Insect cells platforms for fast production of Pseudo-Typed VLPs for drug and vaccine development

Expression systems capable of delivering high concentrations of membrane proteins in their native structure are essential in the vaccine field as well as in drug discovery. In this work, we took advantage of insect cell expression and site-specific gene integration based on flipase-mediated cassette exchange (FMCE) technology to generate cell platforms for efficient production of membrane proteins on the surface of a protein scaffold, namely enveloped virus-like particles (VLPs). The expression of membrane proteins concomitantly with capsid proteins of enveloped viruses (e.g. HIV Gag or influenza M1) will enable their capturing in lipid rafts of the cellular plasma membrane and their display on the surface of budding VLPs, thus providing a native conformation for downstream assays. Parental insect Sf-9 and High Five cells were randomly tagged with GFP-fused Gag or M1 proteins and FACS enriched with cells tagged in genomic “hot-spots” supporting high expression. A linker including a Flp recognition target (FRT) site was used to allow posterior removal of the marker gene from the particle through cassette exchange. By confocal microscopy we could observe that Gag localizes preferentially at the plasma membrane whereas M1 disperses within the cell. Upon promoting Flp-mediated recombination in the tagging populations, cassette exchange was well succeeded, allowing to recover cells tagged in loci supporting FMCE. We are currently evaluating the capability of both core proteins as scaffolds to display GPCRs (e.g. beta-2 adrenergic receptor) and Influenza HA proteins. For the latter, we will present recent results on the feasibility of combining stable and baculovirus-mediated expression of HA in insect High Five cells for production of multi-HA influenza enveloped VLPs towards the development of an “universal” vaccine. This strategy surpasses standard methods for production of multivalent Influenza VLPs such as coinfections or the use of larger, unstable vectors. Overall, modular insect cells platforms are being generated to be readily adaptable for production of a broad range of VLP-based vaccines as well as receptor display particles for drug screening or antibody discovery. Acknowledgments: Funding from European Commission (Project EDUFLUVAC; Grant nr. 602640) and Fundação para a Ciência e a Tecnologia through the project EXPL/BBB-BIO/1541/2013 and PhD fellowships SFRH/BD/86744/2012 and SFRH/BD/90564/2012

Double dermal sinuses: a case study

<p>Abstract</p> <p>Introduction</p> <p>Dermal sinus tracts are rare congenital lesions located in the midline characterized by a cutaneous pit or dimple. They occur all along the midline neuroaxis, from the nasion and occipital area down to the lumbar and sacral regions, most frequently in the lumbar and lumbosacral region.</p> <p>Case presentation</p> <p>Here we report a 5-year-old girl who presented with occasional headache. There were two dimples, one on the dorsal aspect of her head and another on her neck.</p> <p>Conclusion</p> <p>Dermal sinuses are almost always singular and the co-existence of double dermal sinuses has not been reported previously.</p

Evaluation of urinary cysteinyl leukotrienes as biomarkers of severity and putative therapeutic targets in COVID-19 patients

Background Cysteinyl leukotrienes (CysLT) are potent inflammation-promoting mediators, but remain scarcely explored in COVID-19. We evaluated urinary CysLT (U-CysLT) relationship with disease severity and their usefulness for prognostication in hospitalized COVID-19 patients. The impact on U-CysLT of veno-venous extracorporeal membrane oxygenation (VV-ECMO) and of comorbidities such as hypertension and obesity was also assessed. Methods Blood and spot urine were collected in severe (n = 26), critically ill (n = 17) and critically ill on VV-ECMO (n = 17) patients with COVID-19 at days 1-2 (admission), 3-4, 5-8 and weekly thereafter, and in controls (n = 23) at a single time point. U-CysLT were measured by ELISA. Routine markers, prognostic scores and outcomes were also evaluated. Results U-CysLT did not differ between groups at admission, but significantly increased along hospitalization only in critical groups, being markedly higher in VV-ECMO patients, especially in hypertensives. U-CysLT values during the first week were positively associated with ICU and total hospital length of stay in critical groups and showed acceptable area under curve (AUC) for prediction of 30-day mortality (AUC: 0.734, p = 0.001) among all patients. Conclusions U-CysLT increase during hospitalization in critical COVID-19 patients, especially in hypertensives on VV-ECMO. U-CysLT association with severe outcomes suggests their usefulness for prognostication and as therapeutic targets.This work was supported by a RESEARCH 4 COVID-19 grant (project 519, reference number: 613690173) from FCT-Fundacao para a Ciencia e a Tecnologia (special support for rapid implementation projects for innovative response solutions to COVID-9 pandemic). CS-P is a recipient of a Ph.D. fellowship from FCT and MedInUP (UI/BD/150816/2020). P-PT was supported by a research contract within the scope of the RIFF-HEART project funded by FEDER via COMPETE, Portugal 2020-Operational Programme for Competitiveness and Internationalization (POCI) (POCI-01-0145-FEDER-032188) and by FCT (PTDC/MEC-CAR/32188/2017). Open access funding provided by FCT|FCCN (b-on)

Cell functional enviromics: Unravelling the function of environmental factors

<p>Abstract</p> <p>Background</p> <p>While functional genomics, focused on gene functions and gene-gene interactions, has become a very active field of research in molecular biology, equivalent methodologies embracing the environment and gene-environment interactions are relatively less developed. Understanding the function of environmental factors is, however, of paramount importance given the complex, interactive nature of environmental and genetic factors across multiple time scales.</p> <p>Results</p> <p>Here, we propose a systems biology framework, where the function of environmental factors is set at its core. We set forth a "reverse" functional analysis approach, whereby cellular functions are reconstructed from the analysis of dynamic envirome data. Our results show these data sets can be mapped to less than 20 core cellular functions in a typical mammalian cell culture, while explaining over 90% of flux data variance. A functional enviromics map can be created, which provides a template for manipulating the environmental factors to induce a desired phenotypic trait.</p> <p>Conclusion</p> <p>Our results support the feasibility of cellular function reconstruction guided by the analysis and manipulation of dynamic envirome data.</p

