Expression of spidroin proteins in the silk glands of golden orb-weaver spiders.

The expression of spidroins in the major ampullate, minor ampullate, flagelliform, and tubuliform silk glands of Trichonephila clavipes spiders was analyzed using proteomics analysis techniques. Spidroin peptides were identified and assigned to different gene products based on sequence concurrence when compared with the whole genome of the spider. It was found that only a relatively low proportion of the spidroin genes are expressed as proteins in any of the studied glands. In addition, the expression of spidroin genes in different glands presents a wide range of patterns, with some spidroins being found in a single gland exclusively, while others appear in the content of several glands. The combination of precise genomics, proteomics, microstructural, and mechanical data provides new insights both on the design principles of these materials and how these principles might be translated for the production of high-performance bioinspired artificial fibers.

Differential N- and O-glycosylation signatures of HIV-1 Gag virus-like particles and coproduced extracellular vesicles.

Human immunodeficiency virus 1 (HIV-1) virus-like particles (VLPs) are nanostructures derived from the self-assembly and cell budding of Gag polyprotein. Mimicking the native structure of the virus and being noninfectious, they represent promising candidates for the development of new vaccines as they elicit a strong immune response. In addition to this, the bounding membrane can be functionalized with exogenous antigens to target different diseases. Protein glycosylation depends strictly on the production platform and expression system used and the displayed glycosylation patterns may influence downstream processing as well as the immune response. One of the main challenges for the development of Gag VLP production bioprocess is the separation of VLPs and coproduced extracellular vesicles (EVs). In this study, porous graphitized carbon separation method coupled with mass spectrometry was used to characterize the N- and O- glycosylation profiles of Gag VLPs produced in HEK293 cells. We identified differential glycan signatures between VLPs and EVs that could pave the way for further separation and purification strategies to optimize downstream processing and move forward in VLP-based vaccine production technology.

Knowledge about Pain in Spanish Nursing Students.

BACKGROUND: All nurses should receive training and education regarding pain as part of their pre-graduate stage, as its assessment and appropriate management when treating patients largely depends on them. With the right knowledge it is possible to reduce its high prevalence, as well as the serious consequences it can lead to. AIM: To determine the level of knowledge and attitudes towards pain of final-year nursing students in Spain. METHODS: Descriptive cross-sectional study using a convenience sample of five Spanish universities during the academic year 2020-2021. The Spanish version of the Knowledge and Attitudes Survey Regarding Pain (KASRP) was used. In addition, socio-demographic variables such as age, sex, relationship status, employment status, and the number of dependants were collected. The specific palliative or oncology subjects of each university was also assessed. RESULTS: A total of 224 questionnaires were collected. One of the nursing universities obtained the best score in the KASRP (59.75%) which was significant (p = .001). This university was the only one that offers specific subjects in palliative or oncologic care. A training deficit in aspects related to pain assessment and pharmacologic concepts was detected. We found no relationship between the KASRP and the different sociodemographic variables. CONCLUSIONS: Specific training in palliative care improves the students' knowledge regarding pain, although the results did not reach an acceptable minimum. The universities' training programs for Spanish students need to be adapted in order to achieve better results.

Experiences and mediating factors in nurses' responses to electronic device alarms: A phenomenological study.

AIM: This study aims to explore the experiences and mediating factors of nurses' responses to electronic device alarms in critical care units (CCUs). BACKGROUND: Alarm fatigue occasionally has adverse consequences for patient safety. METHODS: This qualitative study was designed and analysed following Giorgi's descriptive phenomenological approach. Seventeen nurses were theoretically sampled, reaching information saturation. Semistructured interviews were used to collect the data. RESULTS: Three central themes explained nurses' experiences: general perceptions about alarms (basic equipment of the CCU), strategies to reduce false alarms (training in the configuration of monitors, customization of the alarms to fit he patient's condition. teamwork and taking advantage of the development of technology) and key elements of the response to alarms (information about patient's condition, nurses' clinical experience, type of CCU, 'cry-wolf' phenomenon and nurse/patient ratio). CONCLUSIONS: To reduce false alarms, nurses need further postgraduate training, training on monitors and customizing alarms to fit the patient's health status. The complex process of deciding to respond to an alarm includes environmental, professional variables and patient status. IMPLICATIONS FOR NURSING MANAGEMENT: Nurse managers should ensure that nurses have sufficient experience and training in the CCU, improve the nurse/patient ratio, promote teamwork and ensure that the devices are the latest generation.

