Isolation of Adeno-associated Viral Vectors Through a Single-step and Semi-automated Heparin Affinity Chromatography Protocol.

Adeno-associated virus (AAV) has become an increasingly valuable vector for in vivo gene delivery and is currently undergoing human clinical trials. However, the commonly used methods to purify AAVs make use of cesium chloride or iodixanol density gradient ultracentrifugation. Despite their advantages, these methods are time-consuming, have limited scalability, and often result in vectors with low purity. To overcome these constraints, researchers are turning their attention to chromatography techniques. Here, we present an optimized heparin-based affinity chromatography protocol that serves as a universal capture step for the purification of AAVs. This method relies on the intrinsic affinity of AAV serotype 2 (AAV2) for heparan sulfate proteoglycans. Specifically, the protocol entails the co-transfection of plasmids encoding the desired AAV capsid proteins with those of AAV2, yielding mosaic AAV vectors that combine the properties of both parental serotypes. Briefly, after the lysis of producer cells, a mixture containing AAV particles is directly purified following an optimized single-step heparin affinity chromatography protocol using a standard fast protein liquid chromatography (FPLC) system. Purified AAV particles are subsequently concentrated and subjected to comprehensive characterization in terms of purity and biological activity. This protocol offers a simplified and scalable approach that can be performed without the need for ultracentrifugation and gradients, yielding clean and high viral titers.

Improving Organizational Commitment among Healthcare Employees in Angola: The Role of Psychological Capital and Perceived Transformational Leadership.

While previous studies conducted in sub-Saharan African countries have focused on verifying standards of clinical care and assessing challenges faced by healthcare professionals, the present study fills a gap in the literature in that it explores the factors that may drive the organizational commitment of healthcare professionals in Angola. This study aimed to analyze the relationship between psychological capital and organizational commitment through perceived transformational leadership. Therefore, using the quantitative methodology, a self-report questionnaire was applied to 342 healthcare professionals (174 male, 168 female) from different public and private hospitals located in three large cities in Angola. The results confirmed that psychological capital is positively related to affective commitment and that perceived transformational leadership is a mediating variable of this relationship. Therefore, this study highlights the role of psychological capital and perceived transformational leadership in improving affective commitment in challenging environments.

Viral-based animal models in polyglutamine disorders.

Polyglutamine disorders are a complex group of incurable neurodegenerative disorders caused by an abnormal expansion in the trinucleotide cytosine-adenine-guanine tract of the affected gene. To better understand these disorders, our dependence on animal models persists, primarily relying on transgenic models. In an effort to complement and deepen our knowledge, researchers have also developed animal models of polyglutamine disorders employing viral vectors. Viral vectors have been extensively used to deliver genes to the brain, not only for therapeutic purposes but also for the development of animal models, given their remarkable flexibility. In a time- and cost-effective manner, it is possible to use different transgenes, at varying doses, in diverse targeted tissues, at different ages, and in different species, to recreate polyglutamine pathology. This paper aims to showcase the utility of viral vectors in disease modelling, share essential considerations for developing animal models with viral vectors, and provide a comprehensive review of existing viral-based animal models for polyglutamine disorders.

Portuguese Global Medicines Access Index 2021: An Indicator to Measure Access to Hospital Medicines.

OBJECTIVES: Access to innovative and effective medication is a citizen's right. The main objectives of this study were to build an indicator to measure access to medicines within hospitals, the Global Medicines Access Index, and to identify the main existing barriers. METHODS: Cross-sectional study carried out in Portuguese National Health Service hospitals. A consensus methodology (expert panel of 7 members) was used to define which dimensions should be included in the index and the weighting that each should take. The panel identified 6 dimensions: access to innovative medicines, proximity distribution, shortages, access to medicines before financing decision, value-based healthcare, and access to medication depending on cost/funding. Data were collected through an electronic questionnaire (September 2021). RESULTS: The response rate was 61.2%. Most hospitals used medicines with and without marketing authorization before the funding decision. Monitoring and generating evidence of new therapies results is still insufficient. The identified barriers were the administrative burden as the major barrier in purchasing medicines, with a relevant impact on shortages of medicines. Most respondents (87%) had a proximity distribution program, mainly implemented in the pandemic context, and the price/funding model was only identified by 10% as a barrier to access. The 2021 Global Medicines Access Index was 66%. Shortages and value-based use of medicines were the dimensions that had more influence in lowering the index value. CONCLUSIONS: The new formula used to obtain a unique and multidimensional index for access to hospital medicines seems to be more sensitive and objective and will be used to monitor access.

How Can Out-of-Hospital Cardiac Arrest (OHCA) Data Collection in Slovenia Be Improved?

Background and Objectives: The prevalence of out-of-hospital cardiac arrest (OHCA) has been established as a significant contributor to mortality rates in developed nations. Due to the challenges associated with conducting controlled randomized trials, there exists a necessity for the collection of high-quality data to enhance the comprehension of the impact of interventions. Several nations have initiated efforts to gather information pertaining to out-of-hospital cardiac arrest (OHCA). The Republic of Slovenia has been collecting data from interventions; however, the variables and data attributes have not yet been standardized to comply with international standards. This lack of conformity poses a challenge in making comparisons or drawing inferences. The aim of this study is to identify how to better gather OHCA data in Slovenia. Materials and methods: The Utstein resuscitation registry protocol (UP) was compared to the Slovenian data points that must be gathered in accordance with the Rules on Emergency Medical Service (REMS) during interventions. In addition, we have proposed alternative measures of digitization to enhance pre-hospital data. Results: Missing data points and attribute mismatches were detected in Slovenia. Eight data points necessitated by the UP are gathered in several databases (hospitals, the National Institute of Public Health, dispatch services, intervention reports from first responders, and defibrillator files), but not in the mandated protocols based on REMS. Two data points have variables that do not match those of the UP. A total of 16 data points according to UP are currently not being collected in Slovenia. The advantages and potential drawbacks of digitizing emergency medical services have been discussed. Conclusions: The study has identified gaps in the methods employed for collecting data on OHCA in Slovenia. The assessment conducted serves as a basis for enhancing the process of data collection, integrating quality control measures across the nation, and establishing a nationwide registry for out-of-hospital cardiac arrest (OHCA) in Slovenia.

Gut microbiota profile of COVID-19 patients: Prognosis and risk stratification (MicroCOVID-19 study).

Background: Gut microbiota is intrinsically associated with the immune system and can promote or suppress infectious diseases, especially viral infections. This study aims to characterize and compare the microbiota profile of infected patients with SARS-CoV-2 (milder or severe symptoms), non-infected people, and recovered patients. This is a national, transversal, observational, multicenter, and case-control study that analyzed the microbiota of COVID-19 patients with mild or severe symptoms at home, at the hospital, or in the intensive care unit, patients already recovered, and healthy volunteers cohabiting with COVID-19 patients. DNA was isolated from stool samples and sequenced in a NGS platform. A demographic questionnaire was also applied. Statistical analysis was performed in SPSS. Results: Firmicutes/Bacteroidetes ratios were found to be significantly lower in infected patients (1.61 and 2.57) compared to healthy volunteers (3.23) and recovered patients (3.89). Furthermore, the microbiota composition differed significantly between healthy volunteers, mild and severe COVID-19 patients, and recovered patients. Furthermore, Escherichia coli, Actinomyces naeslundii, and Dorea longicatena were shown to be more frequent in severe cases. The most common COVID-19 symptoms were linked to certain microbiome groups. Conclusion: We can conclude that microbiota composition is significantly affected by SARS-CoV-2 infection and may be used to predict COVID-19 clinical evolution. Therefore, it will be possible to better allocate healthcare resources and better tackle future pandemics.

A new protocol for whole-brain biodistribution analysis of AAVs by tissue clearing, light-sheet microscopy and semi-automated spatial quantification.

