Therapeutic Effect of Alpha Lipoic Acid in a Rat Preclinical Model of Preeclampsia: Focus on Maternal Signs, Fetal Growth and Placental Function.

Chronic hypertension is a major risk factor for preeclampsia (PE), associated with significant maternal and neonatal morbidity. We previously demonstrated that pregnant stroke-prone spontaneously hypertensive rats (SHRSP) display a spontaneous PE-like phenotype with distinct placental, fetal, and maternal features. Here, we hypothesized that supplementation with alpha lipoic acid (ALA), a potent antioxidant, during early pregnancy could ameliorate the PE phenotype in this model. To test this hypothesis, timed pregnancies were established using 10 to 12-week-old SHRSP females (n = 19-16/group), which were assigned to two treatment groups: ALA (injected intraperitoneally with 25 mg/kg body weight ALA on gestation day (GD1, GD8, and GD12) or control, receiving saline following the same protocol. Our analysis of maternal signs showed that ALA prevented the pregnancy-dependent maternal blood pressure rise (GD14 blood pressure control 169.3 ± 19.4 mmHg vs. 146.1 ± 13.4 mmHg, p = 0.0001) and ameliorated renal function, as noted by the increased creatinine clearance and improved glomerular histology in treated dams. Treatment also improved the fetal growth restriction (FGR) phenotype, leading to increased fetal weights (ALA 2.19 ± 0.5 g vs. control 1.98 ± 0.3 g, p = 0.0074) and decreased cephalization indexes, indicating a more symmetric fetal growth pattern. This was associated with improved placental efficiency, decreased oxidative stress marker expression on GD14, and serum soluble fms-like tyrosine kinase 1 (sFlt1) levels on GD20. In conclusion, ALA supplementation mitigated maternal signs and improved placental function and fetal growth in SHRSP pregnancies, emerging as a promising therapy in pregnancies at high risk for PE.

Virtual Experience Toolkit: An End-to-End Automated 3D Scene Virtualization Framework Implementing Computer Vision Techniques.

Virtualization plays a critical role in enriching the user experience in Virtual Reality (VR) by offering heightened realism, increased immersion, safer navigation, and newly achievable levels of interaction and personalization, specifically in indoor environments. Traditionally, the creation of virtual content has fallen under one of two broad categories: manual methods crafted by graphic designers, which are labor-intensive and sometimes lack precision; traditional Computer Vision (CV) and Deep Learning (DL) frameworks that frequently result in semi-automatic and complex solutions, lacking a unified framework for both 3D reconstruction and scene understanding, often missing a fully interactive representation of the objects and neglecting their appearance. To address these diverse challenges and limitations, we introduce the Virtual Experience Toolkit (VET), an automated and user-friendly framework that utilizes DL and advanced CV techniques to efficiently and accurately virtualize real-world indoor scenarios. The key features of VET are the use of ScanNotate, a retrieval and alignment tool that enhances the precision and efficiency of its precursor, supported by upgrades such as a preprocessing step to make it fully automatic and a preselection of a reduced list of CAD to speed up the process, and the implementation in a user-friendly and fully automatic Unity3D application that guides the users through the whole pipeline and concludes in a fully interactive and customizable 3D scene. The efficacy of VET is demonstrated using a diversified dataset of virtualized 3D indoor scenarios, supplementing the ScanNet dataset.

Supramolecular Complex of Cucurbit[7]uril with Diketopyrrolopyrole Dye: Fluorescence Boost, Biolabeling and Optical Microscopy.

New photostable and bright supramolecular complexes based on cucurbit[7]uril (CB7) host and diketopyrrolopyrole (DPP) guest dyes having two positively charged 4-(trimethylammonio)phenyl groups were prepared; with spectra (H2O, abs. / emission max. 480 / 550 nm; e ~ 19 000, tfl > 4 ns), strong binding with hosts (~560 nM Kd) and a linker affording fluorescence detection of bioconjugates with antibody and nanobody. Combination of protein-functionalized DPP dye with CB7 improves photostability and affords up to 12-fold emission gain. Two-color confocal and stimulated emission depletion (STED) microscopy with 595 nm or 655 nm STED depletion lasers shows that the presence of CB7 not only leads to improved brightness and image quality, but also results in DPP becoming cell-permeable.

