Conventional and microfluidic methods: Design and optimization of lipid-polymeric hybrid nanoparticles for gene therapy.

Gene therapy holds significant promise as a therapeutic approach for addressing a diverse range of diseases through the suppression of overexpressed proteins and the restoration of impaired cell functions. Developing a nanocarrier that can efficiently load and release genetic material into cells remains a challenge. The primary goal of this study is to develop formulations aimed to enhance the therapeutic potential of GapmeRs through technological approaches. To this end, lipid-polymeric hybrid nanoparticles (LPHNPs) with PLGA, DC-cholesterol, and DOPE-mPEG2000 were produced by conventional single-step nanoprecipitation (SSN) and microfluidic (MF) methods. The optimized nanoparticles by SSN have a size of 149.9 ± 18.07 nm, a polydispersity index (PdI) of 0.23 ± 0.02, and a zeta potential of (ZP) of 29.34 ± 2.44 mV, while by MF the size was 179.8 ± 6.3, a PdI of 0.24 ± 0.01, and a ZP of 32.25 ± 1.36 mV. Furthermore, LPHNPs prepared with GapmeR-protamine by both methods exhibit a high encapsulation efficiency of approximately 90%. The encapsulated GapmeR is completely released in 24 h. The LPHNP suspensions are stable for up to 6 h in 10% FBS at pH 5.4 and 7.4. By contrast, LPHNPs remain stable in suspension in 4.5% albumin at pH 7.4 for 24 h. Additionally, LPHNPs were successfully freeze-dried using trehalose in the range of 2.5-5% as cryoprotectant The LPHNPs produced by MF and SSN increase, 6 and 12 fold respectively, GapmeR cell uptake, and both of them reduce by 60-70% expression of Tob1 in 48 h.Our study demonstrates the efficacy of the developed LPHNPs as carriers for oligonucleotide delivery, offering valuable insights for their scale up production from a conventional bulk methodology to a high-throughput microfluidic technology.

The Alk receptor tyrosine kinase regulates Sparkly, a novel activity regulating neuropeptide precursor in the Drosophila central nervous system.

Numerous roles for the Alk receptor tyrosine kinase have been described in Drosophila, including functions in the central nervous system (CNS), however the molecular details are poorly understood. To gain mechanistic insight, we employed Targeted DamID (TaDa) transcriptional profiling to identify targets of Alk signaling in the larval CNS. TaDa was employed in larval CNS tissues, while genetically manipulating Alk signaling output. The resulting TaDa data were analyzed together with larval CNS scRNA-seq datasets performed under similar conditions, identifying a role for Alk in the transcriptional regulation of neuroendocrine gene expression. Further integration with bulk and scRNA-seq datasets from larval brains in which Alk signaling was manipulated identified a previously uncharacterized Drosophila neuropeptide precursor encoded by CG4577 as an Alk signaling transcriptional target. CG4577, which we named Sparkly (Spar), is expressed in a subset of Alk-positive neuroendocrine cells in the developing larval CNS, including circadian clock neurons. In agreement with our TaDa analysis, overexpression of the Drosophila Alk ligand Jeb resulted in increased levels of Spar protein in the larval CNS. We show that Spar protein is expressed in circadian (clock) neurons, and flies lacking Spar exhibit defects in sleep and circadian activity control. In summary, we report a novel activity regulating neuropeptide precursor gene that is regulated by Alk signaling in the Drosophila CNS.

Global Water Challenges in Sub-Saharan Africa and how to strengthen science-policy dialogues on transboundary governance and cooperation.

The United Nations Water Conference 2023 highlighted the need for concrete actions to boost integrated water resources management for achieving the Sustainable Development Goals and called for strategies to enhance cooperation among stakeholders. Technical cooperation between countries and institutions in transboundary systems, e.g., on environmental data collection, is an effective way to promote international diplomacy and prevent disputes between riparian states. Still, establishing collaborations to inform bilateral dialogues on the identification of environmental challenges, their causes, and development priorities may be a difficult task in itself. This is particularly true in the African context because of limited resources and lack of data. In this paper, we analyse the case of nine transboundary river basins in Sub-Saharan Africa to identify which water-management challenges are perceived as most important by the different riparian countries from a policy and scientific perspective. Our insights are based on the most up-to-date scientific papers, open access reports and technical literature, river basin authority's strategy papers, projects' summary reports, and national policy documents. We also complement these sources with the pieces of information we gained through collaborations with regional and local experts, and management bodies (such as river basin authorities). We highlight the current water-related conflicts and the gap between the priorities identified by the scientific community and different riparian countries on how to tackle hydro-climatic change and improve food and energy security, human and environmental health. Based on our experience, we discuss some keys to building trust among stakeholders, strengthening cooperation, and identifying shared water-governance measures in transboundary river basins. They are: (i) connect science and policy to provide sound knowledge for the right questions, (ii) value local knowledge and exploit the complementarity of different perspectives, (iii) consider multiple spatial scales and multi-level stakeholders to leave no one behind, (iv) promote a culture which values trade-offs and handles complexity, and (v) co-create data and knowledge to facilitate stakeholder dialogue from problem definition to intervention identification.

A mild increase in nutrient signaling to mTORC1 in mice leads to parenchymal damage, myeloid inflammation and shortened lifespan.

The mechanistic target of rapamycin complex 1 controls cellular anabolism in response to growth factor signaling and to nutrient sufficiency signaled through the Rag GTPases. Inhibition of mTOR reproducibly extends longevity across eukaryotes. Here we report that mice that endogenously express active mutant variants of RagC exhibit multiple features of parenchymal damage that include senescence, expression of inflammatory molecules, increased myeloid inflammation with extensive features of inflammaging and a ~30% reduction in lifespan. Through bone marrow transplantation experiments, we show that myeloid cells are abnormally activated by signals emanating from dysfunctional RagC-mutant parenchyma, causing neutrophil extravasation that inflicts additional inflammatory damage. Therapeutic suppression of myeloid inflammation in aged RagC-mutant mice attenuates parenchymal damage and extends survival. Together, our findings link mildly increased nutrient signaling to limited lifespan in mammals, and support a two-component process of parenchymal damage and myeloid inflammation that together precipitate a time-dependent organ deterioration that limits longevity.

Correction: University engagement of dental students related to educational environment: A transnational study.

[This corrects the article DOI: 10.1371/journal.pone.0259524.].

miRNome profiling of extracellular vesicles in severe COVID-19 patients and identification of predictors of mortality.

BACKGROUND: Extracellular vesicles (EVs), containing microRNAs (miRNAs) and other molecules, play a central role in intercellular communication, especially in viral infections caused by SARS-CoV-2. This study explores the miRNA profiles in plasma-derived EVs from severe COVID-19 patients referred to controls, identifying potential mortality miRNA predictors. METHODS: A prospective study was carried out, including 36 severe COVID-19 patients and 33 non-COVID-19 controls. EVs-derived miRNAs were sequenced, and bioinformatics and differential expression analysis between groups were performed. The plasma miRNA profile of an additional cohort of severe COVID-19 patients (n=32) and non-COVID-19 controls (n=12) was used to compare with our data. Survival analysis was used to identify potential mortality predictors among the SDE miRNAs in EVs. RESULTS: Severe COVID-19 patients showed 50 significantly differentially expressed (SDE) miRNAs in plasma-derived EVs. These miRNAs were associated with pathways related to inflammation and cell adhesion. Fifteen of these plasma-derived EVs miRNAs were also SDE in the plasma of severe patients vs controls. Two miRNAs, hsa-miR-1469 and hsa-miR-6124, were identified as strong mortality predictors with an área under the ROC Curve (AUC) of 0.938. CONCLUSION: : This research provides insights into the role of miRNAs found within EVs in severe COVID-19 and their potential as clinical biomarkers for mortality.

Cattel and Blumgart Anastomosis: Comparison of Pancreatojejunostomy Techniques in Patients with Pancreatic Cancer.

BACKROUND: Pancreaticojejunal anastomosis has a high risk of pancreatic leakage, which several surgical techniques have been described. Our main objective is to determine what is the association of postoperative pancreatic leakage in patients undergoing pancreaticoduodenectomy according to the pancreaticojejunal anastomosis technique used (Cattel vs Blumgart)? MATERIAL AND METHODS: Historical cohort, all cancer patients undergoing pancreaticoduodenectomy were included. From April 2020 to May 2021, Cattel technique was used; from June 2021 to June 2022, Blumgart technique was used. All procedures were performed by a single board-certified surgical oncologist at Oncology Hospital. RESULTS: 24 patients (11 men and 13 women). The first 9 procedures were performed with the Cattel technique, starting from procedure 10, the Blumgart technique was used. No differences were observed regarding the consistency of the pancreas (p=0.28) or the size of the duct (p=0.51) between the two groups. Pancreatic leakage was observed in 100% with the Cattel technique and in 6.7% with the Blumgart technique (p= 0.0001). An amylase value was observed in the left drainage at 6 days > 200 U/L with the Cattel technique and < 200 U/L with Blumgart (p=0.0001). Intraoperative bleeding (Cattel 1200 ml vs. Blumgart 400 ml, p= 0.03) and 90-day mortality (Cattel 33.3% vs. Blumgart 0%, Log-Rank: 0.022) also showed a difference in favor of Blumgart technique. Blumgart technique is a protective factor for the presence of pancreatic leakage (RR 0.06, 95% CI 0.01-0.44, p=0.0001). DISCUSSION: A lower risk of pancreatic leakage was found using the Blumgart technique, compared to the Cattel technique.

Generational Exploration on Aesthetic Medicine Products: Influence of Social Media, Reviews, and Brand Identity-GEMS Bond Survey.

INTRODUCTION: The study investigates the impact of social media reviews and brand identity on consumer preferences in the non-surgical aesthetics products across different generations. It highlights the evolving landscape of aesthetic medicine and surgery, driven by technological advancements and a cultural shift towards individual well-being. The research aims to explore the interplay between generational preferences, the influence of social media, and the role of brand identity in shaping consumer decisions. METHODS: A cross-sectional study design was employed, with a sample size of > 5000 participants stratified across various generational cohorts. The study utilized an online questionnaire to capture both quantitative and qualitative insights into consumer behaviour, with statistical analysis performed to identify patterns and relationships. RESULTS: Analysis of 5850 responses revealed distinct generational preferences and behaviours. Social media engagement varied significantly across generations, with younger cohorts placing a higher emphasis on online reviews. The study also found that brand identity's influence is diminishing in decision-making processes, with consumers increasingly relying on peer reviews and social media content. CONCLUSION: The findings highlight a pivotal shift in the non-surgical aesthetics consumer market, emphasizing the growing importance of social media and peer reviews over traditional brand identities. Importantly, the study underscores the critical need for integrating patient safety and evidence-based practice within marketing strategies. As consumer preferences evolve towards valuing transparency and authenticity, non-surgical aesthetics providers must prioritize these elements, ensuring that their services are not only appealing but also grounded in safety and scientific validity. LEVEL OF EVIDENCE IV: "This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 ."

Ceftazidime/avibactam resistance is associated with PER-3-producing ST309 lineage in Chilean clinical isolates of non-carbapenemase producing Pseudomonas aeruginosa.