Recombinant adeno-associated virus (rAAV) has become one of the most promising gene delivery systems for both in vitro and in vivo applications. However, a key challenge is the lack of suitable imaging technologies to evaluate delivery, biodistribution and tropism of rAAVs and efficiently monitor disease amelioration promoted by AAV-based therapies at a whole-organ level with single-cell resolution. Therefore, we aimed to establish a new pipeline for the biodistribution analysis of natural and new variants of AAVs at a whole-brain level by tissue clearing and light-sheet fluorescence microscopy (LSFM). To test this platform, neonatal C57BL/6 mice were intravenously injected with rAAV9 encoding EGFP and, after sacrifice, brains were processed by standard immunohistochemistry and a recently released aqueous-based clearing procedure. This clearing technique required no dedicated equipment and rendered highly cleared brains, while simultaneously preserving endogenous fluorescence. Moreover, three-dimensional imaging by LSFM allowed the quantitative analysis of EGFP at a whole-brain level, as well as the reconstruction of Purkinje cells for the retrieval of valuable morphological information inaccessible by standard immunohistochemistry. In conclusion, the pipeline herein described takes the AAVs to a new level when coupled to LSFM, proving its worth as a bioimaging tool in tropism and gene therapy studies.

Time course of lung ultrasound findings in patients with COVID-19 pneumonia and cardiac dysfunction.

BACKGROUND: Lung ultrasound (LUS) is a valuable tool to predict and monitor the COVID-19 pneumonia course. However, the influence of cardiac dysfunction (CD) on LUS findings remains to be studied. Our objective was to determine the effect of CD on LUS in hospitalized patients with COVID-19 pneumonia. MATERIAL AND METHODS: Fifty-one patients with COVID-19 pneumonia participated in the study. Focused echocardiography (FoCUS) was carried out on day 1 to separate patients into two groups depending on whether they had FoCUS signs of CD (CD+ vs CD-). LUS scores, based on the thickness of the pleural line, the B-line characteristics, and the presence or not of consolidations, were obtained three times along the patient's admission (D1, D5, D10) and compared between CD+ and CD- patients. A correlation analysis was carried out between LUS scores and the ratio of the arterial partial pressure of oxygen to the fraction of the inspired oxygen (P/F ratio). RESULTS: Twenty-two patients were CD+ and 29 patients were CD-. Among the CD+ patients, 19 were admitted to the intensive care unit (ICU), seven received invasive mechanical ventilation (IMV), and one did not survive. Among the CD- patients, 11 were admitted to the ICU, one received IMV and seven did not survive. CD+ patients showed a significantly lower P/F ratio than CD- patients. However, LUS scores showed no between-group differences, except for fewer subpleural consolidations in the upper quadrants of CD+ than on CD- patients. CONCLUSION: In patients with COVID-19, CD contributed to a worse clinical course, but it did not induce significant changes in LUS. Our findings suggest that pathophysiological factors other than those reflected by LUS may be responsible for the differences in clinical condition between CD+ and CD- patients.

Changing perspectives in times of crisis. The impact of COVID-19 on territorial accessibility.

The link between transport and land use in urban areas has always been characterized by a slow evolution process. COVID-19 brought, suddenly and unexpectedly, severe changes to the trip structure within urban areas, as several restrictions were combined with individual health fears. This study addresses the impact of the COVID-19 pandemic in the territory of Porto Greater Urban Area, in Portugal, measured under a structural accessibility approach. This was evaluated through a simulation model, combining different destination restrictions in three alternative scenarios during the pandemic and post-COVID, as well as the definition of four different personas, with distinct risk aversion to infections and telecommuting patterns. The results, presented as the spatial configuration of different mobility environments, foster a critical reflection on their implication for future transportation and land use policies. This pandemic has shown that the territory behaves differently under a critical lockdown scenario, where active modes gain predominance to satisfy most travel needs, signalling a potential ability to enforce more sustainable mobility habits. Still, as the territorial configuration tends to the previous state of equilibrium as restrictions are lifted, particularly for non-telecommuters, the need for acting quickly is reinforced. While the growth of telecommuting can induce additional challenges to the management of urban mobility systems, most policy recommendations that were valid in the past will maintain its relevance, as non telecommuters will retain previous travel habits.

Psychological Capital Profiles and Their Relationship With Internal Learning in Teams of Undergraduate Students.

This study aims to analyze the relationship between psychological capital profiles and internal learning in teams. The participants in this study were 480 undergraduate students. We performed a cluster analysis using the SPSS and yielded four distinct psychological capital profiles. The student profile with the highest scores in self-efficacy, optimism, hope, and resilience (Profile 2-Fully PsyCap) exhibited also the highest scores of internal learning in teams. On the other hand, the student profile with the lowest scores in self-efficacy, optimism, hope, and resilience (Profile 1- Empty PsyCap) presented the lowest scores of internal learning in teams. It is also noteworthy that there was no significant relationship between the profile with a positive combination between self-efficacy and hope (profile 4) and the profile that presents the optimism as the only positive psychological capability (profile 3), in the way they relate to internal learning in teams, which led us to reject the second hypothesis of the study. This study reinforces the role of psychological capital in academic settings and suggests that psychological capital profiles can affect internal learning in teams differentially.

ULK overexpression mitigates motor deficits and neuropathology in mouse models of Machado-Joseph disease.

Machado-Joseph disease (MJD) is a fatal neurodegenerative disorder clinically characterized by prominent ataxia. It is caused by an expansion of a CAG trinucleotide in ATXN3, translating into an expanded polyglutamine (polyQ) tract in the ATXN3 protein, that becomes prone to misfolding and aggregation. The pathogenesis of the disease has been associated with the dysfunction of several cellular mechanisms, including autophagy and transcription regulation. In this study, we investigated the transcriptional modifications of the autophagy pathway in models of MJD and assessed whether modulating the levels of the affected autophagy-associated transcripts (AATs) would alleviate MJD-associated pathology. Our results show that autophagy is impaired at the transcriptional level in MJD, affecting multiple AATs, including Unc-51 like autophagy activating kinase 1 and 2 (ULK1 and ULK2), two homologs involved in autophagy induction. Reinstating ULK1/2 levels by adeno-associated virus (AAV)-mediated gene transfer significantly improved motor performance while preventing neuropathology in two in vivo models of MJD. Moreover, in vitro studies showed that the observed positive effects may be mainly attributed to ULK1 activity. This study provides strong evidence of the beneficial effect of overexpression of ULK homologs, suggesting these as promising instruments for the treatment of MJD and other neurodegenerative disorders.

miRNA-Mediated Knockdown of ATXN3 Alleviates Molecular Disease Hallmarks in a Mouse Model for Spinocerebellar Ataxia Type 3.

Spinocerebellar ataxia type 3 (SCA3) is a neurodegenerative disorder caused by the expansion of a CAG repeat in the ATXN3 gene. This mutation leads to a toxic gain of function of the ataxin-3 protein, resulting in neuronal dysfunction and atrophy of specific brain regions over time. As ataxin-3 is a dispensable protein in rodents, ataxin-3 knockdown by gene therapy may be a powerful approach for the treatment of SCA3. In this study, we tested the feasibility of an adeno-associated viral (AAV) vector carrying a previously described artificial microRNA against ATXN3 in a striatal mouse model of SCA3. Striatal injection of the AAV resulted in good distribution throughout the striatum, with strong dose-dependent ataxin-3 knockdown. The hallmark intracellular ataxin-3 inclusions were almost completely alleviated by the microRNA-induced ATXN3 knockdown. In addition, the striatal lesion of dopamine- and cAMP-regulated neuronal phosphoprotein (DARPP-32) in the SCA3 mice was rescued by ATXN3 knockdown, indicating functional rescue of neuronal signaling and health upon AAV treatment. Together, these data suggest that microRNA-induced ataxin-3 knockdown is a promising therapeutic strategy in the treatment of SCA3.

Positive Psychological Capital as a Predictor of Satisfaction With the Fly-In Fly-Out Model.

