Do intangible factors enhance sociocultural productivity and economy in world heritage sites?

Measuring the sociocultural productivity of heritage sites remains an ongoing issue for international organizations concerned with the conservation and promotion of traditional sites. The productivity of these locations is not only affected by tangible elements but also by intangible factors, such as the emotions generated by the experiences. For this purpose, 597 employees of hotels in these historical locations who had visited one of the 14 heritage sites in Spain assessed what role emotions play in this contribution. The methodology used was the application of structural equations. Several conclusions have been drawn utilizing the SmartPLS 4 software. The first is that the generation of positive emotions comes exclusively from cultural and historical dynamization and not from technological advances or an eagerness to learn. The second is that both the application of technological advances and cultural dynamization have a direct impact on productivity.

Molecular basis for transposase activation by a dedicated AAA+ ATPase.

Transposases drive chromosomal rearrangements and the dissemination of drug-resistance genes and toxins1-3. Although some transposases act alone, many rely on dedicated AAA+ ATPase subunits that regulate site selectivity and catalytic function through poorly understood mechanisms. Using IS21 as a model transposase system, we show how an ATPase regulator uses nucleotide-controlled assembly and DNA deformation to enable structure-based site selectivity, transposase recruitment, and activation and integration. Solution and cryogenic electron microscopy studies show that the IstB ATPase self-assembles into an autoinhibited pentamer of dimers that tightly curves target DNA into a half-coil. Two of these decamers dimerize, which stabilizes the target nucleic acid into a kinked S-shaped configuration that engages the IstA transposase at the interface between the two IstB oligomers to form an approximately 1 MDa transpososome complex. Specific interactions stimulate regulator ATPase activity and trigger a large conformational change on the transposase that positions the catalytic site to perform DNA strand transfer. These studies help explain how AAA+ ATPase regulators-which are used by classical transposition systems such as Tn7, Mu and CRISPR-associated elements-can remodel their substrate DNA and cognate transposases to promote function.

Proteomic analysis of endothelial cells and extracellular vesicles in response to indoxyl sulfate: Mechanisms of endothelial dysfunction in chronic kidney disease.

AIMS: Cardiovascular pathology is the main cause of death in chronic kidney disease (CKD) patients. CKD is associated with the accumulation of uremic toxins in the bloodstream, and indoxyl sulfate (IS) is one of the most abundant uremic toxins found in the blood of CKD patients. We conducted an in vitro study to assess the mechanisms underlying the IS-induced endothelial dysfunction that could lead to cardiovascular diseases. We also studied their extracellular vesicles (EVs) owing to their capacity to act as messengers that transmit signals through their cargo. MAIN METHODS: EVs were characterized by nanoparticle tracking analysis, transmission electron microscopy, flow cytometry, and tetraspanin expression. Cell lysates and isolated EVs were analyzed using liquid chromatography coupled with mass spectrometry, followed by Gene Set Enrichment Analysis to identify the altered pathways. KEY FINDINGS: Proteomic analysis of endothelial cells revealed that IS causes an increase in proteins related to adipogenesis, inflammation, and xenobiotic metabolism and a decrease in proliferation. Extracellular matrix elements, as well as proteins associated with myogenesis, response to UV irradiation, and inflammation, were found to be downregulated in IS-treated EVs. Fatty acid metabolism was also found to be increased along with adipogenesis and inflammation observed in cells. SIGNIFICANCE: The treatment of endothelial cells with IS increased the expression of proteins related to adipogenesis, inflammation, and xenobiotic metabolism and was less associated with proliferation. Furthermore, EVs from cells treated with IS may mediate endothelial dysfunction, since they present fewer extracellular matrix elements, myogenesis, inflammatory factors, and proteins downregulated in response to UV radiation.

A proposed modification to the Kellgren and Lawrence classification for knee osteoarthritis using a compartment-specific approach.