Deficiency in origin licensing proteins impairs cilia formation: implications for the aetiology of meier-gorlin syndrome

Mutations in ORC1, ORC4, ORC6, CDT1, and CDC6, which encode proteins required for DNA replication origin licensing, cause Meier-Gorlin syndrome (MGS), a disorder conferring microcephaly, primordial dwarfism, underdeveloped ears, and skeletal abnormalities. Mutations in ATR, which also functions during replication, can cause Seckel syndrome, a clinically related disorder. These findings suggest that impaired DNA replication could underlie the developmental defects characteristic of these disorders. Here, we show that although origin licensing capacity is impaired in all patient cells with mutations in origin licensing component proteins, this does not correlate with the rate of progression through S phase. Thus, the replicative capacity in MGS patient cells does not correlate with clinical manifestation. However, ORC1-deficient cells from MGS patients and siRNA-mediated depletion of origin licensing proteins also have impaired centrosome and centriole copy number. As a novel and unexpected finding, we show that they also display a striking defect in the rate of formation of primary cilia. We demonstrate that this impacts sonic hedgehog signalling in ORC1-deficient primary fibroblasts. Additionally, reduced growth factor-dependent signaling via primary cilia affects the kinetics of cell cycle progression following cell cycle exit and re-entry, highlighting an unexpected mechanism whereby origin licensing components can influence cell cycle progression. Finally, using a cell-based model, we show that defects in cilia function impair chondroinduction. Our findings raise the possibility that a reduced efficiency in forming cilia could contribute to the clinical features of MGS, particularly the bone development abnormalities, and could provide a new dimension for considering developmental impacts of licensing deficiency

Sulforaphane restores cellular glutathione levels and reduces chronic periodontitis neutrophil hyperactivity in vitro

The production of high levels of reactive oxygen species by neutrophils is associated with the local and systemic destructive phenotype found in the chronic inflammatory disease periodontitis. In the present study, we investigated the ability of sulforaphane (SFN) to restore cellular glutathione levels and reduce the hyperactivity of circulating neutrophils associated with chronic periodontitis. Using differentiated HL60 cells as a neutrophil model, here we show that generation of extracellular O2 . - by the nicotinamide adenine dinucleotide (NADPH) oxidase complex is increased by intracellular glutathione depletion. This may be attributed to the upregulation of thiol regulated acid sphingomyelinase driven lipid raft formation. Intracellular glutathione was also lower in primary neutrophils from periodontitis patients and, consistent with our previous findings, patients neutrophils were hyper-reactive to stimuli. The activity of nuclear factor erythroid-2-related factor 2 (Nrf2), a master regulator of the antioxidant response, is impaired in circulating neutrophils from chronic periodontitis patients. Although patients' neutrophils exhibit a low reduced glutathione (GSH)/oxidised glutathione (GSSG) ratio and a higher total Nrf2 level, the DNA-binding activity of nuclear Nrf2 remained unchanged relative to healthy controls and had reduced expression of glutamate cysteine ligase catalytic (GCLC), and modifier (GCLM) subunit mRNAs, compared to periodontally healthy subjects neutrophils. Pre-treatment with SFN increased expression of GCLC and GCM, improved intracellular GSH/GSSG ratios and reduced agonist-activated extracellular O2 . - production in both dHL60 and primary neutrophils from patients with periodontitis and controls. These findings suggest that a deficiency in Nrf2-dependent pathways may underpin susceptibility to hyper-reactivity in circulating primary neutrophils during chronic periodontitis. © 2013 Dias et al

A New Endemic Focus of Chagas Disease in the Northern Region of Veraguas Province, Western Half Panama, Central America

Background: Chagas disease was originally reported in Panama in 1931. Currently, the best knowledge of this zoonosis is restricted to studies done in historically endemic regions. However, little is known about the distribution and epidemiology of Chagas disease in other rural areas of the country. Methods and Findings: A cross-sectional descriptive study was carried out between May 2005 – July 2008 in four rural communities of the Santa Fe District, Veraguas Province. The study included an entomologic search to collect triatomines, bloodmeal type identification and infection rate with trypanosomes in collected vectors using a dot- blot and PCR analysis, genotyping of circulating Trypanosoma cruzi (mini-exon gene PCR analysis) and the detection of chagasic antibodies among inhabitants. The vector Rhodnius pallescens was more frequently found in La Culaca and El Pantano communities (788 specimens), where it was a sporadic household visitor. These triatomines presented darker coloration and larger sizescompared with typical specimens collected in Central Panama. Triatoma dimidiata was more common in Sabaneta de El Macho (162 specimens). In one small sub-region (El Macho), 60 % of the houses were colonized by this vector. Of the examined R. pallescens, 54.7.0 % (88/161) had fed on Didelphis marsupialis, and 24.6 % (34/138) of T. dimidiata specimens collected inside houses were positive for human blood. R. pallescens presented an infection index with T. cruzi of 17.7 % (24/ 136), with T. rangeli of 12.5 % (17/136) and 50.7 % (69/136) were mixed infections. In 117 T. dimidiata domestic specimens th

The substructure of three repetitive DNA regions of Schistosoma haematobium group species as a potential marker for species recognition and interbreeding detection

The file attached is the Published/publisher’s pdf version of the article.© The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated