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Background Research infrastructures are facilities or resources that have proven fundamental for supporting scientific research and innovation. However, they are also known to be very expensive in their establishment, operation and maintenance. As by far the biggest share of these costs is always borne by public funders, there is a strong interest and indeed a necessity to develop alternative business models for such infrastructures that allow them to function in a more sustainable manner that is less dependent on public financing. Methods In this article, we describe a feasibility study we have undertaken to develop a potentially sustainable business model for a vaccine research and development (R&D) infrastructure. The model we have developed integrates two different types of business models that would provide the infrastructure with two different types of revenue streams which would facilitate its establishment and would be a measure of risk reduction. For the business model we are proposing, we have undertaken an ex ante impact assessment that estimates the expected impact for a vaccine R&D infrastructure based on the proposed models along three different dimensions: health, society and economy. Results Our impact assessment demonstrates that such a vaccine R&D infrastructure could achieve a very significant socio-economic impact, and so its establishment is therefore considered worthwhile pursuing. Conclusions The business model we have developed, the impact assessment and the overall process we have followed might also be of interest to other research infrastructure initiatives in the biomedical field.

The first exposure to influenza is presumed to shape the B-cell antibody repertoire, leading to preferential enhancement of the initially formed responses during subsequent exposure to viral variants. Here, we investigated whether this principle remains applicable when there are large genetic and antigenic differences between primary and secondary influenza virus antigens. Because humans usually have a complex history of influenza virus exposure, we conducted this investigation in influenza-naive cynomolgus macaques. Two groups of six macaques were immunized four times with influenza virus-like particles (VLPs) displaying either one (monovalent) or five (pentavalent) different hemagglutinin (HA) antigens derived from seasonal H1N1 (H1N1) strains. Four weeks after the final immunization, animals were challenged with pandemic H1N1 (H1N1pdm09). Although immunization resulted in robust virus-neutralizing responses to all VLP-based vaccine strains, there were no cross-neutralization responses to H1N1pdm09, and all animals became infected. No reductions in viral load in the nose or throat were detected in either vaccine group. After infection, strong virus-neutralizing responses to H1N1pdm09 were induced. However, there were no increases in virus-neutralizing titers against four of the five H1N1 vaccine strains; and only a mild increase was observed in virus-neutralizing titer against the influenza A/Texas/36/91 vaccine strain. After H1N1pdm09 infection, both vaccine groups showed higher virus-neutralizing titers against two H1N1 strains of intermediate antigenic distance between the H1N1 vaccine strains and H1N1pdm09, compared with the naive control group. Furthermore, both vaccine groups had higher HA-stem antibodies early after infection than the control group. In conclusion, immunization with VLPs displaying HA from antigenically distinct H1N1 variants increased the breadth of the immune response during subsequent H1N1pdm09 challenge, although this phenomenon was limited to intermediate antigenic variants.

ABSTRACT Objective: To assess predictive factors for improved diagnostic accuracy with the use of radial-probe EBUS (RP-EBUS). Methods: This was a retrospective review of consecutive patients undergoing RP-EBUS between February of 2012 and January of 2020. Parameters including the presence of a bronchus sign on CT scans, the position of the radial EBUS probe, lesion size, lesion location, and lesion type were analyzed in relation to two defined outcomes (final diagnosis or no diagnosis). Univariate analysis was used in order to explore the individual effects of each parameter on diagnostic accuracy. Multivariate logistic regression was performed to identify significant predictors of diagnostic accuracy. Results: RP-EBUS was used for diagnostic purposes in 101 patients. The lesion was < 3 cm in size in 59 patients (58.4%) and predominantly solid in 60.3%. There was a positive correlation between radial EBUS probe position and diagnostic accuracy (p = 0.036), with 80.9% of the patients showing a bronchus sign on CT scans. Furthermore, 89% of the patients showed a bronchus sign on CT scans and a correlation with diagnostic accuracy (p = 0.030), with 65.8% of the lesions being located in the left/right upper lobe (p = 0.046). When the radial EBUS probe was within the target lesion, the diagnostic yield was = 80.8%. When the probe was adjacent to the lesion, the diagnostic yield was = 19.2%. A bronchus sign on CT scans was the only parameter that independently influenced diagnostic accuracy (adjusted OR, 3.20; 95% CI, 1.081-9.770; p = 0.036). Conclusions: A bronchus sign on CT scans is a powerful predictor of successful diagnosis by RP-EBUS.

