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Abstract Christensenella minuta DSM 22607 has recently been suggested as a potential microbiome-based therapy for inflammatory bowel disease (IBD) because it displays strong anti-inflammatory effects both in vitro and in vivo. Here, we aimed to decipher the mechanism(s) underlying the DSM 22607-mediated beneficial effects on the host in a mouse model of chemically induced acute colitis. We observed that C. minuta plays a key role in the preservation of the epithelial barrier and the management of DNBS-induced inflammation by inhibiting interleukin (IL)-33 and Tumor necrosis factor receptor superfamily member 8 (Tnfrsf8) gene expression. We also showed that DSM 22607 abundance was positively correlated with Akkermansia sp. and Dubosiella sp. and modulated microbial metabolites in the cecum. These results offer new insights into the biological and molecular mechanisms underlying the beneficial effects of C. minuta DSM 22607 by protecting the intestinal barrier integrity and regulating inflammation.

IntroductionThe aim of this work was to characterize a new strain of Ligilactobacillus salivarius (CNCM I-4866) (CNCM I-4866) to address its potential as probiotic with a special focus on intestinal inflammation. Potential anti-inflammatory abilities of this strain were evaluated through in vivo and in vitro experiments.MethodsFirstly, the strain was tested in a murine acute inflammation colitis model induced by DNBS. In vitro characterization was then performed with diverse tests: modulation capability of intestinal permeability; study of the impact on immunity profile through cytokines dosage; capacity to inhibit pathogens and adhere to intestinal cells lines. Production of metabolites, antibiotic resistance and survival to gastro-intestinal tract conditions were also tested.ResultsIn vitro assay has shown a reduction of colonic damage and markers of inflammation after treatment with CNCM I-4866. Transcriptomic analysis performed on colons showed the capacity of the strain to down-regulate pro-inflammatory cytokines. L. salivarius CNCM I-4866 exerted anti-inflammatory profile by reducing IL-8 production by TNF-α stimulated cell and modulated cytokines profile on peripheral blood mononuclear cells (PBMC). It protected intestinal integrity by increasing trans-epithelial electrical resistance (TEER) on Caco-2 TNF-α inflamed cells. Additionally, L. salivarius CNCM I-4866 displayed inhibition capacity on several intestinal pathogens and adhered to eukaryotic cells. Regarding safety and technical concerns, CNCM I-4866 was highly resistant to 0.3% of bile salts and produced mainly L-lactate. Finally, strain genomic characterization allowed us to confirm safety aspect of our strain, with no antibiotic gene resistance found.DiscussionTaken together, these results indicate that L. salivarius CNCM I-4866 could be a good probiotic candidate for intestinal inflammation, especially with its steady anti-inflammatory profile.

Abstract Microbiome-based therapies for inflammatory bowel diseases offer a novel and promising therapeutic approach. The human commensal bacteria of the species Christensenella minuta (C. minuta) have been reported consistently missing in patients affected by Crohn’s disease (CD) and have been documented to induce anti-inflammatory effects in human epithelial cells, supporting their potential as a novel biotherapy. This work aimed at selecting the most promising strain of C. minuta for future development as a clinical candidate for CD therapy. Here, we describe a complete screening process combining in vitro and in vivo assays to conduct a rational selection of a live strain of C. minuta with strong immunomodulatory properties. Starting from a collection of 32 strains, a panel of in vitro screening assays was used to narrow it down to five preclinical candidates that were further screened in vivo in an acute TNBS-induced rat colitis model. The most promising candidate was validated in vivo in two mouse models of colitis. The validated clinical candidate strain, C. minuta DSM 33715, was then fully characterized. Hence, applying a rationally designed screening algorithm, a novel strain of C. minuta was successfully identified as the most promising clinical candidate for CD.

El ecosistema mediático digital se ha estandarizado como referencia preeminente de ocio y entretenimiento. Su exponencial oferta resulta accesible de forma inmediata, ubicua y constante a través de dispositivos conectados, y ello ha diluido la efectividad de los sistemas de orientación sobre la idoneidad o inconveniencia de contenidos para niños y adolescentes. Esta investigación revisa, ordena y sistematiza los criterios e indicadores de los modelos de clasificación por edades en la televisión, el cine, las plataformas de streaming de video y los videojuegos utilizados en España, Reino Unido, Francia, Alemania, Italia y Portugal, seis países europeos con entornos socioculturales y estructuras de mercado asimilables. El estudio constata notables diferencias en la definición y categorización según los medios y territorios; además, pone de relieve la necesidad de redefinir las bases semánticas, estéticas y técnicas sobre las que construir un modelo común de recomendaciones compatible con todos los contenidos digitales, al margen del tipo de soporte o la tecnología de distribución; sólo así se podrá promover un consumo activo que fomente el pensamiento crítico y la educación mediática digital en niños y jóvenes.

Abstract Christensenellaceae is a family of subdominant commensal bacteria found in humans. It is thought to play an important role in gut health by maintaining microbial symbiosis. Indeed, these bacteria occur at significantly lower levels or are absent in individuals suffering from inflammatory bowel diseases (IBDs). Here, we explored if type species Christensenella minuta (strain: DSM 22607) could have the potential to help treat IBDs. We assessed key properties displayed by the bacterium using a combination of in vitro and in vivo assays. We found that while C. minuta is a strict anaerobe, it is also oxygen tolerant. Additionally, we observed that the species produces high levels of acetate and moderate levels of butyrate. We performed deep phenotyping using Biolog microarrays. Using human intestinal cell lines, we discovered that C. minuta demonstrated strong anti-inflammatory activity, resulting in reduced levels of proinflammatory IL-8 cytokines via the inhibition of the NF-κB signaling pathway. Furthermore, C. minuta protected intestinal epithelial integrity in vitro. Finally, in two distinct animal models of acute colitis, C. minuta prevented intestinal damage, reduced colonic inflammation, and promoted mucosal healing. Together, these results indicate that C. minuta has potent immunomodulatory properties, underscoring its potential use in innovative microbiome-based IBD biotherapies.

Faecalibacterium prausnitzii is a commensal bacterium, ubiquitous in the gastrointestinal tracts of animals and humans. This species is a functionally important member of the microbiota and studies suggest it has an impact on the physiology and health of the host. F. prausnitzii is the only identified species in the genus Faecalibacterium, but a recent study clustered strains of this species in two different phylogroups. Here, we propose the existence of distinct species in this genus through the use of comparative genomics. Briefly, we performed analyses of 16S rRNA gene phylogeny, phylogenomics, whole genome Multi-Locus Sequence Typing (wgMLST), Average Nucleotide Identity (ANI), gene synteny, and pangenome to better elucidate the phylogenetic relationships among strains of Faecalibacterium. For this, we used 12 newly sequenced, assembled, and curated genomes of F. prausnitzii, which were isolated from feces of healthy volunteers from France and Australia, and combined these with published data from 5 strains downloaded from public databases. The phylogenetic analysis of the 16S rRNA sequences, together with the wgMLST profiles and a phylogenomic tree based on comparisons of genome similarity, all supported the clustering of Faecalibacterium strains in different genospecies. Additionally, the global analysis of gene synteny among all strains showed a highly fragmented profile, whereas the intra-cluster analyses revealed larger and more conserved collinear blocks. Finally, ANI analysis substantiated the presence of three distinct clusters—A, B, and C—composed of five, four, and four strains, respectively. The pangenome analysis of each cluster corroborated the classification of these clusters into three distinct species, each containing less variability than that found within the global pangenome of all strains. Here, we propose that comparison of pangenome subsets and their associated α values may be used as an alternative approach, together with ANI, in the in silico classification of new species. Altogether, our results provide evidence not only for the reconsideration of the phylogenetic and genomic relatedness among strains currently assigned to F. prausnitzii, but also the need for lineage (strain-based) differentiation of this taxon to better define how specific members might be associated with positive or negative host interactions.

Farmers experience whole-body vibrations when they drive tractors. Among the various factors that influence the vibrations to which the driver is exposed are terrain roughness, tractor speed, tire type and pressure, rear axle width, and tractor seat height above the ground. In this paper the influence of tractor seat height above the ground on the lateral vibrations to which the tractor driver is exposed is studied by means of a geometrical and an experimental analysis. Both analyses show that: (i) lateral vibrations experienced by a tractor driver increase linearly with tractor-seat height above the ground; (ii) lateral vibrations to which the tractor driver is exposed can equal or exceed vertical vibrations; (iii) in medium-size tractors, a feasible 30 cm reduction in the height of the tractor seat, which represents only 15% of its current height, will reduce the lateral vibrations by around 20%; and (iv) vertical vibrations are scarcely influenced by tractor-seat height above the ground. The results suggest that manufacturers could increase the comfort of tractors by lowering tractor-seat height above the ground, which will reduce lateral vibrations.

ABSTRACT Faecalibacterium prausnitzii is an anti-inflammatory commensal bacterium identified on the basis of human clinical data. The mechanisms underlying its beneficial effects are still unknown. Gnotobiotic mice harboring F. prausnitzii (A2-165) and Escherichia coli (K-12 JM105) were subjected to 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced acute colitis. The inflammatory colitis scores and a gas chromatography-time of flight (GC/TOF) mass spectrometry-based metabolomic profile were monitored in blood, ileum, cecum, colon, and feces in gnotobiotic mice. The potential anti-inflammatory metabolites were tested in vitro. We obtained stable E. coli and F. prausnitzii-diassociated mice in which E. coli primed the gastrointestinal tract (GIT), allowing a durable and stable establishment of F. prausnitzii. The disease activity index, histological scores, myeloperoxidase (MPO) activity, and serum cytokine levels were significantly lower in the presence of F. prausnitzii after TNBS challenge. The protective effect of F. prausnitzii against colitis was correlated to its implantation level and was linked to overrepresented metabolites along the GIT and in serum. Among 983 metabolites in GIT samples and serum, 279 were assigned to known chemical reactions. Some of them, belonging to the ammonia (α-ketoglutarate), osmoprotective (raffinose), and phenolic (including anti-inflammatory shikimic and salicylic acids) pathways, were associated with a protective effect of F. prausnitzii, and the functional link was established in vitro for salicylic acid. We show for the first time that F. prausnitzii is a highly active commensal bacterium involved in reduction of colitis through in vivo modulation of metabolites along the GIT and in the peripheral blood. IMPORTANCE Inflammatory bowel diseases (IBD) are characterized by low proportions of F. prausnitzii in the gut microbiome. This commensal bacterium exhibits anti-inflammatory effects through still unknown mechanisms. Stable monoassociated rodents are actually not a reproducible model to decipher F. prausnitzii protective effects. We propose a new gnotobiotic rodent model providing mechanistic clues. In this model, F. prausnitzii exhibits protective effects against an acute colitis and a protective metabolic profile is linked to its presence along the digestive tract. We identified a molecule, salicylic acid, directly involved in the protective effect of F. prausnitzii. Targeting its metabolic pathways could be an attractive therapeutic strategy in IBD.

In yeast, cytokinesis requires coordination between nuclear division, acto-myosin ring contraction, and septum synthesis. We studied the role of the Schizosaccharomyces pombe Bgs1p and Cfh3p proteins during cytokinesis under stress conditions. Cfh3p formed a ring in the septal area that contracted during mitosis; Cfh3p colocalized and co-immunoprecipitated with Cdc15p, showing that Cfh3p interacted with the contractile acto-myosin ring. In a wild-type strain, a significant number of contractile rings collapsed under stress conditions and this number increased dramatically in the cfh3Δ, bgs1cps1-191, and cfh3Δ bgs1/cps1-191. Our results show that after osmotic shock Cfh3p is essential for the stability of the (1,3) glucan synthase Bgs1p in the septal area, but not at the cell poles. Finally, cells adapted to stress; they repaired their contractile rings and re-localized Bgs1p to the cell surface some time after osmotic shock. A detailed analysis of the cytokinesis machinery in the presence of KCl revealed that the actomyosin ring collapsed before Bgs1p was internalized, and that it was repaired before Bgs1p re-localized to the cell surface. In the cfh3Δ, bgs1/cps1-191, and cfh3Δ bgs1/cps1-191 mutants, which have reduced glucan synthesis, the damage produced to the ring had stronger consequences, suggesting that an intact primary septum contributes to ring stability. The results show that the contractile actomyosin ring is very sensitive to stress, and that cells have efficient mechanisms to remedy the damage produced in this structure.