Purpose: Since Kellgren and Lawrence (KL) originally classified knee osteoarthritis, several authors have reported varying levels of reliability and a lack of uniformity in the use of this classification system. We propose several modifications to the KL classification including the use of a compartment-specific approach that we hypothesize will lead to a better understanding of knee OA while maintaining an adequate interobserver and intraobserver reliability. Methods: We propose the addition of the lateral and skyline-view radiographs to the standard anteroposterior (AP) and lateral projections in the evaluation. Also suggest a more precise definition of the evaluated parameters; the addition of the subchondral cancellous bone as parameter of evaluation; and the assessment of medial tibiofemoral compartment (MTFC), lateral tibiofemoral compartment (LTFC) and patellofemoral compartment (PFC) separately resulting in a compartment-specific KL staging score rather than a single overall KL score. Six evaluators (two knee surgeons, two radiologists and two knee fellows) used the modified KL classification to classify 230 randomly selected knees on two separate occasions. Reliabilities were assessed by calculating Krippendorff's ⍺ coefficients. Results: Two hundred and ten knees were included for final evaluation and analyses (53% left knees; 65% females; mean age 56 years old). Average interobserver reliability was moderate for all compartments (0.51 for the MTFC; 0.51 for the LTFC; and 0.56 for the PFC). Average intraobserver reliability was substantial for all compartments (0.63 for the MTFC; 0.65 for the LTFC; and 0.7 for the PFC). Experienced evaluators showed a higher intraobserver reliability than less-experienced evaluators. Conclusions: A modified compartment-specific KL classification enables a practical and detailed description of knee OA involvement and demonstrates acceptable interobserver and intraobserver reliability. Level of Evidence: Level III.

What role geoparks play improving the health and well-being of senior tourists?

In recent years geoparks, helped by governmental policies, have become tourist destinations especially among senior visitors. The paper aimed to analyse whether geoparks contribute to improving the health of tourists older than 65 years and what were their main motives to visit geoparks. The data were collected from 398 senior tourists who visited the Villuerca- Ibores-Jara Geopark (Spain) in 2023, presenting our results using SmartPLS version 4. The results showed that senior tourists are very interested in visiting this geopark for psychotherapeutic reasons, given its high environmental and geological interest. In addition, they consider geoparks as spaces where they can socialise, which is beneficial considering the isolation that many often experience during the year. These findings are highly relevant for public authorities to protect, maintain and promote geoparks among senior tourists.

Plasma glycocalyx pattern: a mirror of endothelial damage in chronic kidney disease.

Background: Endothelial damage and cardiovascular disease complicate chronic kidney disease (CKD). The increased atherogenicity observed in patients with CKD can be linked to microinflammation and endothelial damage. Circulating endothelial glycocalyx degradation products, such as perlecan and decorin, tend to be elevated in CKD. We aimed to explore the association between the plasma perlecan and decorin levels and this pro-inflammatory and atherogenic state by studying monocyte subpopulations and intracellular adhesion molecule (ICAM)-1 expression in patients with CKD. Methods: We studied 17 healthy controls, 23 patients with advanced CKD, 25 patients on haemodialysis, 23 patients on peritoneal dialysis and 20 patients who underwent kidney transplantation. Perlecan and decorin levels were evaluated using enzyme-linked immunosorbent assays, and the monocyte phenotype was analysed using direct immunofluorescence and flow cytometry. Results: The plasma perlecan levels were higher in patients with CKD than in the healthy controls. These levels were associated with a higher prevalence of ICAM-1+ monocytes. Conversely, patients with advanced CKD (pre-dialysis) had higher plasma decorin levels, which were associated with a reduced ICAM-1 expression per monocyte. Conclusions: Elevated perlecan levels in CKD may be associated with a higher prevalence of ICAM-1+ monocytes and a pro-inflammatory phenotype. Elevated decorin levels may act as a negative regulator of ICAM-1 expression in monocytes. Therefore, perlecan and decorin may be related to inflammation and monocyte activation in CKD and may act as potential markers of endothelial damage.

Do the emotions of tourist agents contribute to improving the sustainable planning of a territory?

The sustainability of a territory is achieved through orderly, balanced and harmonious planning over time. Sustainable tourism planning must incorporate the emotions of interest groups. Based on a scale of negative and positive emotions that has already been validated, a participatory study of a qualitative nature has been developed with 118 hotel managers from the region of Extremadura, in the south-west of Spain. In addition, another quantitative research study has been carried out, using a longitudinal exploratory model analyzed in three phases throughout the years 2021 and 2022, using the SEM-PLS methodology. The objective is to detect if the II Tourism Plan (2021-2023) can influence the willingness of hotel managers to participate, and if this participation generates emotions that enrich the planning process of the tourist authorities. The results highlight the importance of completing decision making (cognitive part) with the measurement of emotions (sensitive part) of private agents to involve them in the planning process.