Adeno-associated viruses (AAVs) are one of the most promising tools for gene therapy applications. These vectors are purified using affinity and ion exchange chromatography, typically using packed beds of resin adsorbents. This leads to diffusion and pressure drop limitations that affect process productivity. Due to their high surface area and porosity, electrospun nanofiber adsorbents offer mass transfer and flow rate advantages over conventional chromatographic media. The present work investigated the use of affinity cellulose-based nanofiber adsorbents for adeno-associated virus serotype 5 (AAV5) capture, evaluating dynamic binding capacity, pressure drop, and AAV5 recovery at residence times (RT) less than 5 s. The dynamic binding capacity was found to be residence time-dependent, but nevertheless higher than 1.0 × 1014 TP mL−1 (RT = 1.6 s), with a pressure drop variation of 0.14 MPa obtained after loading more than 2,000 column volumes of clarified AAV5 feedstock. The single affinity chromatography purification step using these new affinity adsorbents resulted in 80% virus recovery, with the removal of impurities comparable to that of bead-based affinity adsorbents. The high binding capacity, virus recovery and reduced pressure drop observed at residence times in the sub-minute range can potentially eliminate the need for prior concentration steps, thereby reducing the overall number of unit operations, process time and costs.

Virus-like particles (VLPs) are excellent platforms for the development of influenza vaccine candidates. Nonetheless, their characterization is challenging due to VLPs’ unique biophysical and biochemical properties. To cope with such complexity, multiple analytical techniques have been developed to date (e.g., single-particle analysis, thermal stability, or quantification assays), most of which are rarely used or have been successfully demonstrated for being applicable for virus particle characterization. In this study, several biophysical and biochemical methods have been evaluated for thorough characterization of monovalent and pentavalent influenza VLPs from diverse groups (A and B) and subtypes (H1 and H3) produced in insect cells using the baculovirus expression vector system (IC-BEVS). Particle size distribution and purity profiles were monitored during the purification process using two complementary technologies — nanoparticle tracking analysis (NTA) and tunable resistive pulse sensing (TRPS). VLP surface charge at the selected process pH was also assessed by this last technique. The morphology of the VLP (size, shape, and presence of hemagglutinin spikes) was evaluated using transmission electron microscopy. Circular dichroism was used to assess VLPs’ thermal stability. Total protein, DNA, and baculovirus content were also assessed. All VLPs analyzed exhibited similar size ranges (90–115 nm for NTA and 129–141 nm for TRPS), surface charges (average of −20.4 mV), and morphology (pleomorphic particles resembling influenza virus) exhibiting the presence of HA molecules (spikes) uniformly displayed on M1 protein scaffold. Our data shows that HA titers and purification efficiency in terms of impurity removal and thermal stability were observed to be particle dependent. This study shows robustness and generic applicability of the tools and methods evaluated, independent of VLP valency and group/subtype. Thus, they are most valuable to assist process development and enhance product characterization.

Lentiviral vectors (LVs) are excellent tools for gene transfer into mammalian cells. It is noteworthy that the first gene therapy treatment using LVs was approved for commercialization in 2017. The G glycoprotein from rhabdovirus vesicular stomatitis virus (VSV-G) is the glycoprotein most used to pseudotype LVs, due to its high efficiency in transducing several cell types and its resistance to viral vector purification and storage conditions. However, VSV-G expression induces cytotoxicity, which limits LV production to short periods. As alternative to VSV-G, γ-retrovirus glycoproteins (4070A derived, GaLV derived, and RD114 derived) have been used to pseudotype both γ-retroviral vectors (RVs) and LVs. These glycoproteins do not induce cytotoxicity, allowing the development of stable LV producer cells. Additionally, these LV pseudotypes present higher transduction efficiencies of hematopoietic stem cells when compared to VSV-G. Here, new 4070A-, RD114-TR-, and GaLV-TR-derived glycoproteins were developed with the aim of improving its cytoplasmic tail R-peptide cleavage and thus increase LV infectious titers. The new glycoproteins were tested in transient LV production using the wild-type or the less active T26S HIV-1 protease. The GaLV-TR-derived glycoproteins were able to overcome titer differences observed between LV production using wild-type and T26S protease. Additionally, these glycoproteins were even able to increase LV titers, evidencing its potential as an alternative glycoprotein to pseudotype LVs. Keywords: lentiviral vectors, gene therapy, pseudotyping, envelope glycoproteins, GalV, 4070A, RD114, lentiviral, protease

Resumén Las amiloidosis son un grupo poco común de enfermedades que resultan de la deposición extracelular de amiloide, un material fibrilar derivado de varias proteínas precursoras que se autoensamblan con una conformación de hoja β cruzada anormal altamente ordenada. Los autores presentan el caso de una mujer de 76 años con anemia y deterioro de la función renal, que desarrolla lesiones nodulares intermitentes en la lengua y lesiones hemorrágicas en la mucosa oral. El estudio etiológico permitió el diagnóstico de amiloidosis de cadenas ligeras. Abstract The amyloidoses are a rare group of diseases that result from extracellular deposition of amyloid, a fibrillar material derived from various precursor proteins that self-assemble with highly ordered abnormal cross β-sheet conformation. The authors present the case of a 76-year-old woman with anemia and worsening of renal function, who develops intermittent nodular lesions on the tongue and hemorrhagic lesions on the oral mucosa. The etiological study allowed the diagnosis of light chain amyloidosis.

Resumén Los quistes ováricos son una condición muy común en mujeres, alrededor del 7% de las mujeres reportan tener un quiste ovárico en algún momento de su vida.1 Presentamos el caso de una mujer de 78 años, referida a nuestra consulta para el estudio de la ascitis. La TC abdominopélvica reveló un tumor quístico ovárico masivo, que fue sometido a cirugía y drenaje de aproximadamente 30L (40Kg). Abstract Ovarian cysts are a very commonly encountered condition in females, about 7% of women are reported to have an ovarian cyst at any point in their life.1 We present the case of a 78-year-old woman, referred to our consult for the study of ascites. The Abdominal-pelvic CT revealed a massive ovarian cystic tumor, that was submitted to surgery and drainage of approximately 30L (40Kg).

Weber-Christian disease is a skin condition that features recurring inflammation in the subcutaneous fat layer and systemic symptoms. The disease is a diagnosis of exclusion characterized by a lobular panniculitis without vasculitis in histopathology. The authors report a 80-year-old man with constitutional symptoms and relapsing nonsuppurative nodular panniculitis. The purpose of the case described is to emphasize that rare diseases may occur, therefore their diagnosis can only be made with previous medical knowledge and the delay, or even the absence, in the diagnosis of these pathologies could interfere with the quality of life of patients.

Abstract Background Defined as a headache lasting at least 15 days per month, chronic headache is reported by 3% of the general population, and a substantial proportion of them are refractory to current therapies. Occipital nerve stimulation (ONS) is a treatment option, but is still considered as a last resort treatment especially because of its invasive nature and the cost associated. Some reviews reported a limited efficacy of ONS for the treatment of migraines, with a high risk of complications. However, results reporting its efficacy and safety on other headache disorders are unclear. The aim of this review is to assess the efficacy and safety of ONS in regards to non-migrainous chronic headaches. Methods We will conduct a systematic review and meta-analysis of studies evaluating the use of ONS in comparison to sham stimulation or the best available treatment in patients with chronic headache. MEDLINE, CINHAL, EMBASE, PsycINFO, ECRI Institute Library, WIKISTIM, the Cochrane Library databases, and clinical trial registries will be searched for eligible studies. The review will include adult patients diagnosed with chronic headache excluding migraine. Two independent reviewers will process to the screening of studies according to titles, abstracts, and then full texts. The primary outcome is the overall reduction of head pain severity. The secondary outcomes are rates of reduction in the severity of head pain, headache frequency, and duration, use of medication, impairment, quality of life, healthcare utilization, return to work, and adverse events. Extracted data will include patients’ and procedure characteristics, details on comparative treatment or sham, and clinical outcomes. The risk of bias of the studies will be also independently assessed using the Cochrane risk of bias tools. Discussion This systematic review will allow us to better evaluate the potential role of ONS for the treatment of patients with chronic headache that are refractory to less invasive therapies. It will help to determine the degree of safety of ONS. Moreover, it will help to design and conduct future randomized controlled trials focused on patients who may better respond to such treatment. Systematic review registration PROSPERO CRD42019121623

Abstract Hepatitis C virus (HCV) infects 3% of world population being responsible for nearly half a million deaths annually urging the need for a prophylactic vaccine. Retrovirus like particles are commonly used scaffolds for antigens presentation being the core of diverse vaccine candidates. The immunogenicity of host proteins naturally incorporated in retrovirus was hypothesized to impact the performance of retrovirus based vaccines. In this work, the capacity of engineered retrovirus like particles devoided of host protein CD81 to display HCV envelope antigens was compared to non-engineered particles. A persistent inability of CD81 negative VLPs to incorporate HCV E2 protein as a result from the inefficient transport of HCV E2 to the plasma membrane, was observed. This work enabled the identification of a CD81-mediated transport of HCV E2 while stressing the importance of host proteins for the development of recombinant vaccines.