Characterization of HIV-1 virus-like particles and determination of Gag stoichiometry for different production platforms.

The importance of developing new vaccine technologies towards versatile platforms that can cope with global virus outbreaks has been evidenced with the most recent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. Virus-like particles (VLPs) are a highly immunogenic, safe, and robust approach that can be used to base several vaccine candidates on. Particularly, HIV-1 Gag VLPs is a flexible system comprising a Gag core surrounded by a lipid bilayer that can be modified to present diverse types of membrane proteins or antigens against several diseases, like influenza, dengue, West Nile virus, or human papillomavirus, where it has been proven successful. The size distribution and structural characteristics of produced VLPs vary depending on the cell line used to produce them. In this study, we established an analytical method of characterization for the Gag protein core and clarified the current variability of Gag stoichiometry in HIV-1 VLPs depending on the cell-based production platform, directly determining the number of Gag molecules per VLP in each case. Three Gag peptides have been validated to quantify the number of monomers using parallel reaction monitoring, an accurate and fast, mass-spectrometry-based method that can be used to assess the quality of the produced Gag VLPs regardless of the cell line used. An average of 3617 ± 17 monomers per VLP was obtained for HEK293, substantially varying between platforms, including mammalian and insect cells. This offers a key advantage in quantification and quality control methods to characterize VLP production at a large scale to accelerate new recombinant vaccine production technologies.

Metabolic engineering of HEK293 cells to improve transient transfection and cell budding of HIV-1 virus-like particles.

HIV-1 Gag virus-like particles (VLPs) are promising candidates for the development of future vaccines. Recent viral outbreaks have manifested the need of robust vaccine production platforms able to adapt to new challenges while achieving mass production capacity. For the rapid production of VLPs, the method of transient gene expression (TGE) have proved highly efficient. Based on a previous characterization of the HEK293 cell line upon transient transfection using multiplexed quantitative proteomics, molecular production bottlenecks and metabolic pathways likely to be optimized were identified. In this study, these molecular components and metabolic pathways have been explored and modulated via transient metabolic engineering using approaches like design of experiments to fully exploit and optimize VLP production, transfection and budding efficiency. Upon overexpression of endosomal sorting complex required for transport accessory proteins like NEDD4L and CIT, VLP production increased 3.3 and 2.9-fold, respectively. Overexpression of glycosphingolipid precursor enzyme UGCG improved transfection efficiency by 17% and knocking-down the Gag-binding protein CNP improved 2.5-fold VLP specific productivity. Combining CNP inhibition and UGCG overexpression further improved budding efficiency by 37.3%. Modulating VLP production and accessory pathways like intracellular budding, demonstrated the potential of metabolic engineering to optimize and intensify the development of robust production platforms for future vaccines.

Multiplexed Quantitative Proteomic Analysis of HEK293 Provides Insights into Molecular Changes Associated with the Cell Density Effect, Transient Transfection, and Virus-Like Particle Production.

The production of virus-like particles (VLPs) has gained importance over the last few years owing to the benefits they provide compared to conventional vaccines. The biopharmaceutical industry is currently searching for safer candidates based on VLPs for new and existing vaccines and implementing new methods of manufacturing, thus allowing a more sustainable, effective, and species-specific production. Despite achieving lower yields compared to traditional platforms, the use of mammalian cells provides the right post-translational modifications, and consequently, the intensification of bioprocesses using mammalian cell platforms has become a matter of pressing concern. One of the methods subjected to intensification is transient gene expression, which has been proven to be highly effective regarding VLP production for preclinical or even clinical trials. In this work, a multiplexed quantitative proteomic approach has been applied to study the molecular characteristics of HEK293 cell cultures when growing at cell densities higher than 4 × 106 cells/mL and to study the effects related to cell transfection and VLP production. The obtained results revealed a set of functional and metabolic profiles of HEK293 under these three different conditions that allowed the identification of physiological bottlenecks regarding VLP production. Regarding the cell density effect, molecular alterations in the cell biology were proposed to help explain the difficulty for the cells to be transfected at higher densities. In addition, an overall disruption of cellular homeostasis after transfection was observed based on altered biological processes, and after identifying potential pathways liable to be optimized via metabolic engineering, different solutions were proposed to improve VLP production.

Molecular Characterization of the Coproduced Extracellular Vesicles in HEK293 during Virus-Like Particle Production.

Vaccine therapies based on virus-like particles (VLPs) are currently in the spotlight due to their potential for generating high immunogenic responses while presenting fewer side effects than conventional vaccines. These self-assembled nanostructures resemble the native conformation of the virus but lack genetic material. They are becoming a promising platform for vaccine candidates against several diseases due to the ability of modifying their membrane with antigens from different viruses. The coproduction of extracellular vesicles (EVs) when producing VLPs is a key phenomenon currently still under study. In order to characterize this extracellular environment, a quantitative proteomics approach has been carried out. Three conditions were studied: non-transfected, transfected with an empty plasmid as control, and transfected with a plasmid coding for HIV-1 Gag polyprotein. A shift in EV biogenesis has been detected upon transfection, changing the production from large to small EVs. Another remarkable trait found was the presence of DNA being secreted within vesicles smaller than 200 nm. Studying the protein profile of these biological nanocarriers, it was observed that EVs were reflecting an overall energy homeostasis disruption via mitochondrial protein deregulation. Also, immunomodulatory proteins like ITGB1, ENO3, and PRDX5 were identified and quantified in VLP and EV fractions. These findings provide insight on the nature of the VLP extracellular environment defining the characteristics and protein profile of EVs, with potential to develop new downstream separation strategies or using them as adjuvants in viral therapies.

Quality of life in elderly people after a hip fracture: a prospective study.

BACKGROUND: Hip fracture is an important social and medical problem due to its increasing prevalence, the consequences for health and the economic impact on the health care system, but there is no doubt that it also has repercussions on health-related quality of life (HRQoL). Hence the importance of understanding and determining the impact of the condition on everyday life from the perspective of the patient's physical, emotional and social well-being. PURPOSE: To determine the impact of hip fracture on HRQoL of people over the age of 65 1 month after surgery, related factors and the effects on functional ability and mood. METHODS: Prospective observational study conducted in the traumatology units of two university hospitals in the province of Cáceres with consecutive sampling of all patients over the age of 65 admitted for hip fracture surgery during the study period. Sociodemographic and clinical data were recorded at the time of admission and prospectively at the follow-up visit 1 month later. Clinical, social, quality of life (EQ-5D-), basic functional and instrumental capacity (Barthel Index (BI) and Lawton & Brody Scale), and geriatric depression (Yesavage) variables were collected. RESULTS: The study included 224 patients with a median age of 84.6 years (SD ± 6.1), 76.3% were female. Charlson's comorbidity was 5.3 (SD ± 1.2). The EQ-5D index decreased from 0.62 (SD ± 0.35) to 0.16 at 1 month follow up (SD ± 0.20) p <  0.001. The mean Visual Analog Scale (VAS) score of EQ-5D decreased from 72.8 (SD ±15.8) to 48.3 (SD ± 17.2) p <  0.001. All dimensions of EQ-5D showed a significant reduction from the time of pre-fracture status to 1 month after surgery. Independent factors associated with HRQoL 1 month after surgery were pre-fracture status Barthel Index score, Lawton and Brody scale, presence of depression, and type of surgery. CONCLUSIONS: After a hip fracture, patients experience considerable deterioration in their HRQoL, especially in self-care, daily activities, and mobility. There is also a significant decline in functional capacity for both the basic and instrumental activities of daily living. One month after surgery, HRQoL is a long way from pre-fracture levels.

Segmental fat-free and fat mass measurements by bioelectrical impedance analysis in 2,224 healthy Spanish women aged 18-85 years.

OBJECTIVES: This study provides updated data on body composition in adult Spanish women. METHODS: We considered data, including height and weight, from a survey conducted on a total of 4,013 adult women between 2009 and 2010. A subgroup of 2,224 women completed a bioelectrical body impedance analysis of body composition using a Tanita Body Composition Analyzer (Model no. BF-418). RESULTS: Total fat mass (FM) gradually increased between 18 and 74 years of age and decreased thereafter. FM increased in both legs between 65 and 74 years of age (5.69 ± 1.4 Kg and 5.66 ± 1.4 Kg for right and left legs, respectively) and decreased thereafter. FM in the right arm increased until 44 years of age (1.22 ± 2.6 Kg), decreased to 1.19 ± 0.5 Kg between 45 and 54 years of age, and increased to 1.54 ± 0.63 from 65 to 74 years of age. FM in the left arm increased constantly until it reached a peak of 1.63 ± 0.6 Kg between 65 and 74 years of age and decreased thereafter. FM increased in the trunk throughout life (peaks at 13.27±3.8 Kg) until subjects reached between 65 and 74 years of age. Fat free mass increased until 44 years of age (42.42 ± 4.17 Kg) and decreased thereafter. The prevalence of overweight/obesity significantly increased with age in the entire sample. CONCLUSIONS: Our results indicate that age-related increases in weight were at least partially due to increased adiposity.

[LIMB SHAKING: DESCRIPTION OF A CASE FROM A NURSING PERSPECTIVE]. / Limb shaking: DESCRIPCIÓN DE UN CASO DESDE LA PERSPECTIVA ENFERMERA.

INTRODUCTION: Limb shaking, which was described by MillerFisher in 1962, is characterized by involuntary, irregular, stereotyped a hemibody triggered by the contralateral hemisphere hypoperfusion. It is associated with an occlusion or stenosis preoclusive of the extracranial internal carotid artery (ICA) contralateral to the movements, and poor circulation contralateral. This causes ischemia resulting in typical clinical manifestations of stroke and these abnormal movements. OBJECTIVE: To describe a case of limb shaking. MATERIALS AND METHODS: 59 years old man, with cardiovascular risk factors, who go to the Emergency room with symptoms and motor dysphasia and sudden loss of strength in right limbs, with distal brachial predominance. Admitted to Stroke Unit for neurological and hemodynamic monitoring, which coincides with the beginning of the sitting have an episode of these involuntary movements. RESULTS: Diagnostic tests confirm a left frontal cortical ischemic stroke. The EEG shows a normal background bioelectric activity. The angio-MRI and angiography showed a left ICA pseudoocclusion. DISCUSSON: A diagnosis of limb shaking based in the clinical examination and additional tests, which confirm the finding of a left ICA pseudo-occlusion and refractory to antiepileptic treatment. CONCLUSION: The limb shaking is a rare syndrome, which must be recognized and differentiated early from other processes to treat it properly. Treatment is aimed at restoring cerebral blood flow through the ischemic hemisphere revascularization.

Quantitative ultrasound measurements of the calcaneus and hand phalanges in elderly Spanish men: relationship with peripheral bone mineral density of the hand phalanges.

OBJECTIVES: The aims of this pilot study were to describe quantitative ultrasound (US) measurements and peripheral bone mineral density (BMD) of the hand phalanges on dual-energy x-ray absorptiometry and to examine the correlations between them in elderly Spanish men. METHODS: We studied 199 healthy men (mean age ± SD, 73.31 ± 5.10 years). The participants were not taking any medications, and they reported no diseases, including diseases that are associated with abnormalities in mineral metabolism. Phalangeal and calcaneal quantitative US measurements and phalangeal BMD measurements were performed in all participants. RESULTS: A bivariate correlation analysis showed no association between quantitative US assessments at the phalanges or the calcaneus (P = .409). After adjustment for potential confounders, the correlation between phalangeal BMD and phalangeal quantitative US measurements was r = 0.417 (P < .0001), and the correlation for calcaneal quantitative US was r = 0.26 (P = .001). Further adjustment by percentage of body fat increased quantitative US correlations with phalangeal BMD: r = 0.450 (P < .0001) at the phalanges; r = 0.291 (P = .001) at the calcaneus. CONCLUSIONS: There is a small correlation between quantitative US measurements at the calcaneus and phalangeal BMD that increases to a moderate level with quantitative US measurements at the phalanges in elderly Spanish men.

Unravelling the essential elements for recombinant adeno-associated virus (rAAV) production in animal cell-based platforms.

Recombinant adeno-associated viruses (rAAVs) stand at the forefront of gene therapy applications, holding immense significance for their safe and efficient gene delivery capabilities. The constantly increasing and unmet demand for rAAVs underscores the need for a more comprehensive understanding of AAV biology and its impact on rAAV production. In this literature review, we delved into AAV biology and rAAV manufacturing bioprocesses, unravelling the functions and essentiality of proteins involved in rAAV production. We discuss the interconnections between these proteins and how they affect the choice of rAAV production platform. By addressing existing inconsistencies, literature gaps and limitations, this review aims to define a minimal set of genes that are essential for rAAV production, providing the potential to advance rAAV biomanufacturing, with a focus on minimizing the genetic load within rAAV-producing cells.