Introduction: Ceftazidime/avibactam (CZA) is indicated against multidrug-resistant Pseudomonas aeruginosa, particularly those that are carbapenem resistant. CZA resistance in P. aeruginosa producing PER, a class A extended-spectrum ß-lactamase, has been well documented in vitro. However, data regarding clinical isolates are scarce. Our aim was to analyze the contribution of PER to CZA resistance in non-carbapenemase-producing P. aeruginosa clinical isolates that were ceftazidime and/or carbapenem non-susceptible. Methods: Antimicrobial susceptibility was determined through agar dilution and broth microdilution, while bla PER gene was screened through PCR. All PER-positive isolates and five PER-negative isolates were analyzed through Whole Genome Sequencing. The mutational resistome associated to CZA resistance was determined through sequence analysis of genes coding for PBPs 1b, 3 and 4, MexAB-OprM regulators MexZ, MexR, NalC and NalD, AmpC regulators AmpD and AmpR, and OprD porin. Loss of bla PER-3 gene was induced in a PER-positive isolate by successive passages at 43°C without antibiotics. Results: Twenty-six of 287 isolates studied (9.1%) were CZA-resistant. Thirteen of 26 CZA-resistant isolates (50%) carried bla PER. One isolate carried bla PER but was CZA-susceptible. PER-producing isolates had significantly higher MICs for CZA, amikacin, gentamicin, ceftazidime, meropenem and ciprofloxacin than non-PER-producing isolates. All PER-producing isolates were ST309 and their bla PER-3 gene was associated to ISCR1, an insertion sequence known to mobilize adjacent DNA. PER-negative isolates were classified as ST41, ST235 (two isolates), ST395 and ST253. PER-negative isolates carried genes for narrow-spectrum ß-lactamases and the mutational resistome showed that all isolates had one major alteration in at least one of the genes analyzed. Loss of bla PER-3 gene restored susceptibility to CZA, ceftolozane/tazobactam and other ß-lactamsin the in vitro evolved isolate. Discussion: PER-3-producing ST309 P. aeruginosa is a successful multidrug-resistant clone with blaPER-3 gene implicated in resistance to CZA and other ß-lactams.

LINC00662 Promotes Aggressive Traits by Modulating OCT4 Expression through miR-335-5p in Gallbladder Cancer Cells.

Long non-coding RNAs (lncRNAs) are nucleotide sequences that participate in different biological processes and are associated with different pathologies, including cancer. Long intergenic non-protein-coding RNA 662 (LINC00662) has been reported to be involved in different cancers, including colorectal, prostate, and breast cancer. However, its role in gallbladder cancer has not yet been described. In this article, we hypothesize that LINC00662 has an important role in the acquisition of aggressiveness traits such as a stem-like phenotype, invasion, and chemoresistance in gallbladder cancer. Here, we show that LINC00662 is associated with larger tumor size and lymph node metastasis in patients with gallbladder cancer. Furthermore, we show that the overexpression of LINC00662 promotes an increase in CD133+/CD44+ cell populations and the expression of stemness-associated genes. LINC00662 promotes greater invasive capacity and the expression of genes associated with epithelial-mesenchymal transition. In addition, the expression of LINC00662 promotes resistance to cisplatin and 5-fluorouracil, associated with increased expression of chemoresistance-related ATP-binding cassette (ABC) transporters in gallbladder cancer (GBC) cell lines. Finally, we show that the mechanism by which LINC00662 exerts its function is through a decrease in microRNA 335-5p (miR-335-5p) and an increase in octamer-binding transcription factor 4 (OCT4) in GBC cells. Thus, our data allow us to propose LINC00662 as a biomarker of poor prognosis and a potential therapeutic target for patients with GBC.

Population assessment of health system performance in 16 countries.

Objective: To demonstrate how the new internationally comparable instrument, the People's Voice Survey, can be used to contribute the perspective of the population in assessing health system performance in countries of all levels of income. Methods: We surveyed representative samples of populations in 16 low-, middle- and high-income countries on health-care utilization, experience and confidence during 2022-2023. We summarized and visualized data corresponding to the key domains of the World Health Organization universal health coverage framework for health system performance assessment. We examined correlation with per capita health spending by calculating Pearson coefficients, and within-country income-based inequities using the slope index of inequality. Findings: In the domain of care effectiveness, we found major gaps in health screenings and endorsement of public primary care. Only one in three respondents reported very good user experience during health visits, with lower proportions in low-income countries. Access to health care was rated highest of all domains; however, only half of the populations felt secure that they could access and afford high-quality care if they became ill. Populations rated the quality of private health systems higher than that of public health systems in most countries. Only half of respondents felt involved in decision-making (less in high-income countries). Within countries, we found statistically significant pro-rich inequalities across many indicators. Conclusion: Populations can provide vital information about the real-world function of health systems, complementing other system performance metrics. Population-wide surveys such as the People's Voice Survey should become part of regular health system performance assessments.