Replacing batch unit operations of biopharmaceuticals by continuous manufacturing is a maturing concept, with periodic counter-current chromatography (PCC) favoured to replace batch chromatography. Continuous affinity capture of adeno-associated virus (AAV) using PCC has the potential to cope with the high doses required for AAV therapies thanks to its inherent high throughput. The implementation of continuous AAV affinity capture using a four-column PCC process is described herein. First, elution buffer screening was used to optimize virus recovery. Second, breakthrough curves were generated and described using a mechanistic model, which was later used to characterize the loading zone of the PCC. The experimental runs achieved a stable cyclic steady state yielding virus recoveries in line with the optimized batch process (>82%), with almost a three-fold improvement in productivity. The PCC affinity capture process developed here can bolster further improvements to process economics and manufacturing footprint, thereby contributing to the integrated continuous manufacturing concept.

Metabolism plays an essential role in cell fate decisions. However, the methods used for metabolic characterization and for finding potential metabolic regulators are still based on characterizing cellular metabolic steady-state which is dependent on the extracellular environment. In this work, we hypothesized that the response dynamics of intracellular metabolic pools to extracellular stimuli is controlled in a cell type-specific manner. We applied principles of process dynamics and control to human induced pluripotent stem cells (hiPSC) and human neural stem cells (hNSC) subjected to a sudden extracellular glutamine step. The fold-changes of steady-states and the transient profiles of metabolic pools revealed that dynamic responses were reproducible and cell type-specific. Importantly, many amino acids had conserved dynamics and readjusted their steady state concentration in response to the increased glutamine influx. Overall, we propose a novel methodology for systematic metabolic characterization and identification of potential metabolic regulators.

The use of non-standard culture conditions has proven efficient to increase cell performance and recombinant protein production in different cell hosts. However, the establishment of high-producing cell populations through adaptive laboratory evolution (ALE) has been poorly explored, in particular for insect cells. In this study, insect High Five cells were successfully adapted to grow at a neutral culture pH (7.0) through ALE for an improved production of influenza hemagglutinin (HA)-displaying virus-like particles (VLPs). A stepwise approach was used for the adaptation process, in which the culture pH gradually increased from standard 6.2 to 7.0 (ΔPh = 0.2–0.3), and cells were maintained at each pH value for 2–3 weeks until a constant growth rate and a cell viability over 95% were observed. These adapted cells enabled an increase in cell-specific HA productivity up to three-fold and volumetric HA titer of up to four-fold as compared to non-adapted cells. Of note, the adaptation process is the element driving increased specific HA productivity as a pH shift alone was inefficient at improving productivities. The production of HA-VLPs in adapted cells was successfully demonstrated at the bioreactor scale. The produced HA-VLPs show the typical size and morphology of influenza VLPs, thus confirming the null impact of the adaptation process and neutral culture pH on the quality of HA-VLPs produced. This work strengthens the potential of ALE as a bioprocess engineering strategy to improve the production of influenza HA-VLPs in insect High Five cells.

Os cuidados de saúde apresentam uma complexidade crescente. Face à diversidade de novas tecnologias e fármacos disponíveis, é essencial uma abordagem por uma equipa multidisciplinar para que esses cuidados possam ser optimizados e os riscos possam ser minimizados.

Mucins are high-molecular weight glycoproteins (0.25-20 MDa) containing one or more domains that are heavily O-glycosylated. Their implications as targets for cancer treatment have increased the interest in these glycoproteins, mainly in the fields of vaccines and antibodies. However, mucins present high heterogeneity, posing challenges that affect purification processes and quality control analysis. In that sense, it is necessary to develop and improve downstream processes and analytical methods to characterize these products. Here a tool based on biolayer interferometry analysis to improve mucin's detection and quantification in a fast, simple and label free-way is presented. Taking advantage of lectin recognition of mucins' carbohydrate structures, several lectins were evaluated and immobilized on streptavidin biosensors. Different assay conditions were optimized and the most suitable lectin, Aleuria aurantia lectin (AAL), was selected. Bovine Submaxillary Gland and human MUC5B mucins were used as proof of concept and were successfully detected and quantified at different stages of purification. High sensitivity levels were achieved with LOD and LOQ of 3.8 μg mL-1 and 11.7 μg mL-1 for BSM, and 0.2 μg mL-1 and 0.6 μg mL-1 for MUC5B. AAL binding specificity was also confirmed with fucose competition assays. Our method represents an advance on mucins detection and quantification since the existing methods present several disadvantages for process development. Hereafter, it can be applied to the optimization of new or already established downstream processes for mucins' purification.