Unlocking DOE potential by selecting the most appropriate design for rAAV optimization.

Producing recombinant adeno-associated virus (rAAV) for gene therapy via triple transfection is an intricate process involving many cellular interactions. Each of the different elements encoded in the three required plasmids-pHelper, pRepCap, and pGOI-plays a distinct role, affecting different cellular pathways when producing rAAVs. The required expression balance emphasizes the critical need to fine-tune the concentration of all these different elements. The use of design of experiments (DOE) to find optimal ratios is a powerful method to streamline the process. However, the choice of the DOE method and design construction is crucial to avoid misleading results. In this work, we examined and compared four distinct DOE approaches: rotatable central composite design (RCCD), Box-Behnken design (BBD), face-centered central composite design (FCCD), and mixture design (MD). We compared the abilities of the different models to predict optimal ratios and interactions among the plasmids and the transfection reagent. Our findings revealed that blocking is essential to reduce the variability caused by uncontrolled random effects and that MD coupled with FCCD outperformed all other approaches, improving volumetric productivity 109-fold. These outcomes underscore the importance of selecting a model that can effectively account for the biological context, ultimately yielding superior results in optimizing rAAV production.

Extracellular vesicle depletion and UGCG overexpression mitigate the cell density effect in HEK293 cell culture transfection.

The hitherto unexplained reduction of cell-specific productivity in transient gene expression (TGE) at high cell density (HCD) is known as the cell density effect (CDE). It currently represents a major challenge in TGE-based bioprocess intensification. This phenomenon has been largely reported, but the molecular principles governing it are still unclear. The CDE is currently understood to be caused by the combination of an unknown inhibitory compound in the extracellular medium and an uncharacterized cellular change at HCD. This study investigates the role of extracellular vesicles (EVs) as extracellular inhibitors for transfection through the production of HIV-1 Gag virus-like particles (VLPs) via transient transfection in HEK293 cells. EV depletion from the extracellular medium restored transfection efficiency in conditions that suffer from the CDE, also enhancing VLP budding and improving production by 60%. Moreover, an alteration in endosomal formation was observed at HCD, sequestering polyplexes and preventing transfection. Overexpression of UDP-glucose ceramide glucosyltransferase (UGCG) enzyme removed intracellular polyplex sequestration, improving transfection efficiency. Combining EV depletion and UGCG overexpression improved transfection efficiency by ∼45% at 12 × 106 cells/mL. These results suggest that the interaction between polyplexes and extracellular and intracellular vesicles plays a crucial role in the CDE, providing insights for the development of strategies to mitigate its impact.

Internalization of PEI-based complexes in transient transfection of HEK293 cells is triggered by coalescence of membrane heparan sulfate proteoglycans like Glypican-4.

Polymer-cationic mediated gene delivery is a well-stablished strategy of transient gene expression (TGE) in mammalian cell cultures. Nonetheless, its industrial implementation is hindered by the phenomenon known as cell density effect (CDE) that limits the cell density at which cultures can be efficiently transfected. The rise in personalized medicine and multiple cell and gene therapy approaches based on TGE, make more relevant to understand how to circumvent the CDE. A rational study upon DNA/PEI complex formation, stability and delivery during transfection of HEK293 cell cultures has been conducted, providing insights on the mechanisms for polyplexes uptake at low cell density and disruption at high cell density. DNA/PEI polyplexes were physiochemically characterized by coupling X-ray spectroscopy, confocal microscopy, cryo-transmission electron microscopy (TEM) and nuclear magnetic resonance (NMR). Our results showed that the ionic strength of polyplexes significantly increased upon their addition to exhausted media. This was reverted by depleting extracellular vesicles (EVs) from the media. The increase in ionic strength led to polyplex aggregation and prevented efficient cell transfection which could be counterbalanced by implementing a simple media replacement (MR) step before transfection. Inhibiting and labeling specific cell-surface proteoglycans (PGs) species revealed different roles of PGs in polyplexes uptake. Importantly, the polyplexes uptake process seemed to be triggered by a coalescence phenomenon of HSPG like glypican-4 around polyplex entry points. Ultimately, this study provides new insights into PEI-based cell transfection methodologies, enabling to enhance transient transfection and mitigate the cell density effect (CDE).