Valproate decreases transgenerationally blood pressure by affecting thyrotropin-releasing hormone promoter DNA methylation and gene expression in spontaneously hypertensive rat.

Central TRH, a neuropeptide, is involved in cardiovascular regulation. We demonstrated that the overexpression of diencephalic TRH (dTRH) in SHR rats can be prevented by antisense treatment, normalizing blood pressure (BP). Valproate (VPA) is an inhibitor of histone deacetylases (HDAC) which modulates gene expression through epigenetic modifications such as DNA methylation. AIMS: Study the role of HDAC inhibition in the regulation of dTRH gene expression and its effect on the pathogenesis of hypertension. MAIN METHODS: We treated 7-weeks-old male and female SHR and WKY rats with VPA for 10 weeks and evaluated BP, dTRH mRNA and methylation gene status. KEY FINDINGS: VPA attenuated the elevated BP and dTRH mRNA expression characteristic of SHR. Indeed, we found a significant 62% reduction in dTRH mRNA expression in the SHR + VPA group compared to control SHR. The decrease TRH mRNA expression induced by VPA was confirmed "in vitro" in a primary neuron culture using trichostatin A. With methylation specific PCR we demonstrated a significant increase in TRH promoter DNA methylation level in SHR + VPA group compared to control SHR. After 2 weeks of treatment interruption, rats were mated. Although they did not receive any treatment, the offspring born from VPA-treated SHR parents showed similar changes in BP, dTRH expression and methylation status, implying a transgenerational inheritance. Our findings suggest that dTRH modulation by epigenetics mechanism affects BP and could be inherited by the next generation in SHR rats.

Inteligencia artificial. Aplicación en las imágenes cardiovasculares y la prevención cardiovascular / Artificial Intelligence in Cardiovascular Imaging and Cardiovascular Prevention

RESUMEN La inteligencia artificial (IA) está basada en programas computacionales que pueden imitar el pensamiento humano y automatizar algunos procesos. En el ámbito médico se está estudiando hace más de 50 años, pero en los últimos años el crecimiento ha sido exponencial. El campo de las imágenes cardiovasculares es particularmente atractivo para aplicarla, dado que, guiadas por IA, personas no expertas pueden adquirir imágenes completas, automatizar procesos y mediciones, orientar diagnósticos, detectar hallazgos no visibles al ojo humano, realizar diagnósticos oportunistas de afecciones no buscadas en el estudio índice pero evaluables a través de las imágenes disponibles, o identificar patrones de asociación dentro de una gran cantidad de datos como fuente de generación de hipótesis. En el campo de la prevención cardiovascular, la IA se ha aplicado en diferentes escenarios con fines diagnósticos, pronósticos y terapéuticos en el manejo de algunos factores de riesgo cardiovascular, como las dislipidemias o la hipertensión arterial. Si bien existen limitaciones con el uso de la IA tales como el costo, la accesibilidad y la compatibilidad de los programas, la validez externa de los resultados en determinadas poblaciones, o algunos aspectos éticos-legales (privacidad de los datos), esta tecnología está en crecimiento vertiginoso y posiblemente revolucione la práctica médica actual.

ABSTRACT Artificial intelligence (AI) is based on computer programs that imitate human thinking and automate certain processes. Artificial intelligence has been studied in the medical field for over 50 years, but in recent years, its growth has been exponential. The field of cardiovascular imaging is particularly attractive since AI can guide non-experts in image acquisition, automate processes and measurements, guide diagnoses, detect findings not visible to the human eye, make opportunistic diagnoses of unexpected conditions in the index test, or identify patterns of association within a large amount of data as a source of hypothesis generation. In the field of cardiovascular prevention, AI has been used for diagnostic, prognostic, and therapeutic purposes in managing cardiovascular risk factors such as dyslipidemia and hypertension. While there are limitations to the use of AI, such as cost, accessibility, compatibility of programs, external validity of results in certain populations, and ethical-legal aspects such as data privacy, this technology is rapidly growing and is likely to revolutionize current medical practice.

Photoactivatable Xanthone (PaX) Dyes Enable Quantitative, Dual Color, and Live-Cell MINFLUX Nanoscopy.