Introduction: The spinal cord stimulation (SCS) has been described as a valuable neuromodulating procedure in the management of chronic and medically untreated neuropathic pain. Although, many studies have discussed the use of this technique, a question still remains regarding its efficacy in different medical conditions with different etiology in the long term. The aim of this paper is to discuss the risks, complications, cost-effectiveness and results of SCS in patients affected by chronic neuropathic pain based on the comprehensive literature review. Methods: Bibliographic search of references from 1950 to 2016 using the databases MEDLINE, LILACS, SciELO, PubMed, and applied language as selection criteria, choosing preferably recent articles written in Portuguese, Spanish or English. Results: Based on literature review, SCS is a safe, reversible, adjustable and nondestructive surgical procedure demonstrating a significant effect in the reduction of pain intensity and improvement in quality of life in these patients. Furthermore, in spite of the initial high cost to its application, SCS has been associated with lower rates of complications and high rates of cost-effectiveness when compared to standard therapies. Conclusion: Although used in medical conditions with different etiology, the procedure is still an effective and a cost-effective approach to neuropathic pain, mainly in patients affected by failed back pain syndrome (FBSS) and complex regional pain syndrome (CRPS).

The purification of virus particles and viral vectors for vaccine and gene therapy applications is gaining increasing importance in order to deliver a fast, efficient, and reliable production process. Ultrafiltration (UF) is a widely employed unit operation in bioprocessing and its use is present in several steps of the downstream purification train of biopharmaceuticals. However, to date few studies have thoroughly investigated the performance of several membrane materials and cut-offs for virus concentration/diafiltration. The present study aimed at developing a novel class of UF cassettes for virus concentration/diafiltration. A detailed study was conducted to evaluate the effects of (i) membrane materials, namely polyethersulfone (PES), regenerated cellulose (RC), and highly cross-linked RC (xRC), (ii) nominal cut-off, and (iii) UF device geometry at different production scales. The results indicate that the xRC cassettes with a cut-off of approximately 500 kDa are able to achieve a 10-fold concentration factor with 100% recovery of particles with a process time twice as fast as that of a commercially available hollow fiber. DNA and host cell protein clearances, as well as hydraulic permeability and fouling behavior, were also assessed.

Latency-associated nuclear antigen (LANA) mediates γ2-herpesvirus genome persistence and regulates transcription. We describe the crystal structure of the murine gammaherpesvirus-68 LANA C-terminal domain at 2.2 Å resolution. The structure reveals an alpha-beta fold that assembles as a dimer, reminiscent of Epstein-Barr virus EBNA1. A predicted DNA binding surface is present and opposite this interface is a positive electrostatic patch. Targeted DNA recognition substitutions eliminated DNA binding, while certain charged patch mutations reduced bromodomain protein, BRD4, binding. Virus containing LANA abolished for DNA binding was incapable of viable latent infection in mice. Virus with mutations at the charged patch periphery exhibited substantial deficiency in expansion of latent infection, while central region substitutions had little effect. This deficiency was independent of BRD4. These results elucidate the LANA DNA binding domain structure and reveal a unique charged region that exerts a critical role in viral latent infection, likely acting through a host cell protein(s).

Baculoviruses are insect viruses extensively exploited as eukaryotic protein expression vectors. Molecular biology studies have provided exciting discoveries on virus-host interactions, but the application of omic high throughput techniques on the baculovirus-insect cell system has been hampered by the lack of host genome sequencing. While a broader, systems level analysis of biological responses to infection is urgently needed, recent advances on proteomic studies have yielded new insights on the impact of infection on the host cell. These works are reviewed and critically assessed in the light of current biological knowledge of the molecular biology of baculoviruses and insect cells.

