for the normal function of the ER. The closest homologue of Spf1 is the vacuolar P-type ATPase Ypk9 that influences Mn2+ homeostasis. However in vitro reconstitution assays with Spf1 have not yielded insight into its transport specificity. Here we took an in vivo approach to detect the direct and indirect effects of deleting SPF1. We found a specific reduction in the luminal concentration of Mn2+ in ∆spf1 cells and an increase following it’s overexpression. In agreement with the observed loss of luminal Mn2+ we could observe concurrent reduction in many Mn2+-related process in the ER lumen. Conversely, cytosolic Mn2+-dependent processes were increased. Together, these data support a role for Spf1p in Mn2+ transport in the cell. We also demonstrate that the human sequence homologue, ATP13A1, is a functionally conserved orthologue. Since ATP13A1 is highly expressed in developing neuronal tissues and in the brain, this should help in the study of Mn2+-dependent neurological disorders

Digital Mazes and Spatial Reasoning: Using Colour and Movement to Explore the 4th Dimension

This chapter focuses on innovative developments of four-dimensional digital mazes, examining how these mazes tap into the ideas of mathematician and fiction writer Charles Hinton (1853-1907) who wrote extensively on perception of a 4th geometric dimension. Hinton treats mathematical objects as physical and material movements, and draws on non-Euclidean geometry to argue for a virtual dimension to matter. I discuss recent attempts to build digital mazes that develop spatial sense in four dimensions, and show how these are directly linked to Hinton’s ideas. I focus on how colour and movement in digital environments are used to develop a distinctive kind of spatial sense. This chapter sheds light on innovative uses of digital software for developing student spatial sense. My aim is to explicate the new materialism of Charles Hinton, contribute to discussions about the nature of spatial sense and spatial reasoning, and to point to possible directions for future research on inventive approaches to geometry