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.

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).

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.

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.

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.

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.

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.

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.

Long-Term Dietary and Physical Activity Interventions in the School Setting and Their Effects on BMI in Children Aged 6-12 Years: Meta-Analysis of Randomized Controlled Clinical Trials.

Preventive actions and potential obesity interventions for children are mainly researched throughout the school period, either as part of the school curricula or after regular school hours, via interventions mostly lasting less than 12 months. We aimed to perform a meta-analysis on randomized controlled clinical trials to evaluate the evidence of the efficacy of long-term school-based interventions in the management of childhood obesity in terms of BMI from a dietary and physical activity-based approach. Eleven randomized controlled clinical trials were examined using the random effects model, and the results showed that there were no significant effects associated with physical activity + nutrition intervention in school children aged 6-12 years, with a pooled standardized mean difference (SMD) (95% CI) of -0.00 (-0.05, 0.04). No effects were observed after subgroup analysis based on the intervention length. The findings from our study indicate that long-term school-based interventions on physical activity and dietary habits received by children aged 6-12 years seem to have no effect on BMI. However, the promotion of such interventions should not be discouraged, as they promote additional positive health outcomes for other domains of children's health.

Health-Related Quality of Life in Spanish Women with Eating Disorders.

People with eating disorders show impaired health-related quality of life (HRQoL). We aimed to investigate the relative role of physical and mental factors and stage of change as possible predictors of HRQoL in a group of Spanish women (n = 124) with eating disorders. For this purpose, initial and follow-up data were obtained after 6 months from patients attending an outpatient treatment unit for eating disorders. The determinants of the physical and mental domains of the Medical Outcomes Survey Short-form Health Survey (SF-36) questionnaire were investigated in the total sample and separately based on the eating disorder diagnosis by multiple linear regression. Lower scores in the physical component of the SF-36 questionnaire were associated with the presence of a higher body mass index (BMI) at follow-up as well as a higher score in the "action" component of the Attitudes towards Change in Eating Disorders Questionnaire (ACTA). Conversely, a higher index in the EuroQoL-5D overall quality of life questionnaire (EQ-5D) and the presence of obsessive compulsive disorder were associated with a higher score in the physical dimension. The instrument used demonstrated the ability to assess changes associated with the physical component of these patients over the period studied, and the analysis provided more information and specific data on different aspects of HRQoL, thus allowing a more detailed analysis of the information.

Impact of a Postoperative Intervention Educational Program on the Quality of Life of Patients with Hip Fracture: A Randomized, Open-Label Controlled Trial.

The objective of this study was to determine the impact of a postoperative educational intervention program on the health-related quality of life (HRQoL) of patients with hip fracture using a controlled clinical trial in a randomized, multicenter study. In total, 102 patients (45.5%) from trauma units at the two University Hospitals of the province of Cáceres received the educational program, whereas 122 (54.5%) did not. Patients were consecutively included in either an intervention or a control group. Patients from the intervention group received an educational program during admission and the postoperative period. Patients from the control group did not receive any educational program. These patients were managed according to routine protocols. The patients were predominantly female (76.3%), aged 84.6 years (SD 6.1). All dimensions in both groups at 12 months showed a significant decrease with respect to baseline, except for bodily pain in both groups (p = 0.447; p = 0.827) and social functioning in the intervention group (p = 0.268). Patients receiving the educational program showed higher levels in the dimensions of the Mental Component Summary (MCS-12) (p = 0.043), vitality (p = 0.010), and social functioning (p < 0.001), as well as in the dimensions of the SF-12 health survey questionnaire of HRQoL 12 months after surgery. In conclusion, our study of the intervention group showed that there were significant improvements in MCS-12, vitality, and social function dimensions compared to the control group.

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.

Nursing Genetic Research: New Insights Linking Breast Cancer Genetics and Bone Density.