Spondylodiskitis is a rare condition in adults but with high index of morbidity and mortality due to difficult diagnosis. The insidious evolution and the unspecificity of symptoms interfere with prompt treatment, being the diagnosis based primarily on a high index of suspicion. There are two major groups of spondylodiskitis: pyogenic, being Staphylococcus aureus the most frequent etiological pathogen, and granulomatous, caused mainly by Mycobacterium tuberculosis and Brucella melitensis, both endemic in Portugal. The authors describe two clinical cases of spondylodiskitis, where the etiological investigation proved a challenge. In both cases the combination of clinical and imagiological data contributed to the appropriate empirical treatment, with a favorable clinical outcome.

A presente reunião de reflexão teve como objectivo debater aspectos referentes à realidade portuguesa no âmbito do diagnóstico e tratamento de doentes com sarcomas. Durante a reunião foram apresentados dados relativos à doença, foram identificadas potenciais vantagens e riscos do tratamento farmacológico e foram debatidos vários aspectos relativos à abordagem dos sarcomas em Portugal. Todos os intervenientes participaram activamente na reunião com apresentações formais pré-concebidas para o efeito ou contribuindo com a sua opinião. Este documento foi objecto de parecer por parte dos participantes na reunião, sendo a sua redacção final resultado de um extenso processo de consulta com o objectivo de obter um texto no qual todos os participantes se revissem.

Previous studies have reported the production of malformed virus-like-particles (VLP) in recombinant host systems. Here we computationally investigate the case of a large triple-layered rotavirus VLP (RLP). In vitro assembly, disassembly and reassembly data provides strong evidence of microscopic reversibility of RLP assembly. Light scattering experimental data also evidences a slow and reversible assembly untypical of kinetic traps, thus further strengthening the fidelity of a thermodynamically controlled assembly. In silico analysis further reveals that under favourable conditions particles distribution is dominated by structural subunits and completely built icosahedra, while other intermediates are present only at residual concentrations. Except for harshly unfavourable conditions, assembly yield is maximised when proteins are provided in the same VLP protein mass composition. The assembly yield decreases abruptly due to thermodynamic equilibrium when the VLP protein mass composition is not obeyed. The latter effect is more pronounced the higher the Gibbs free energy of subunit association is and the more complex the particle is. Overall this study shows that the correct formation of complex multi-layered VLPs is restricted to a narrow range of association energies and protein concentrations, thus the choice of the host system is critical for successful assembly. Likewise, the dynamic control of intracellular protein expression rates becomes very important to minimize wasted proteins.

The successful implementation of human embryonic stem cells (hESCs)-based technologies requires the production of relevant numbers of well-characterized cells and their efficient long-term storage. In this study, cells were microencapsulated in alginate to develop an integrated bioprocess for expansion and cryopreservation of pluripotent hESCs. Different three-dimensional (3D) culture strategies were evaluated and compared, specifically, microencapsulation of hESCs as: i) single cells, ii) aggregates and iii) immobilized on microcarriers. In order to establish a scalable bioprocess, hESC-microcapsules were cultured in stirred tank bioreactors.The combination of microencapsulation and microcarrier technology resulted in a highly efficient protocol for the production and storage of pluripotent hESCs. This strategy ensured high expansion ratios (an approximately twenty-fold increase in cell concentration) and high cell recovery yields (>70%) after cryopreservation. When compared with non-encapsulated cells, cell survival post-thawing demonstrated a three-fold improvement without compromising hESC characteristics.Microencapsulation also improved the culture of hESC aggregates by protecting cells from hydrodynamic shear stress, controlling aggregate size and maintaining cell pluripotency for two weeks.This work establishes that microencapsulation technology may prove a powerful tool for integrating the expansion and cryopreservation of pluripotent hESCs. The 3D culture strategy developed herein represents a significant breakthrough towards the implementation of hESCs in clinical and industrial applications.

Baculovirus infection of Spodoptera frugiperda cells is a system of choice to produce a range of recombinant proteins, vaccines and, potentially, gene therapy vectors. While baculovirus genomes are well characterized, the genome of S. frugiperda is not sequenced and the virus-host molecular interplay is sparsely known. Herein, we describe the application of stable isotope labeling by amino acids in cell culture (SILAC) to obtain the first comparative proteome quantitation of S. frugiperda cells during growth and early baculovirus infection. The proteome coverage was maximized by compiling a search database with protein annotations from insect species. Of interest were differentially proteins related to energy metabolism, endoplasmic reticulum and oxidative stress, yet not investigated in the scope of baculovirus infection. Further, the reduced expression of key viral-encoded proteins early in the infection cycle is suggested to be related with decreased viral replication at high cell density culture. These findings have implications for virological research and improvement of baculovirus-based bioprocesses.