The single-molecule localization concept MINFLUX has triggered a reevaluation of the features of fluorophores for attaining nanometer-scale resolution. MINFLUX nanoscopy benefits from temporally controlled fluorescence ("on"/"off") photoswitching. Combined with an irreversible switching behavior, the localization process is expected to turn highly efficient and quantitative data analysis simple. The potential in the recently reported photoactivable xanthone (PaX) dyes is recognized to extend the list of molecular switches used for MINFLUX with 561 nm excitation beyond the fluorescent protein mMaple. The MINFLUX localization success rates of PaX560 , PaX+560, and mMaple are quantitatively compared by analyzing the effective labeling efficiency of endogenously tagged nuclear pore complexes. The PaX dyes prove to be superior to mMaple and on par with the best reversible molecular switches routinely used in single-molecule localization microscopy. Moreover, the rationally designed PaX595 is introduced for complementing PaX560 in dual color 561 nm MINFLUX imaging based on spectral classification and the deterministic, irreversible, and additive-independent nature of PaX photoactivation is showcased in fast live-cell MINFLUX imaging. The PaX dyes meet the demands of MINFLUX for a robust readout of each label position and fill the void of reliable fluorophores dedicated to 561 nm MINFLUX imaging.

Synthesis of Thioxanthone 10,10-Dioxides and Sulfone-Fluoresceins via Pd-Catalyzed Sulfonylative Homocoupling.

Our report describes the facile and scalable preparation of 9H-thioxanthen-9-one 10,10-dioxides via Pd-catalyzed sulfonylative homocoupling of the appropriately substituted benzophenones. This transformation provides a straightforward route to previously unreported sulfone-fluoresceins and -fluorones. Several examples of these red fluorescent dyes have been prepared, characterized, and evaluated as live-cell permeant labels compatible with super-resolution fluorescence microscopy with 775 nm stimulated emission depletion.

Bioorthogonal Caging-Group-Free Photoactivatable Probes for Minimal-Linkage-Error Nanoscopy.

Here we describe highly compact, click compatible, and photoactivatable dyes for super-resolution fluorescence microscopy (nanoscopy). By combining the photoactivatable xanthone (PaX) core with a tetrazine group, we achieve minimally sized and highly sensitive molecular dyads for the selective labeling of unnatural amino acids introduced by genetic code expansion. We exploit the excited state quenching properties of the tetrazine group to attenuate the photoactivation rates of the PaX, and further reduce the overall fluorescence emission of the photogenerated fluorophore, providing two mechanisms of selectivity to reduce the off-target signal. Coupled with MINFLUX nanoscopy, we employ our dyads in the minimal-linkage-error imaging of vimentin filaments, demonstrating molecular-scale precision in fluorophore positioning.

Photoactivatable Carbo- and Silicon-Rhodamines and Their Application in MINFLUX Nanoscopy.

New photoactivatable fluorescent dyes (rhodamine, carbo- and silicon-rhodamines [SiR]) with emission ranging from green to far red have been prepared, and their photophysical properties studied. The photocleavable 2-nitrobenzyloxycarbonyl unit with an alpha-carboxyl group as a branching point and additional functionality was attached to a polycyclic and lipophilic fluorescent dye. The photoactivatable probes having the HaloTagTM amine (O2) ligand bound with a dye core were obtained and applied for live-cell staining in stable cell lines incorporating Vimentin (VIM) or Nuclear Pore Complex Protein NUP96 fused with the HaloTag. The probes were applied in 2D (VIM, NUP96) and 3D (VIM) MINFLUX nanoscopy, as well as in superresolution fluorescence microscopy with single fluorophore activation (VIM, live-cell labeling). Images of VIM and NUPs labeled with different dyes were acquired and their apparent dimensions and shapes assessed on a lower single-digit nanometer scale. Applicability and performance of the photoactivatable dye derivatives were evaluated in terms of photoactivation rate, labeling and detection efficiency, number of detected photons per molecule and other parameters related to MINFLUX nanoscopy.

Accelerated MINFLUX Nanoscopy, through Spontaneously Fast-Blinking Fluorophores.