Abstract Autoimmune thyroid diseases (AITD) such as Graves’ disease (GD) or Hashimoto’s thyroiditis (HT) are organ-specific diseases that involve complex interactions between distinct components of thyroid tissue. Here, we use spatial transcriptomics to explore the molecular architecture, heterogeneity and location of different cells present in the thyroid tissue, including thyroid follicular cells (TFCs), stromal cells such as fibroblasts, endothelial cells, and thyroid infiltrating lymphocytes. We identify damaged antigen-presenting TFCs with upregulated CD74 and MIF expression in thyroid samples from AITD patients. Furthermore, we discern two main fibroblast subpopulations in the connective tissue including ADIRF+ myofibroblasts, mainly enriched in GD, and inflammatory fibroblasts, enriched in HT patients. We also demonstrate an increase of fenestrated PLVAP+ vessels in AITD, especially in GD. Our data unveil stromal and thyroid epithelial cell subpopulations that could play a role in the pathogenesis of AITD.

ABSTRACTAccumulation of Lewy bodies in dopaminergic neurons is associated to Parkinson disease (PD). The main component of Lewy bodies appears to be aggregates of alpha-synuclein (α-syn). Several mutations of the gene encoding this protein promote its aggregation. Thus, clustering of α-syn is considered a central event in the onset of PD. An old theory also postulates that mitochondrial dysfunction represents another cause of PD pathogenesis. However, the impact of α-syn aggregates on mitochondria remains poorly understood considering the technical difficulties to discriminate between the different forms of α-syn. In this punctum, we describe our recent work in which we used a newly developed optogenetic tool to control the aggregation of α-syn and examine the impact on mitochondria. This work revealed that α-syn aggregates dynamically interact with mitochondria, triggering their depolarization and leading to cardiolipin translocation to the surface of mitochondria and mitophagy.Abbreviations: α-syn: alpha-synuclein; BNIP3L: BCL2/adenovirus E1B 19 kDa protein-interacting protein 3-like; FUNDC1: FUN14 domain-containing protein 1; IMM: inner mitochondrial membrane; LIPA: light-induced protein aggregation; OMM: outer mitochondrial membrane; PD: Parkinson disease; SNc: substantia nigra par compacta;

Summary: Rod-shaped fission yeast grows through cell wall expansion at poles and septum, synthesized by essential glucan synthases. Bgs1 synthesizes the linear β(1,3)glucan of primary septum at cytokinesis. Linear β(1,3)glucan is also present in the wall poles, suggesting additional Bgs1 roles in growth polarity. Our study reveals an essential collaboration between Bgs1 and Tea1-Tea4, but not other polarity factors, in controlling growth polarity. Simultaneous absence of Bgs1 function and Tea1-Tea4 causes complete loss of growth polarity, spread of other glucan synthases, and spherical cell formation, indicating this defect is specifically due to linear β(1,3)glucan absence. Furthermore, linear β(1,3)glucan absence induces actin patches delocalization and sterols spread, which are ultimately responsible for the growth polarity loss without Tea1-Tea4. This suggests strong similarities in Bgs1 functions controlling actin structures during cytokinesis and polarized growth. Collectively, our findings unveil that cell wall β(1,3)glucan regulates polarized growth, like the equivalent extracellular matrix in neuronal cells.