A Systematic Review on the Reinforcement Loop in Aesthetic Medicine and Surgery: The Interplay of Social Media, Self-Perception, and Repeat Procedures.

INTRODUCTION: This systematic review delves into the impact of social media on self-perception and the escalating interest in clinical aesthetic procedures, proposing that social media significantly influences beauty standards and increases demand for aesthetic enhancements. METHOD: Following PRISMA guidelines, a mixed-method analysis of 34 articles was conducted, sourced from various databases, focusing on social media's psychological effects on clinical aesthetics decisions. The studies encompassed a broad spectrum, including qualitative, quantitative, and mixed methodologies, reflecting diverse geographical and cultural perspectives. RESULTS: The quantitative analysis demonstrated a strong positive correlation between social media usage and the consideration of aesthetic procedures (r=0.45, p<0.001), indicating a significant impact. Specific findings included a large effect size (Cohen's d=0.8) for the relationship between time spent on social media and the desire for aesthetic enhancements. Individuals spending more than 3 hours per day on social media platforms were twice as likely to consider aesthetic procedures compared to those with less usage, with a 95% confidence interval indicating robustness in these findings. CONCLUSION: Confirming the reinforcing effect of social media on aesthetic decision-making, this study highlights the complex interplay between digital media exposure, altered self-perception, and the increased inclination towards aesthetic procedures. It suggests a critical need for practitioners to carefully navigate the digital influence on patient's desires, reinforcing the significance of understanding psychological motivations and societal pressures in clinical aesthetics. This comprehensive analysis offers pivotal insights for clinical practice and ongoing research into social media's role in contemporary beauty standards. LEVEL OF EVIDENCE III: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Executive summary: Quality standards of care units for people with sexual and gender diversity.

Comprehensive biopsychosocial care for people with gender incongruence (ICD 11) who are transgender (trans) or gender diverse is a complex process in which the quality of the medical transition can only be guaranteed after a multidisciplinary approach, through teams that integrate professionals with training and experience not only in medicine but also in diversity and gender identity. Based on this, the Gonad, Identity and Sexual Differentiation working group of the Spanish Society of Endocrinology and Nutrition (GT-GIDSEEN) has established minimum care requirements that aim to guarantee adequate health care for these people by professionals. A position paper has been produced and is available at https://www.seen.es/portal/documentos/estandares-calidad-gidseen-2024.

Harnessing extracellular vesicle membrane for gene therapy: EVs-biomimetic nanoparticles.

One of the main concerns in oligonucleotide-based therapeutics is achieving a successful cell targeting while avoiding drug degradation and clearance. Nanoparticulated drug delivery systems have emerged as a way of overcoming these issues. Among them, membrane-coated nanoparticles are of increasing relevance mainly due to their enhanced cellular uptake, immune evasion and biocompatibility. In this study, we designed and elaborated a simple and highly tuneable biomimetic drug delivery nanosystem based on a polymeric core surrounded by extracellular vesicles (EVs)-derived membranes. This strategy should allow the nanosystems to benefit from the properties conferred by the membrane proteins present in EVs membrane, key paracrine mediators. The developed systems were able to successfully encapsulate the required oligonucleotides. Also, their characterisation through already well standardised methods (dynamic light scattering, transmission electron microscopy and nanoparticle tracking analysis) and by fluorescence cross-correlation spectroscopy (FCCS) showed the desired core-shell structure. The cellular uptake using different cell types further confirmed the coating though an enhancement in cell internalisation of the developed biomimetic nanoparticles. This study brings up new possibilities for GapmeR delivery as it might be a base for the development of new delivery systems for gene therapy.