The introduction of MINFLUX nanoscopy allows single molecules to be localized with one nanometer precision in as little as one millisecond. However, current applications have so far focused on increasing this precision by optimizing photon collection, rather than minimizing the localization time. Concurrently, commonly used fluorescent switches are specifically designed for stochastic methods (e.g., STORM), optimized for a high photon yield and rather long on-times (tens of milliseconds). Here, accelerated MINFLUX nanoscopy with up to a 30-fold gain in localization speed is presented. The improvement is attained by designing spontaneously blinking fluorescent markers with remarkably fast on-times, down to 1-3 ms, matching the iterative localization process used in a MINFLUX microscope. This design utilizes a silicon rhodamine amide core, shifting the spirocyclization equilibrium toward an uncharged closed form at physiological conditions and imparting intact live cell permeability, modified with a fused (benzo)thiophene spirolactam fragment. The best candidate for MINFLUX microscopy (also suitable for STORM imaging) is selected through detailed characterization of the blinking behavior of single fluorophores, bound to different protein tags. Finally, optimization of the localization routines, customized to the fast blinking times, renders a significant speed improvement on a commercial MINFLUX microscope.

Photoactivatable Large Stokes Shift Fluorophores for Multicolor Nanoscopy.

We designed caging-group-free photoactivatable live-cell permeant dyes with red fluorescence emission and ∼100 nm Stokes shifts based on a 1-vinyl-10-silaxanthone imine core structure. The proposed fluorophores undergo byproduct-free one- and two-photon activation, are suitable for multicolor fluorescence microscopy in fixed and living cells, and are compatible with super-resolution techniques such as STED (stimulated emission depletion) and PALM (photoactivated localization microscopy). Use of photoactivatable labels for strain-promoted tetrazine ligation and self-labeling protein tags (HaloTag, SNAP-tag), and duplexing of an imaging channel with another large Stokes shift dye have been demonstrated.

Diagnostic yield of computed tomography guided fine-needle aspiration for pancreatic cancer.

Fine-needle aspiration (FNA) for the histological diagnosis of occupying lesions in the pancreas as opposed to tru-cut needle biopsy to obtain tissue for analysis has been associated with a lower incidence of post-procedure complications, with almost immediate recovery and no need for hospital stay. Nevertheless, the question of the diagnostic effectiveness of percutaneous computed axial tomography (CT)-guided FNA in solid pancreatic lesions has been raised. The aim of this study was to confirm the diagnostic effectivity of percutaneous CT-guided FNA in pancreatic space-occupying lesions and to assess short-term complications. All percutaneous CT-guided FNA with real-time monitoring, performed between April 2010 and December 2015, were retrospectively analyzed. In all cases 21-gauge needles were used. All FNA were performed in the presence of a pathologist who immediately stained and reported as adequate for analysis in all cases. The diagnosis was confirmed by histopathological evaluation. Of 54 FNA performed, final histopathological evaluation revealed neoplastic cells compatible with adenocarcinoma in 52 patients (96%) and was negative for neoplastic cells in two patients (4%). The sensitivity was 94%, and the specificity 100%. Post-FNA morbidity was observed in four patients, consisting of epigastric pain in two and abdominal wall hematoma in two other patients. Percutaneous CT-guided FNA of pancreatic space-occupying lesions was found to be a good, minimally invasive and safe method with low morbidity. The presence of the pathologist in the procedure allowed for immediate cytological diagnosis.