Diversos tipos de ooides y oncoides se encuentran en travertinos y tobas a lo largo de 3 km del Barranco de Azuaje (Gran Canaria). Los ooides muestran formas esféricas a elipsoidales, de 0,1 mm->2 mm de tamaño, y envueltas concéntricas regulares. Los oncoides son de 0,4 mm a varios milímetros en general, con laminación. La mineralogía es aragonítica y aragonítica-calcítica en travertinos, y calcitítica en tobas. Los núcleos son intraclastos, moldes de partes de plantas o son indistinguibles de las envueltas. Las láminas de granos con envueltas aparecen comúnmente pasando a estromatolitos, pero también a otras facies (rafts, tallos colgantes, briófitas, etc.). En general, y considerando las características de núcleos y envueltas, asociaciones de facies y su distribución, los depósitos de ooides y oncoides representarían etapas diferentes de: (a) disponibilidad de partículas sueltas que sirven como núcleos, (b) formación de envueltas, (c) inmovilización de los granos y (d) fosilización del depósito por la formación de costras sobre el mismo. Todas estas etapas estarían relacionadas con condiciones físicas, químicas y biológicas específicas y sus variaciones, relacionadas con la dinámica sedimentaria del barranco.

Sodium hydroxide (NaOH) is increasingly drawing attention as a highly selective etchant for femtosecond laser-modified fused silica. Unprecedented etching contrasts between the irradiated and pristine areas have enabled the fabrication of hollow, high-aspect-ratio structures in the bulk of the material, overcoming the micrometer threshold as the minimum feature size. In this work, we systematically study the effect of NaOH solutions under different etching conditions (etchant concentration, temperature, and etching time) on the tracks created by tightly focused femtosecond laser pulses to assess the best practices for the fabrication of hollow nanostructures in bulk fused silica.

Inertial focusing-based Lab-on-Chip systems represent a promising technology for cell sorting in various applications, thanks to their alignment with the ASSURED criteria recommended by the World Health Organization: Affordable, Sensitive, Specific, User-friendly, Rapid and Robust, Equipment-free, and Delivered. Inertial focusing techniques using spiral microchannels offer a rapid, portable, and easy-to-prototype solution for cell sorting. Various microfluidic devices have been investigated in the literature to understand how hydrodynamic forces influence particle focusing in spiral microchannels. This is crucial for the effective prototyping of devices that allow for high-throughput and efficient filtration of particles of different sizes. However, a clear, comprehensive, and organized overview of current research in this area is lacking. This review aims to fill this gap by offering a thorough summary of the existing literature, thereby guiding future experimentation and facilitating the selection of spiral geometries and materials for cell sorting in microchannels. To this end, we begin with a detailed theoretical introduction to the physical mechanisms underlying particle separation in spiral microfluidic channels. We also dedicate a section to the materials and prototyping techniques most commonly used for spiral microchannels, highlighting and discussing their respective advantages and disadvantages. Subsequently, we provide a critical examination of the key details of inertial focusing across various cross-sections (rectangular, trapezoidal, triangular, hybrid) in spiral devices as reported in the literature.

This study examines the dynamics of limnological parameters of a South American lake located in southern Chile with the objective of predicting chlorophyll-a levels, which are a key indicator of algal biomass and water quality, by integrating combined remote sensing and machine learning techniques. Employing four advanced machine learning models (recurrent neural network (RNNs), long short-term memory (LSTM), recurrent gate unit (GRU), and temporal convolutional network (TCNs)), the research focuses on the estimation of chlorophyll-a concentrations at three sampling stations within Lake Ranco. The data span from 1987 to 2020 and are used in three different cases: using only in situ data (Case 1), using in situ and meteorological data (Case 2), using in situ, and meteorological and satellite data from Landsat and Sentinel missions (Case 3). In all cases, each machine learning model shows robust performance, with promising results in predicting chlorophyll-a concentrations. Among these models, LSTM stands out as the most effective, with the best metrics in the estimation, the best performance was Case 1, with R2 = 0.89, an RSME of 0.32 µg/L, an MAE 1.25 µg/L and an MSE 0.25 (µg/L)2, consistently outperforming the others according to the static metrics used for validation. This finding underscores the effectiveness of LSTM in capturing the complex temporal relationships inherent in the dataset. However, increasing the dataset in Case 3 shows a better performance of TCNs (R2 = 0.96; MSE = 0.33 (µg/L)2; RMSE = 0.13 µg/L; and MAE = 0.06 µg/L). The successful application of machine learning algorithms emphasizes their potential to elucidate the dynamics of algal biomass in Lake Ranco, located in the southern region of Chile. These results not only contribute to a deeper understanding of the lake ecosystem but also highlight the utility of advanced computational techniques in environmental research and management.