The flexibility of markets and international agreements have lured a growing number of companies to expand their business beyond frontiers in search for new markets and a bigger business network. Specifically, expatriates became keystones to implant and promote the so desired expansion into international markets, Particularly, Fly-in fly-out (FIFO) flexpatriates. Although FIFO work practices are widely used, little is known about how to promote these professionals' perceived job satisfaction (JS) across the course of their work cycles. That is why the goal of our research is to test the positive psychological capital (PsyCap) applicability to Portuguese FIFO flexpatriates. In the midst of the positive psychology theories, Luthans et al. (2007b) underline that workers are the psychological capital of any organization. Therefore, the development of the PsyCap becomes crucial and also contributes to the promotion of JS, nowadays a construct intertwined with well-being. As such, we developed and applied a HERO-(hope, self-efficacy, resilience, and optimism)-micro-intervention in order to assess whether it moderated the relationship between a FIFO flexpatriates PsyCap and their JS. The research took place over three distinct moments, both PsyCap and JS were measured before and after the HERO micro-intervention, and again 3 months later. The data collected shows that a positive correlation exists between FIFO flexpatriates PsyCap and JS. Moreover, our results pointed out that the micro-intervention enhanced FIFO flexpatriates PsyCap, and also showed that this increase lasted over (at least) 3 months.

The RNA-binding profile of the splicing factor SRSF6 in immortalized human pancreatic ß-cells.

In pancreatic ß-cells, the expression of the splicing factor SRSF6 is regulated by GLIS3, a transcription factor encoded by a diabetes susceptibility gene. SRSF6 down-regulation promotes ß-cell demise through splicing dysregulation of central genes for ß-cells function and survival, but how RNAs are targeted by SRSF6 remains poorly understood. Here, we define the SRSF6 binding landscape in the human pancreatic ß-cell line EndoC-ßH1 by integrating individual-nucleotide resolution UV cross-linking and immunoprecipitation (iCLIP) under basal conditions with RNA sequencing after SRSF6 knockdown. We detect thousands of SRSF6 bindings sites in coding sequences. Motif analyses suggest that SRSF6 specifically recognizes a purine-rich consensus motif consisting of GAA triplets and that the number of contiguous GAA triplets correlates with increasing binding site strength. The SRSF6 positioning determines the splicing fate. In line with its role in ß-cell function, we identify SRSF6 binding sites on regulated exons in several diabetes susceptibility genes. In a proof-of-principle, the splicing of the susceptibility gene LMO7 is modulated by antisense oligonucleotides. Our present study unveils the splicing regulatory landscape of SRSF6 in immortalized human pancreatic ß-cells.

Persistent or Transient Human ß Cell Dysfunction Induced by Metabolic Stress: Specific Signatures and Shared Gene Expression with Type 2 Diabetes.

Pancreatic ß cell failure is key to type 2 diabetes (T2D) onset and progression. Here, we assess whether human ß cell dysfunction induced by metabolic stress is reversible, evaluate the molecular pathways underlying persistent or transient damage, and explore the relationships with T2D islet traits. Twenty-six islet preparations are exposed to several lipotoxic/glucotoxic conditions, some of which impair insulin release, depending on stressor type, concentration, and combination. The reversal of dysfunction occurs after washout for some, although not all, of the lipoglucotoxic insults. Islet transcriptomes assessed by RNA sequencing and expression quantitative trait loci (eQTL) analysis identify specific pathways underlying ß cell failure and recovery. Comparison of a large number of human T2D islet transcriptomes with those of persistent or reversible ß cell lipoglucotoxicity show shared gene expression signatures. The identification of mechanisms associated with human ß cell dysfunction and recovery and their overlap with T2D islet traits provide insights into T2D pathogenesis, fostering the development of improved ß cell-targeted therapeutic strategies.

Enhancing Internal Learning in Teams: The Role of Network Centrality and Psychological Capital of Undergraduate Students.

This study aims to analyze the mediating role of psychological capital (PsyCap) in the relationship between network centrality and internal learning in teams. A questionnaire was administered to 480 undergraduate students to test this relationship. The results confirmed the positive relationship between network centrality and internal learning in teams, and a mediating role of PsyCap in the relationship between student network centrality and internal learning in teams. This study suggests that it is important to promote centrality in advice networks among undergraduate students. In addition, this study might raise awareness among students, teachers, and public policymakers about the need to promote a socially responsible environment in higher education institutions.

Efflux Pump-Driven Antibiotic and Biocide Cross-Resistance in Pseudomonas aeruginosa Isolated from Different Ecological Niches: A Case Study in the Development of Multidrug Resistance in Environmental Hotspots.

Pseudomonas aeruginosa is an opportunistic pathogen displaying high intrinsic antimicrobial resistance and the ability to thrive in different ecological environments. In this study, the ability of P. aeruginosa to develop simultaneous resistance to multiple antibiotics and disinfectants in different natural niches were investigated using strains collected from clinical samples, veterinary samples, and wastewater. The correlation between biocide and antimicrobial resistance was determined by employing principal component analysis. Molecular mechanisms linking biocide and antimicrobial resistance were interrogated by determining gene expression using RT-qPCR and identifying a potential genetic determinant for co- and cross-resistance using whole-genome sequencing. A subpopulation of P. aeruginosa isolates belonging to three sequence types was resistant against the common preservative benzalkonium chloride and showed cross-resistance to fluoroquinolones, cephalosporins, aminoglycosides, and multidrug resistance. Of these, the epidemiological high-risk ST235 clone was the most abundant. The overexpression of the MexAB-OprM drug efflux pump resulting from amino acid mutations in regulators MexR, NalC, or NalD was the major contributing factor for cross-resistance that could be reversed by an efflux pump inhibitor. This is the first comparison of antibiotic-biocide cross-resistance in samples isolated from different ecological niches and serves as a confirmation of laboratory-based studies on biocide adapted isolates. The isolates from wastewater had a higher incidence of multidrug resistance and biocide-antibiotic cross-resistance than those from clinical and veterinary settings.

Combined transcriptome and proteome profiling of the pancreatic ß-cell response to palmitate unveils key pathways of ß-cell lipotoxicity.

BACKGROUND: Prolonged exposure to elevated free fatty acids induces ß-cell failure (lipotoxicity) and contributes to the pathogenesis of type 2 diabetes. In vitro exposure of ß-cells to the saturated free fatty acid palmitate is a valuable model of lipotoxicity, reproducing features of ß-cell failure observed in type 2 diabetes. In order to map the ß-cell response to lipotoxicity, we combined RNA-sequencing of palmitate-treated human islets with iTRAQ proteomics of insulin-secreting INS-1E cells following a time course exposure to palmitate. RESULTS: Crossing transcriptome and proteome of palmitate-treated ß-cells revealed 85 upregulated and 122 downregulated genes at both transcript and protein level. Pathway analysis identified lipid metabolism, oxidative stress, amino-acid metabolism and cell cycle pathways among the most enriched palmitate-modified pathways. Palmitate induced gene expression changes compatible with increased free fatty acid mitochondrial import and ß-oxidation, decreased lipogenesis and modified cholesterol transport. Palmitate modified genes regulating endoplasmic reticulum (ER) function, ER-to-Golgi transport and ER stress pathways. Furthermore, palmitate modulated cAMP/protein kinase A (PKA) signaling, inhibiting expression of PKA anchoring proteins and downregulating the GLP-1 receptor. SLC7 family amino-acid transporters were upregulated in response to palmitate but this induction did not contribute to ß-cell demise. To unravel critical mediators of lipotoxicity upstream of the palmitate-modified genes, we identified overrepresented transcription factor binding sites and performed network inference analysis. These identified LXR, PPARα, FOXO1 and BACH1 as key transcription factors orchestrating the metabolic and oxidative stress responses to palmitate. CONCLUSIONS: This is the first study to combine transcriptomic and sensitive time course proteomic profiling of palmitate-exposed ß-cells. Our results provide comprehensive insight into gene and protein expression changes, corroborating and expanding beyond previous findings. The identification of critical drivers and pathways of the ß-cell lipotoxic response points to novel therapeutic targets for type 2 diabetes.