Nursing research is expected to provide options for the primary prevention of disease and health promotion, regardless of pathology or disease. Nurses have the skills to develop and lead research that addresses the relationship between genetic factors and health. Increasing genetic knowledge and research capacity through interdisciplinary cooperation as well as the development of research resources, will accelerate the rate at which nurses contribute to the knowledge about genetics and health. There are currently different fields in which knowledge can be expanded by research developed from the nursing field. Here, we present an emerging field of research in which it is hypothesized that genetics may affect bone metabolism. Better insight of genetic factors that are contributing to metabolic bone diseases would allow for focused nursing care and preventive interventions.

Quality of life after hip fracture: a 12-month prospective study.

BACKGROUND: Hip fracture is an important and frequent health problem worldwide. To date, there are still limited studies focused on the analysis of health-related quality of life (HRQOL) after a hip fracture in the Spanish population, especially with long-term follow-up. OBJECTIVE: To determine the HRQOL at 12 months after hip fracture and to identify potential factors associated with HRQOL. DESIGN: Prospective observational study. SETTING: Traumatology units of two university hospitals in province Cáceres (Spain). PARTICIPANTS: A total of 224 patients were admitted to the unit and required immediate surgery due to a hip fracture. METHODS: HRQOL was measured with the EuroQol-5D questionnaire (EQ-5D) and the SF-12 Health Survey. RESULTS: Scores from the visual analog scale EQ-5D decreased significantly (p < 0.001) from 72.8 at baseline to 48.3 after 1 month, to 48.2 after 6 months and to 46.1 after 12 months. The EQ-5D index score showed a similar significant reduction (p < 0.001) from 0.6 to 0.1, 0.3 and 0.3, respectively. Values of the physical component summary (PCS-12) significantly decreased (p < 0.001) from 38.6 at baseline to 31.0, 33.1 and 33.5. The mental component summary (MCS-12) decreased from 46.5 to 44.8 after 6 months (p = 0.022) and 44.3 after 12 months (p = 0.005). Factors potentially associated with HRQOL at 12 months after hip fracture were depression status after 12 months (B = 0-1.876; 95% CI [-2.409 to -1.343]; p < 0.001), functional ambulation classification after 12 months (B = -12.133; 95% CI [-17.970 to -6.297]; p < 0.001), EQ-5D VAS at baseline (B = 0.223; 95% CI [0.115-0.330]; p < 0.001), and age (B = -0.323; 95% CI [-0.594 to -0.053; p = 0.015). CONCLUSIONS: Patients experience a significant impairment in HRQOL H after a hip fracture, especially in self-care, pain/discomfort, usual activities, mobility and anxiety/depression. The decline in the HRQOL is effective the first month and lasts at least 12 months after the surgical intervention.

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.

Olive Oil Consumption and Bone Microarchitecture in Spanish Women.

Olive oil has been demonstrated to enhance various cardiometabolic risk factors. However, to our knowledge, the association between olive oil intake and cortical and trabecular bone microarchitecture has never been evaluated in Spanish women. We aimed to examine the association between olive oil intake and cortical and trabecular bone microarchitecture. We analyzed 523 women aged 50 (9) year, range (23⁻81) year. Participants underwent dual-energy X-ray absorptiometry and peripheral quantitative computed tomography scans. Dietary intake of calcium, vitamin D, energy and olive oil (g/day) were assessed by a self-administered food frequency questionnaire (FFQ). After adjustment for potential confounding factors (calcium (mg/day), vitamin D (µg/day) energy (Kcal/day), age, body mass index (BMI) (kg/m²), menopausal status, and osteoporotic diagnosis (normal, osteopenia, or osteoporosis)), there were significant increases in volumetric bone mineral density (vBMD) (mg/cm³) (p < 0.01) in the group with a higher intake of olive oil. Total, trabecular and cortical bone density were positively correlated with olive oil intake. The dietary intake of olive oil was significantly associated with vBMD in multiple regression analysis; total density: olive oil intake (g/day) standardized ß = 0.185 (p < 0.001), trabecular density: olive oil intake (g/day) standardized ß = 0.186 (p < 0.001) and cortical density olive oil intake (g/day) standardized ß = 0.114 (p = 0.008). We conclude that the dietary intake of olive oil is positively associated with a better vBMD in Spanish women.