Introduction: RET inhibitors with impressive overall response rates are now available for patients with NSCLC, yet the identification of RET fusions remains a difficult challenge. Most guidelines encourage the upfront use of next-generation sequencing (NGS), or alternatively, fluorescence in situ hybridization (FISH) or reverse transcriptase-polymerase chain reaction (RT-PCR) when NGS is not possible or available. Taken together, the suboptimal performance of single-analyte assays to detect RET fusions, although consistent with the notion of encouraging universal NGS, is currently widening some of the clinical practice gaps in the implementation of predictive biomarkers in patients with advanced NSCLC. Methods: This situation prompted us to evaluate several RET assays in a large multicenter cohort of RET fusion–positive NSCLC (n = 38) to obtain real-world data. In addition to RNA-based NGS (the criterion standard method), all positive specimens underwent break-apart RET FISH with two different assays and were also tested by an RT-PCR assay. Results: The most common RET partners were KIF5B (78.9%), followed by CCDC6 (15.8%). The two RET NGS-positive but FISH-negative samples contained a KIF5B(15)-RET(12) fusion. The three RET fusions not identified with RT-PCR were AKAP13(35)-RET(12), KIF5B(24)-RET(9) and KIF5B(24)-RET(11). All three false-negative RT-PCR cases were FISH-positive, exhibited a typical break-apart pattern, and contained a very high number of positive tumor cells with both FISH assays. Signet ring cells, psammoma bodies, and pleomorphic features were frequently observed (in 34.2%, 39.5%, and 39.5% of tumors, respectively). Conclusions: In-depth knowledge of the advantages and disadvantages of the different RET testing methodologies could help clinical and molecular tumor boards implement and maintain sensible algorithms for the rapid and effective detection of RET fusions in patients with NSCLC. The likelihood of RET false-negative results with both FISH and RT-PCR reinforces the need for upfront NGS in patients with NSCLC.

Tissue histopathology, based on hematoxylin and eosin (H&E) staining of thin tissue slices, is the gold standard for the evaluation of the immune reaction to the implant of a biomaterial. It is based on lengthy and costly procedures that do not allow longitudinal studies. The use of non-linear excitation microscopy in vivo, largely label-free, has the potential to overcome these limitations. With this purpose, we develop and validate an implantable microstructured device for the non-linear excitation microscopy assessment of the immune reaction to an implanted biomaterial label-free. The microstructured device, shaped as a matrix of regular 3D lattices, is obtained by two-photon laser polymerization. It is subsequently implanted in the chorioallantoic membrane (CAM) of embryonated chicken eggs for 7 days to act as an intrinsic 3D reference frame for cell counting and identification. The histological analysis based on H&E images of the tissue sections sampled around the implanted microstructures is compared to non-linear excitation and confocal images to build a cell atlas that correlates the histological observations to the label-free images. In this way, we can quantify the number of cells recruited in the tissue reconstituted in the microstructures and identify granulocytes on label-free images within and outside the microstructures. Collagen and microvessels are also identified by means of second-harmonic generation and autofluorescence imaging. The analysis indicates that the tissue reaction to implanted microstructures is like the one typical of CAM healing after injury, without a massive foreign body reaction. This opens the path to the use of similar microstructures coupled to a biomaterial, to image in vivo the regenerating interface between a tissue and a biomaterial with label-free non-linear excitation microscopy. This promises to be a transformative approach, alternative to conventional histopathology, for the bioengineering and the validation of biomaterials in in vivo longitudinal studies.