El uso de la punción-aspiración con aguja fina (PAAF) en el diagnóstico histológico de lesiones ocupantes de páncreas es una alternativa frente al uso de agujas tru-cut en la obtención de tejido para su análisis, con una incidencia más baja de complicaciones y una recuperación casi inmediata sin necesidad de internación. El objetivo fue valorar la efectividad diagnóstica de las PAAF de lesiones ocupantes pancreáticas guiadas por tomografía axial computada (TAC) por vía percutánea, y su tasa de complicaciones a corto plazo. Se analizaron de forma retrospectiva todas las PAAF realizadas mediante guía tomográfica por vía percutánea con control en tiempo real, entre abril 2010 y diciembre 2015. Todas las PAAF se realizaron en presencia de un patólogo que inmediatamente tiñó e informó como adecuado para el análisis. La confirmación diagnóstica se hizo con el análisis anatomopatológico diferido. De las 54 PAAF realizadas, el diagnóstico anatomopatológico informó positivo para células neoplásicas compatible con adenocarcinoma en 52 pacientes (96%) y en otros dos (4%) como negativo para células neoplásicas. La sensibilidad del método fue 94% y la especificidad del 100%. Se registraron 4 casos de morbilidad post punción (2 dolores epigástricos y 2 hematomas de pared abdominal). Las punciones percutáneas de lesiones ocupantes pancreáticas guiadas por TC pueden considerarse un buen método diagnóstico mini invasivo, seguro, con una morbilidad post punción baja. La presencia del patólogo en el procedimiento permitió el diagnóstico citológico inmediato.

Diagnostic yield of computed tomography guided fine-needle aspiration for pancreatic cancer / Rendimiento diagnóstico de la aspiración con aguja fina guiada por tomografía computarizada para el cáncer de páncreas

Abstract Fine-needle aspiration (FNA) for the histological diagnosis of occupying lesions in the pancreas as opposed to tru-cut needle biopsy to obtain tissue for analysis has been associated with a lower incidence of post-procedure complications, with almost immediate recovery and no need for hospital stay. Nev ertheless, the question of the diagnostic effectiveness of percutaneous computed axial tomography (CT)-guided FNA in solid pancreatic lesions has been raised. The aim of this study was to confirm the diagnostic effectivity of percutaneous CT-guided FNA in pancreatic space-occupying lesions and to assess short-term complications. All percutaneous CT-guided FNA with real-time monitoring, performed between April 2010 and December 2015, were retrospectively analyzed. In all cases 21-gauge needles were used. All FNA were performed in the pres ence of a pathologist who immediately stained and reported as adequate for analysis in all cases. The diagnosis was confirmed by histopathological evaluation. Of 54 FNA performed, final histopathological evaluation revealed neoplastic cells compatible with adenocarcinoma in 52 patients (96%) and was negative for neoplastic cells in two patients (4%). The sensitivity was 94%, and the specificity 100%. Post-FNA morbidity was observed in four patients, consisting of epigastric pain in two and abdominal wall hematoma in two other patients. Percutaneous CT-guided FNA of pancreatic space-occupying lesions was found to be a good, minimally invasive and safe method with low morbidity. The presence of the pathologist in the procedure allowed for immediate cytological diagnosis.

Resumen El uso de la punción-aspiración con aguja fina (PAAF) en el diagnóstico histológico de lesiones ocupantes de páncreas es una alternativa frente al uso de agujas tru-cut en la obtención de tejido para su análisis, con una incidencia más baja de complicaciones y una recuperación casi inmediata sin necesidad de internación. El objetivo fue valorar la efectividad diagnóstica de las PAAF de lesiones ocupantes pancreáticas guiadas por tomografía axial computada (TAC) por vía percutánea, y su tasa de complicaciones a corto plazo. Se analizaron de forma retrospectiva todas las PAAF realizadas mediante guía tomográfica por vía percutánea con control en tiempo real, entre abril 2010 y diciembre 2015. Todas las PAAF se realizaron en presencia de un patólogo que inmediatamente tiñó e informó como adecuado para el análisis. La confirmación diagnóstica se hizo con el análisis anatomopatológico diferido. De las 54 PAAF realizadas, el diagnóstico anatomopatológico informó positivo para células neoplásicas compatible con adenocarcinoma en 52 pacientes (96%) y en otros dos (4%) como negativo para células neoplásicas. La sensibilidad del método fue 94% y la especificidad del 100%. Se registraron 4 casos de morbilidad post punción (2 dolores epigástricos y 2 hematomas de pared abdominal). Las punciones percutáneas de lesiones ocupantes pancreáticas guiadas por TC pueden considerarse un buen método diagnóstico mini invasivo, seguro, con una morbilidad post punción baja. La presencia del patólogo en el procedimiento permitió el diagnóstico citológico inmediato.

N-Cyanorhodamines: cell-permeant, photostable and bathochromically shifted analogues of fluoresceins.