Influence of lower limb isokinetic muscle strength and power on the occurrence of falls in community-dwelling older adults: A longitudinal study.

INTRODUCTION: Previous studies have highlighted the association between lower limb muscle strength and falls in older adults. However, a comprehensive understanding of the specific influence of each lower limb muscle group on fall occurrences remains lacking. OBJECTIVE: This study aimed to investigate the impact of knee, ankle, and hip muscle strength and power on falls in older adults, with the goal of identifying which muscle groups are more predictive of fall risk in this population. METHODS: This longitudinal observational study enrolled 94 community-dwelling older adults. Muscle strength and power of the ankle's plantiflexors and dorsiflexors, knee flexors and extensors, and hip flexors, extensors, adductors, and abductors were assessed using a Biodex System 4 Pro® isokinetic dynamometer. Fall occurrences were monitored through monthly telephone contact over a year. RESULTS: Participants, with a median age of 69 years (range 64-74), included 67% women, and 63.8% reported a sedentary lifestyle. Among them, 45,7% of older adults were classified as fallers. Comparative analyses revealed that non-fallers displayed significantly superior isokinetic muscle strength in the hip abductors and adductors, along with higher muscle power in the hip abductors, hip flexors, and knee flexors compared to fallers. Multivariate logistic regression analysis indicated that a 1 Nm/Kg increase in hip abductor strength reduced the chance of a fall by 86.3%, and a 1 Watt increase in hip flexor power reduced the chance of a fall by 3.6%. CONCLUSION: The findings indicate that hip abductor strength and hip flexor power can be considered protective factors against falls in independent older adults in the community. These findings may contribute to developing effective fall-prevention strategies for this population.

Magnification in preclinical procedures: effect on muscle activity and angular deviations of the neck and trunk.

Objectives: This study aimed to assess the effects of different magnification systems on the angular deviations of the neck and trunk and the muscle activities of the upper back and neck during preclinical cavity preparation. Methods: This was an experimental laboratory study, with the angular deviations from the neutral positions of the neck and trunk and the activities of the bilateral upper back (the descending and ascending trapezius) and neck (sternocleidomastoid) muscles as the dependent variables. The independent variables were the different magnification systems used (Simple, Galilean, and Keplerian loupes, with direct vision as the control) and prepared teeth (teeth 16, 26, 36, and 46). A dental mannequin phantom head with artificial resin teeth was used, and Class I cavity preparations for composite resin were performed on teeth 16, 26, 36, and 46 using a 1012 round diamond bur at low speed. To analyze the angular deviations, the postures adopted during the procedure were recorded using a tripod-mounted camera positioned to provide a lateral view of the operator. A trained researcher measured the angular deviations using the software entitled "Software for Postural Assessment"-SAPO (version 0.69). Bilateral muscle activity was assessed using surface electromyography. Descriptive statistical analysis was performed, and after verifying the assumptions of normality and homoscedasticity, two-way analysis of variance and the Tukey and Games-Howell post-hoc tests were used to compare the data (α=0.05). Results: The angular deviation from the neutral position of the neck was found to be significantly higher during cavity preparations performed with the naked eye and the Simple loupe, irrespective of the prepared tooth. With regard to tooth location, the angular deviation of the neck was significantly greater during cavity preparation on teeth 16 and 26, and the angular deviation of the trunk was significantly greater during cavity preparation on tooth 26, regardless of the magnification system used. There were significant differences in right sternocleidomastoid muscle activity between the Simple, Galilean, and Keplerian loupes, with activity being the lowest for the Galilean loupe (p = 0.008). There were no significant differences in left sternocleidomastoid muscle activity between the loupes, regardless of the prepared tooth (p = 0.077). The activities of the bilateral descending trapezius and the right ascending trapezius muscles were significantly lower when the Galilean loupe was used (p < 0.010). Conclusion: These results suggest that the Galilean loupe resulted in lower muscle activity in the neck and back regions and that the Galilean and Keplerian loupes resulted in less angular deviations of the neck and trunk during cavity preparation.