[Les retraités au Canada : portrait comparatif selon le statut d'immigrant].

La retraite au Canada a fait l'objet de plusieurs recherches, mais peu d'études ont comparé le passage de la vie active à la retraite des natifs et des immigrants ainsi que leurs caractéristiques une fois à la retraite, une lacune importante compte tenu de l'augmentation de la part des immigrants parmi les futures cohortes canadiennes de retraités. Cette étude descriptive vise à pallier cette lacune à l'aide des données de l'Enquête sociale générale de 2016. Les résultats montrent, entre autres, que les femmes et les hommes natifs ont plus de chances de prendre leur retraite que les immigrants, quel que soit le groupe d'âge étudié, et que l'âge moyen à la retraite des femmes et hommes immigrants est de deux ans supérieur à celui des natifs. Cette étude suggère que le statut d'immigrant implique une transition vers la retraite différente de celle vécue par les natifs ; différence qui devrait être considérée dans la structure du système de revenus de retraite.

Mechanisms of adaptive hypertrophic cardiac remodeling in a large animal model of premature ventricular contraction-induced cardiomyopathy.

Frequent premature ventricular contractions (PVCs) promoted eccentric cardiac hypertrophy and reduced ejection fraction (EF) in a large animal model of PVC-induced cardiomyopathy (PVC-CM), but the molecular mechanisms and markers of this hypertrophic remodeling remain unexplored. Healthy mongrel canines were implanted with pacemakers to deliver bigeminal PVCs (50% burden with 200-220 ms coupling interval). After 12 weeks, left ventricular (LV) free wall samples were studied from PVC-CM and Sham groups. In addition to reduced LV ejection fraction (LVEF), the PVC-CM group showed larger cardiac myocytes without evident ultrastructural alterations compared to the Sham group. Biochemical markers of pathological hypertrophy, such as store-operated Ca2+ entry, calcineurin/NFAT pathway, ß-myosin heavy chain, and skeletal type α-actin were unaltered in the PVC-CM group. In contrast, pro-hypertrophic and antiapoptotic pathways including ERK1/2 and AKT/mTOR were activated and/or overexpressed in the PVC-CM group, which appeared counterbalanced by an overexpression of protein phosphatase 1 and a borderline elevation of the anti-hypertrophic factor atrial natriuretic peptide. Moreover, the potent angiogenic and pro-hypertrophic factor VEGF-A and its receptor VEGFR2 were significantly elevated in the PVC-CM group. In conclusion, a molecular program is in place to keep this structural remodeling associated with frequent PVCs as an adaptive pathological hypertrophy.

The trinomial health, safety and happiness promote rural tourism.

BACKGROUND: Health and safety protocols have become a requirement to promote rural tourism (PRT). From this perspective, this paper empirically analyses how the health and safety dimensions influence the happiness of hotel managers and rural tourists in the post-Covid 19 era. METHODS: A theory-based structural equation model will be carried out of activation of norms, that measure variables: sanitary, socioeconomic, and safety. Precisely, we will measure how those three attributes affect the managers-guests' health in rural areas and their search for happiness at the rural destination. Based on the above, a field of study has been 215 rural tourist accommodations in the Extremadura region (Spain) and a sample population of 443 guests. Data were organised through the SEM-PLS path modelling. RESULTS: The results achieved statistically show the need to undertake a new model of healthier and safer tourism consumption that values the tourist resources of rural areas, especially nearby and sustainable destinations, based on the guiding principles of safety, health, and happiness. CONCLUSIONS: The first conclusion is that promoting tourist destinations under safe and healthy conditions has become a priority objective in the tourism industry. The second conclusion that follows from the first is that the variables safety and health and the pursuit of happiness are essential factors in promoting tourist destinations for rural hotel managers and rural tourists. The third conclusion related to the first two is that the opportunity that this study provides to develop strategies of an innovative, sustainable, and creative nature based on the relationships of the new trinomial of health, safety and happiness, from the perspective of happiness management.

New mechanisms involved in the development of cardiovascular disease in chronic kidney disease.

Chronic kidney disease (CKD) is a pathology with a high worldwide incidence and an upward trend affecting the elderly. When CKD is very advanced, the use of renal replacement therapies is required to prolong its life (dialysis or kidney transplantation). Although dialysis improves many complications of CKD, the disease does not reverse completely. These patients present an increase in oxidative stress, chronic inflammation and the release of extracellular vesicles (EVs), which cause endothelial damage and the development of different cardiovascular diseases (CVD). CKD patients develop premature diseases associated with advanced age, such as CVD. EVs play an essential role in developing CVD in patients with CKD since their number increases in plasma and their content is modified. The EVs of patients with CKD cause endothelial dysfunction, senescence and vascular calcification. In addition, miRNAs free or transported in EVs together with other components carried in these EVs promote endothelial dysfunction, thrombotic and vascular calcification in CKD, among other effects. This review describes the classic factors and focuses on the role of new mechanisms involved in the development of CVD associated with CKD, emphasizing the role of EVs in the development of cardiovascular pathologies in the context of CKD. Moreover, the review summarized the EVs' role as diagnostic and therapeutic tools, acting on EV release or content to avoid the development of CVD in CKD patients.

Nuevos mecanismos implicados en el desarrollo de la enfermedad cardiovascular en la enfermedad renal crónica / New mechanisms involved in the development of cardiovascular disease in chronic kidney disease

La enfermedad renal crónica (ERC) tiene una alta incidencia mundial y una tendencia ascendente que afecta principalmente a personas de edad avanzada. Cuando la ERC está muy avanzada se requiere el uso de terapias renales sustitutivas para prolongar la vida (diálisis o trasplante renal) y, pese a que la diálisis mejora muchas complicaciones de la ERC, la enfermedad no revierte de manera completa. Estos pacientes presentan un aumento del estrés oxidativo, inflamación crónica y aumento de la liberación de vesículas extracelulares (VE), que provocan daño endotelial y el desarrollo de distintas enfermedades cardiovasculares (ECV). De hecho, los pacientes con ERC desarrollan de forma prematura enfermedades asociadas a una edad avanzada, como es el caso de las ECV. Las VE desempeñan un papel muy importante en el desarrollo de ECV en pacientes con ERC, ya que su número aumenta en el plasma y su contenido se modifica. Las VE de pacientes con ERC generan disfunción endotelial, senescencia y calcificación vascular. Además, los miRNA libres o transportados en las VE junto a otros componentes vehiculados en estas VE promueven disfunción endotelial, eventos trombóticos y calcificación vascular en los pacientes con ERC, entre otros efectos. En esta revisión se describen los factores clásicos y el papel de nuevos mecanismos que intervienen en el desarrollo de la ECV asociada a la ERC, con especial hincapié en el papel de las VE en el desarrollo de enfermedades cardiovasculares en un contexto de ERC. Además, se expone el papel de las VE como herramienta diagnóstica y como diana terapéutica, actuando sobre su liberación o contenido para intentar evitar el desarrollo de ECV en enfermos renales crónicos. (AU)

Chronic kidney disease (CKD) is a pathology with a high worldwide incidence and an upward trend affecting the elderly. When CKD is very advanced, the use of renal replacement therapies is required to prolong its life (dialysis or kidney transplantation). Although dialysis improves many complications of CKD, the disease does not reverse completely. These patients present an increase in oxidative stress, chronic inflammation and the release of extracellular vesicles (EVs), which cause endothelial damage and the development of different cardiovascular diseases (CVD). CKD patients develop premature diseases associated with advanced age, such as CVD. EVs play an essential role in developing CVD in patients with CKD since their number increases in plasma and their content is modified. The EVs of patients with CKD cause endothelial dysfunction, senescence and vascular calcification. In addition, miRNAs free or transported in EVs together with other components carried in these EVs promote endothelial dysfunction, thrombotic and vascular calcification in CKD, among other effects. This review describes the classic factors and focuses on the role of new mechanisms involved in the development of CVD associated with CKD, emphasizing the role of EVs in the development of cardiovascular pathologies in the context of CKD. Moreover, the review summarized the EVs’ role as diagnostic and therapeutic tools, acting on EV release or content to avoid the development of CVD in CKD patients. (AU)

Alterations of sarcoplasmic reticulum-mediated Ca2+ uptake in a model of premature ventricular contraction (PVC)-induced cardiomyopathy.

Premature ventricular contractions (PVCs) are the most frequent ventricular arrhythmias in the overall population. PVCs are known to acutely enhance contractility by the post-extrasystolic potentiation phenomenon, but over time persistent PVCs promote PVC-induced cardiomyopathy (PVC-CM), characterized by a reduction of the left ventricular (LV) ejection fraction. Ca2+ cycling in myocytes commands muscle contraction and in this process, SERCA2 leads the Ca2+ reuptake into the sarcoplasmic reticulum (SR) shaping cytosolic Ca2+ signal decay and muscle relaxation. Altered Ca2+ reuptake can contribute to the contractile dysfunction observed in PVC-CM. To better understand Ca2+ handling using our PVC-CM model (canines with 50% PVC burden for 12 weeks), SR-Ca2+ reuptake was investigated by measuring Ca2+ dynamics and analyzing protein expression. Kinetic analysis of Ca2+ reuptake in electrically paced myocytes showed a ~ 21 ms delay in PVC-CM compared to Sham in intact isolated myocytes, along with a ~ 13% reduction in SERCA2 activity assessed in permeabilized myocytes. Although these trends were not statistically significant between groups using hierarchical statistics, relaxation of myocytes following contraction was significantly slower in PVC-CM vs Sham myocytes. Western blot analyses indicate a 22% reduction in SERCA2 expression, a 23% increase in phospholamban (PLN) expression, and a 50% reduction in PLN phosphorylation in PVC-CM samples vs Sham. Computational analysis simulating a 20% decrease in SR-Ca2+ reuptake resulted in a ~ 22 ms delay in Ca2+ signal decay, consistent with the experimental result described above. In conclusion, SERCA2 and PLB alterations described above have a modest contribution to functional adaptations observed in PVC-CM.

Variabilidad climática y su influencia en la aparición del dengue en provincia Guantánamo

Introducción: El dengue es considerada una de las enfermedades más devastadoras a nivel mundial con un 40 % de la población viviendo en zonas de transmisión y de las más relacionadas con el clima. Objetivo: Determinar la incidencia de la variabilidad climática sobre la infestación por mosquito Aedes aegypti y la aparición del dengue en provincia Guantánamo en el periodo 2017-2019. Método: Se realizó una investigación descriptiva longitudinal, retrospectiva, donde el universo estuvo conformado por 9 municipios y 10 221 casos sospechosos de dengue. Se estudiaron las variables: infestación en depósitos por mosquitos Aedes aegypti según temporada climática, tasa de riesgo de arbovirosis según estratos, casos de dengue, temperatura y precipitación. Los datos fueron obtenidos de los registros meteorológicos mensuales y del departamento de Estadística del Centro Provincial de Higiene, Epidemiología y Microbiología. Se utilizaron frecuencias absolutas y relativas, la media, desviación estándar y coeficiente de correlación de Pearson. Resultados: La temperatura media anual varió 1 °C, los valores medios de humedad relativa y precipitación disminuyeron e impactaron en todos los estratos. La infestación ascendió con la mayor tasa de riesgo de dengue en el estrato de clima tropical de sabana, razón de tasa 0,63 (IC 95 %: 0,59-0,67) P=0,000. La temperatura y precipitación guardaron la mayor relación de asociación lineal con infestación y tasa de riesgo de dengue con coeficiente de correlación lineal R2=0,9 y R2=0,8. Conclusiones: La variabilidad climática ejerce efectos favorecedores sobre la infestación por Aedes aegypti y la transmisión de dengue durante la temporada climática húmeda, con correlación significativa en todos los estratos.

Introduction: Dengue virus is considered one of the most devastating diseases worldwide with 40% of the population living in transmission areas and one of the most climate-related diseases. Objective: To determine the incidence rate of climate variability on Aedes aegypti mosquito infestation and dengue virus appearing in Guantanamo province, period time 2017-2019. Method: A retrospective, longitudinal, descriptive research was carried out, in which 9 municipalities and 10,221 suspected cases of dengue fever were selected (as universe). The variables studied were as follows: infestation in reservoirs by Aedes aegypti mosquitoes according to climatic season, arboviroses risk rate according to stratum, dengue cases, temperature and precipitation. Data were obtained from monthly meteorological records and from the Statistics Department of Centro Provincial de Higiene, Epidemiología y Microbiología. It was used the absolute and relative frequencies, mean frequency, the standard deviation and the Pearson correlation coefficient. Results: Mean annual temperature varied by 1 °C, mean values of relative humidity and precipitation decreased and impacted all stratums. Infestation ascended with the highest dengue risk rate in the tropical savanna climate stratum, rate ratio 0.63 (95 % CI: 0.59-0.67) P=0.000. Temperature and precipitation had the strongest linear association with Aedes aegypti mosquito infestation and dengue risk rate with the linear correlation coefficient of R2=0.9 and R2=0.8. Conclusions: Climatic variability has a favorable effect on Aedes aegypti infestation and dengue transmission during the wet season, with a significant correlation in all stratums.

Introdução: A dengue é considerada uma das doenças mais devastadoras em todo o mundo com 40% da população vivendo em áreas de transmissão e uma das mais relacionadas ao clima. Objetivo: Determinar a incidência da variabilidade climática na infestação pelo mosquito Aedes aegypti e o aparecimento da dengue na província de Guantánamo no período 2017-2019. Método: Realizou-se uma investigação descritiva retrospectiva longitudinal, onde o universo foi constituído por 9 municípios e 10.221 casos suspeitos de dengue. As variáveis estudadas foram: infestação em armazéns por mosquitos Aedes aegypti de acordo com a estação climática, taxa de risco de arbovirose de acordo com estratos, casos de dengue, temperatura e precipitação. Os dados foram obtidos dos registos meteorológicos mensais e do Departamento de Estatística do Centro Provincial de Higiene, Epidemiología y Microbiología. Foram utilizadas frequências absolutas e relativas, média, desvio padrão e coeficiente de correlação de Pearson. Resultados: A temperatura média anual variou 1°C, os valores médios de umidade relativa e precipitação diminuíram e impactaram todos os estratos. A infestação aumentou com a maior taxa de risco de dengue no estrato climático de savana tropical, razão de taxa 0,63 (IC 95%: 0,59-0,67) P=0,000. Temperatura e precipitação tiveram a maior relação de associação linear com infestação de dengue e taxa de risco com coeficiente de correlação linear R2=0,9 e R2=0,8. Conclusões: A variabilidade climática exerce efeitos favoráveis na infestação por Aedes aegypti e na transmissão da dengue durante a estação chuvosa, com correlação significativa em todos os estratos.

Accuracy of machine learning algorithms for the assessment of upper-limb motor impairments in patients with post-stroke hemiparesis: A systematic review and meta-analysis.

BACKGROUND: The assessment of motor function is vital in post-stroke rehabilitation protocols, and it is imperative to obtain an objective and quantitative measurement of motor function. There are some innovative machine learning algorithms that can be applied in order to automate the assessment of upper extremity motor function. OBJECTIVES: To perform a systematic review and meta-analysis of the efficacy of machine learning algorithms for assessing upper limb motor function in post-stroke patients and compare these algorithms to clinical assessment. MATERIAL AND METHODS: The protocol was registered in the International Prospective Register of Systematic Reviews (PROSPERO) database. The review was carried out according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines and the Cochrane Handbook for Systematic Reviews of Interventions. The search was performed using 6 electronic databases. The meta-analysis was performed with the data from the correlation coefficients using a random model. RESULTS: The initial search yielded 1626 records, but only 8 studies fully met the eligibility criteria. The studies reported strong and very strong correlations between the algorithms tested and clinical assessment. The meta-analysis revealed a lack of homogeneity (I2 = 85.29%, Q = 48.15), which is attributable to the heterogeneity of the included studies. CONCLUSION: Automated systems using machine learning algorithms could support therapists in assessing upper extremity motor function in post-stroke patients. However, to draw more robust conclusions, methodological designs that minimize the risk of bias and increase the quality of the methodology of future studies are required.

Structural and functional role of Domain I for the insecticidal activity of the Vip3Aa protein from Bacillus thuringiensis.

Vip3 proteins are produced by Bacillus thuringiensis and are toxic against lepidopterans, reason why the vip3Aa gene has been introduced into cotton and corn to control agricultural pests. Recently, the structure of Vip3 proteins has been determined and consists of a tetramer where each monomer is composed of five structural domains. The transition from protoxin to the trypsin-activated form involves a major conformational change of the N-terminal Domain I, which is remodelled into a tetrameric coiled-coil structure that is thought to insert into the apical membrane of the midgut cells. To better understand the relevance of this major change in Domain I for the insecticidal activity, we have generated several mutants aimed to alter the activity and remodelling capacity of this central region to understand its function. These mutants have been characterized by proteolytic processing, negative staining electron microscopy, and toxicity bioassays against Spodoptera exigua. The results show the crucial role of helix α1 for the insecticidal activity and in restraining the Domain I in the protoxin conformation, the importance of the remodelling of helices α2 and α3, the proteolytic processing that takes place between Domains I and II, and the role of the C-t Domains IV and V to sustain the conformational change necessary for toxicity.

Interaction of Cationic Carbosilane Dendrimers and Their siRNA Complexes with MCF-7 Cells Cultured in 3D Spheroids.

Cationic dendrimers are effective carriers for the delivery of siRNA into cells; they can penetrate cell membranes and protect nucleic acids against RNase degradation. Two types of dendrimers (CBD-1 and CBD-2) and their complexes with pro-apoptotic siRNA (Mcl-1 and Bcl-2) were tested on MCF-7 cells cultured as spheroids. Cytotoxicity of dendrimers and dendriplexes was measured using the live-dead test and Annexin V-FITC Apoptosis Detection Kit (flow cytometry). Uptake of dendriplexes was examined using flow cytometry and confocal microscopy. The live-dead test showed that for cells in 3D, CBD-2 is more toxic than CBD-1, contrasting with the data for 2D cultures. Attaching siRNA to a dendrimer molecule did not lead to increased cytotoxic effect in cells, either after 24 or 48 h. Measurements of apoptosis did not show a high increase in the level of the apoptosis marker after 24 h exposure of spheroids to CBD-2 and its dendriplexes. Measurements of the internalization of dendriplexes and microscopy images confirmed that the dendriplexes were transported into cells of the spheroids. Flow cytometry analysis of internalization indicated that CBD-2 transported siRNAs more effectively than CBD-1. Cytotoxic effects were visible after incubation with 3 doses of complexes for CBD-1 and both siRNAs.

Safety of G2-S16 Polyanionic Carbosilane Dendrimer as Possible HIV-1 Vaginal Microbicide.

The UNAIDS objective for 2020 was 500,000 new HIV-1 infections per year; however, the latest annual reported data confirmed 1.7 million new HIV-1 infections in that year. Those data evidences the need for new prevention strategies and prophylactic treatments. This prevention crisis occurred in spite of the knowledge and availability of efficient prevention strategies. The G2-S16 is a microbicidal polyanionic carbosilane dendrimer currently being tested for topical vaginal application, which has been shown to be efficient in the prevention of HIV-1 infection. However, safety tests were lacked. For this purpose, we injected intravenously G2-S16 dendrimer to CD1 mice, thereby analyzing the hemogram, blood biochemical markers of systemic damage, accumulation in the organs and organ-tissue damage in heart, spleen, kidney, liver and brain. This work shows that even if the G2-S16 dendrimer penetrates the epithelial tissue, it does not cause vaginal irritation or tissue damage. Moreover, the i.v. injection of the G2-S16 dendrimer did not cause a damaging effect on the studied organs and it did not modify the hemogram or the biochemical plasma markers. In conclusion, the G2-S16 dendrimer has a very good safety profile, indicating that this molecule can be a very safe and efficient vaginal microbicide.

Endothelial Senescence and the Chronic Vascular Diseases: Challenges and Therapeutic Opportunities in Atherosclerosis.

Atherosclerosis is probably one of the paradigms of disease linked to aging. Underlying the physiopathology of atherosclerosis are cellular senescence, oxidative stress, and inflammation. These factors are increased in the elderly and from chronic disease patients. Elevated levels of oxidative stress affect cellular function and metabolism, inducing senescence. This senescence modifies the cell phenotype into a senescent secretory phenotype. This phenotype activates immune cells, leading to chronic systemic inflammation. Moreover, due to their secretory phenotype, senescence cells present an increased release of highlighted extracellular vesicles that will change nearby/neighborhood cells and paracrine signaling. For this reason, searching for specific senescent cell biomarkers and therapies against the development/killing of senescent cells has become relevant. Recently, senomorphic and senolityc drugs have become relevant in slowing down or eliminating senescence cells. However, even though they have shown promising results in experimental studies, their clinical use is still yet to be determined.