Fluorescein and its analogues have found only limited use in biological imaging because of the poor photostability and cell membrane impermeability of their O-unprotected forms. Herein, we report rationally designed N-cyanorhodamines as orange- to red-emitting, photostable and cell-permeant fluorescent labels negatively charged at physiological pH values and thus devoid of off-targeting artifacts often observed for cationic fluorophores. In combination with well-established fluorescent labels, self-labelling protein (HaloTag, SNAP-tag) ligands derived from N-cyanorhodamines permit up to four-colour confocal and super-resolution STED imaging in living cells.

Supramolecular Complex of Photochromic Diarylethene and Cucurbit[7]uril: Fluorescent Photoswitching System for Biolabeling and Imaging.

Photoswitchable fluorophores─proteins and synthetic dyes─whose emission is reversibly switched on and off upon illumination, are powerful probes for bioimaging, protein tracking, and super-resolution microscopy. Compared to proteins, synthetic dyes are smaller and brighter, but their photostability and the number of achievable switching cycles in aqueous solutions are lower. Inspired by the robust photoswitching system of natural proteins, we designed a supramolecular system based on a fluorescent diarylethene (DAE) and cucurbit[7]uril (CB7) (denoted as DAE@CB7). In this assembly, the photoswitchable DAE molecule is encapsulated by CB7 according to the host-guest principle, so that DAE is protected from the environment and its fluorescence brightness and fatigue resistance in pure water improved. The fluorescence quantum yield (Φfl) increased from 0.40 to 0.63 upon CB7 complexation. The photoswitching of the DAE@CB7 complex, upon alternating UV and visible light irradiations, can be repeated 2560 times in aqueous solution before half-bleaching occurs (comparable to fatigue resistance of the reversibly photoswitchable proteins), while free DAE can be switched on and off only 80 times. By incorporation of reactive groups [maleimide and N-hydroxysuccinimidyl (NHS) ester], we prepared bioconjugates of DAE@CB7 with antibodies and demonstrated both specific labeling of intracellular proteins in cells and the reversible on/off switching of the probes in cellular environments under irradiations with 355 nm/485 nm light. The bright emission and robust photoswitching of DAE-Male3@CB7 and DAE-NHS@CB7 complexes (without exclusion of air oxygen and addition of any stabilizing/antifading reagents) enabled confocal and super-resolution RESOLFT (reversible saturable optical fluorescence transitions) imaging with apparent 70-90 nm optical resolution.

A general design of caging-group-free photoactivatable fluorophores for live-cell nanoscopy.

The controlled switching of fluorophores between non-fluorescent and fluorescent states is central to every super-resolution fluorescence microscopy (nanoscopy) technique, and the exploration of radically new switching mechanisms remains critical to boosting the performance of established, as well as emerging super-resolution methods. Photoactivatable dyes offer substantial improvements to many of these techniques, but often rely on photolabile protecting groups that limit their applications. Here we describe a general method to transform 3,6-diaminoxanthones into caging-group-free photoactivatable fluorophores. These photoactivatable xanthones (PaX) assemble rapidly and cleanly into highly fluorescent, photo- and chemically stable pyronine dyes upon irradiation with light. The strategy is extendable to carbon- and silicon-bridged xanthone analogues, yielding a family of photoactivatable labels spanning much of the visible spectrum. Our results demonstrate the versatility and utility of PaX dyes in fixed and live-cell labelling for conventional microscopy, as well as the coordinate-stochastic and deterministic nanoscopies STED, PALM and MINFLUX.

Cleavable Linker Incorporation into a Synthetic Dye-Nanobody-Fluorescent Protein Assembly: FRET, FLIM and STED Microscopy.

A bright and photostable fluorescent dye with a disulfide (S-S) linker and maleimide group (Rho594-S2-mal), as cleavable and reactive sites, was synthesized and conjugated with anti-GFP nanobodies (NB). The binding of EGFP (FRET donor) with anti-GFP NB labeled with one or two Rho594-S2-mal residues was studied in vitro and in cellulo. The linker was cleaved with dithiothreitol recovering the donor (FP) signal. The bioconjugates (FP-NB-dye) were applied in FRET-FLIM assays, confocal imaging, and superresolution STED microscopy.