Dietary Intake of Cadmium, Lead and Mercury and Its Association with Bone Health in Healthy Premenopausal Women.

The bone is one of the relevant target organs of heavy metals, and heavy metal toxicity is associated with several degenerative processes, such osteoporosis and bone mineral alterations, that could lead to fractures. We aimed to study a presumed relationship between bone density, evaluated by quantitative bone ultrasound (QUS), dual-energy X-ray absorptiometry (DXA) and peripheral quantitative computed tomography (pQCT) and the dietary intake of cadmium, lead and mercury in healthy premenopausal women. A total of 158 healthy, non-smoking, premenopausal women were incorporated into the study. A validated food frequency questionnaire (FFQ) was administered to assess intake during the preceding seven days. The median predicted dietary cadmium intake among the 158 women studied was 25.29 µg/day (18.62-35.00) and 2.74 µg/kg body weight/week (b.w./w) (1.92-3.83). Dietary lead intake was 43.85 µg/day (35.09-51.45) and 4.82 µg/kg b.w./w (3.67-6.13). The observed dietary mercury intake was 9.55 µg/day (7.18-13.57) and 1.02 µg/kg b.w./w (0.71-1.48). Comparisons, in terms of heavy metal intake, showed no significant results after further adjusting for energy intake. No statistically significant correlations between heavy metal intake and the QUS, DXA and pQCT parameters were observed. Levels of dietary exposure of cadmium, lead and mercury were mostly within the recommendations. We did not find associations between the QUS, DXA and pQCT parameters and the dietary intake of the studied heavy metals in healthy premenopausal women.

Quantitative ultrasound in Spanish children and young adults with cystic fibrosis.

We studied the relationship between bone density and cystic fibrosis in Spanish children and young adults. We measured the phalangeal bone amplitude-dependent speed of sound (Ad-SoS) in 35 patients with cystic fibrosis and in 30 healthy controls matched for gender, age, and body mass index (BMI). Participants were subjects with normal levels of 25(OH) Vitamin D. We found no difference in Ad-SoS between patients and controls. The only difference between the groups was that the patients had a significantly higher daily caloric intake than the controls (p < .05) as a result of the patient group's greater intake of fats (p < .05). There was a positive correlation between Ad-SoS and weight (p < .0001), but after adjusting for potential confounding factors such as age, the correlation was lost. The percentage of ideal weight did not differ between the two groups. We conclude that well-nourished CF patients had similar Ad-SoS to controls.

Methods for nurses to measure body composition / Métodos de enfermagem para a medição da composição corporal / Métodos en enfermería para la medición de la composición corporal

Among the methods available for assessing body composition, traditional methods like hydrodensitometry and skin-fold measurements are well known. In this review, we focus on the impedance and interactance methods, which use systems that are usually inexpensive, easily transportable and simple to operate. We also discuss the usefulness of dual energy X-ray absorptiometry, particularly for the measurement of fat distribution. Nurses need to be skilled in the use of the equipment and familiar with the techniques.

Entre os métodos disponíveis para a determinação da composição corporal, os tradicionais, como a hidrodensitometria e a avaliação de pregas corporais, são bem conhecidos. Para esta revisão, centrou-se nos métodos baseados na impedância e a interactância, caracterizados pela utilização de equipamentos econômicos, fáceis de transportar e manobrar. Também discutiu-se a utilidade da absorciometria dual de raios X, especialmente para a determinação da distribuição da gordura corporal. É importante que o pessoal de enfermagem adquira conhecimentos sobre o uso desse equipamento, bem como que se familiarize com as técnicas descritas.

Entre los métodos disponibles para la determinación de la composición corporal, los tradicionales como la hidrodensitometría y la valoración de pliegues corporales son bien conocidos. En esta revisión nos centramos en los métodos basados en la impedancia y en la interactancia, caracterizados por la utilización de equipos económicos, fáciles de transportar y manejar. También discutimos la utilidad de la absorciometría dual de rayos-X, particularmente para la determinación de la distribución de la grasa corporal. Es importante que el personal de enfermería adquiera competencias en el uso de estos equipamientos y también se familiarice con las técnicas descritas.