Bone mass of Spanish school children: impact of anthropometric, dietary and body composition factors.

The purpose of this study was to: (a) determine the relationship between quantitative ultrasound (QUS) results and anthropometric, dietary and body composition factors and establish reference ranges for amplitude-dependent speed of sound (Ad-SoS) in the phalanges and broadband ultrasound attenuation (BUA) in the calcaneus of children from Extremadura, Spain, and (b) to present reference curves for this population. Healthy children (n = 245), aged 4-16 years, were included (124 girls and 121 boys). Phalangeal and calcaneal QUS measurements were performed using DBM Sonic Bone Profiler and McCue CUBA Clinical ultrasound devices, respectively. Weight, height and body mass index (BMI) were evaluated by anthropometric methods. Fat percentage, fat mass, lean mass (FFM) and total body water (TBWater) were evaluated by bioelectrical impedance measurements using a Holtain body composition analyzer. Food intake was evaluated by a 7-day food record. A gender analysis revealed that Ad-SoS and BUA parameters increased significantly with age and that both positively correlated with age, weight, height, BMI, FFM and TBWater. For both genders, Ad-SoS showed significant and positive correlations with age, weight, height, BMI, FFM, BUA and TBWater.

The cell density effect in animal cell-based bioprocessing: Questions, insights and perspectives.

One of the main challenges in the development of bioprocesses based on cell transient expression is the commonly reported reduction of cell specific productivity at increasing cell densities. This is generally known as the cell density effect (CDE). Many efforts have been devoted to understanding the cell metabolic implications to this phenomenon in an attempt to design operational strategies to overcome it. A comprehensive analysis of the main studies regarding the CDE is provided in this work to better define the elements comprising its cause and impact. Then, examples of methodologies and approaches employed to achieve successful transient expression at high cell densities (HCD) are thoroughly reviewed. A critical assessment of the limitations of the reported studies in the understanding of the CDE is presented, covering the leading hypothesis of the molecular implications. The overall analysis of previous work on CDE may offer useful insights for further research into manufacturing of biologics.

Optimization, Production, Purification and Characterization of HIV-1 GAG-Based Virus-like Particles Functionalized with SARS-CoV-2.

Virus-like particles (VLPs) constitute a promising approach to recombinant vaccine development. They are robust, safe, versatile and highly immunogenic supra-molecular structures that closely mimic the native conformation of viruses without carrying their genetic material. HIV-1 Gag VLPs share similar characteristics with wild-type severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus, making them a suitable platform for the expression of its spike membrane protein to generate a potential vaccine candidate for COVID-19. This work proposes a methodology for the generation of SARS-CoV-2 VLPs by their co-expression with HIV-1 Gag protein. We achieved VLP functionalization with coronavirus spike protein, optimized its expression using a design of experiments (DoE). We also performed the bioprocess at a bioreactor scale followed by a scalable downstream purification process consisting of two clarifications, an ion exchange and size-exclusion chromatography. The whole production process is conceived to enhance its transferability at current good manufacturing practice (cGMP) industrial scale manufacturing. Moreover, the approach proposed could be expanded to produce additional Gag-based VLPs against different diseases or COVID-19 variants.

Micrometric DNA/PEI polyplexes correlate with higher transient gene expression yields in HEK 293 cells.

DNA delivery with polyethylenimine (PEI) has been widely used in the last three decades for the transfection of mammalian cells. Advances in novel characterization techniques at the nanometric scale offer new opportunities to revisit the physicochemical properties of DNA/PEI polyplexes that lead to efficient transfection. In this work, these properties are tuned by studying the synergies between simple parameters such as NaCl concentration, pH and incubation time in the DNA/PEI polyplex preparation protocol by means of Design of Experiments (DoE). By doing so, a model is obtained where an optimal NaCl concentration of 125 mM and an incubation time of 11 min provided the highest transfection yields. Correlation analyses between the physicochemical properties of DNA/PEI polyplexes and the predicted model responses revealed the existence of an optimal degree of aggregation in the pre-complexing solution to attain the highest transfection efficiencies. The presence of these micrometric DNA/PEI polyplex aggregates was confirmed by several nanoparticle characterization techniques including cryo-TEM, DLS and flow virometry. The findings provide a better understanding of the role of DNA/PEI aggregates in transient gene expression approaches, in particular considering that similar complexation protocols and saline solutions are widely used for the transfection of mammalian cell cultures.