Abstract Accumulation of α-synuclein aggregates in the substantia nigra pars compacta is central in the pathophysiology of Parkinson’s disease, leading to the degeneration of dopaminergic neurons and the manifestation of motor symptoms. Although several PD models mimic the pathological accumulation of α-synuclein after overexpression, they do not allow for controlling and monitoring its aggregation. We recently generated a new optogenetic tool by which we can spatiotemporally control the aggregation of α-synuclein using a light-induced protein aggregation system. Using this innovative tool, we aimed to characterize the impact of α-synuclein clustering on mitochondria, whose activity is crucial to maintain neuronal survival. We observed that aggregates of α-synuclein transiently and dynamically interact with mitochondria, leading to mitochondrial depolarization, lower ATP production, mitochondrial fragmentation and degradation via cardiolipin externalization-dependent mitophagy. Aggregation of α-synuclein also leads to lower mitochondrial content in human dopaminergic neurons and in mouse midbrain. Interestingly, overexpression of α-synuclein alone did not induce mitochondrial degradation. This work is among the first to clearly discriminate between the impact of α-synuclein overexpression and aggregation on mitochondria. This study thus represents a new framework to characterize the role of mitochondria in PD.

In the present study, we evaluated the influence of maternal and neonatal factors on the efficiency of the placental transfer of neutralizing antibodies against SARS-CoV-2. Vaccination during pregnancy provides fetal and neonatal protection through the passive transplacental transfer of maternal neutralizing antibodies. To date, little information is available regarding the factors that affect the transfer of antibodies against SARS-CoV-2. A retrospective, cross-sectional, observational, and analytical study was carried out. It was found that several biological factors could be altering transplacental passive immunity after vaccination against COVID-19. In our study population, type 2 diabetes mellitus and chronic hypertension tended to decrease efficiency, while data from women with pre-eclampsia showed better indices compared to those from women with healthy pregnancies. Neonates born prematurely showed lower transfer rates when compared to healthy neonates. The premature rupture of membranes significantly decreased antibody transfer. Taken together, the data suggest that vaccination against COVID-19 during pregnancy is effective even under certain unfavorable clinical conditions for the mother, fetus, and neonate. It is important to create and disseminate immunization strategies in vulnerable populations to reduce maternal and perinatal morbidity and mortality associated with infections preventable by vaccination.

Geopolymer, a novel binder material, possesses the potential to replace conventional cement completely. Recent studies have investigated the incorporation of various nanomaterials (NMs) into geopolymer concrete (GPC) to enhance its properties. Nanotechnology, an emerging field of study, has garnered significant attention in recent decades due to its groundbreaking research and practical applications. This comprehensive review aims to elucidate the effects of nanomaterial inclusion on the workability, strength, durability, and physical and microstructural characteristics of GPC. The properties have been meticulously examined, reviewed, and discussed. Over 190 research and review articles were reviewed, analyzed, and presented to develop a database containing critical properties of GPC modified with different doses and types of NMs. The influence of introducing diverse kinds and doses of NMs on GPC behavior was assessed. Historical trends, current tendencies, challenges, and the benefits and limitations of NM-modified GPC were explored. According to this review, incorporating NMs is promising for developing high-strength or high-performance GPC. It significantly improves mechanical, microstructural, and durability properties by providing additional calcium-silicate-hydrate, calcium-aluminate-silicate-hydrate gel, and nano-filling effects in the GPC matrix. This advancement will enable the construction industry to realize the potential of NM-enhanced GPC successfully. The present review demonstrated that the geopolymer concrete showed enhancements in engineering properties and effectiveness when modified with different types of nanomaterials and tested under different conditions. The current study also presented some challenges and research gaps that should be addressed in future research studies.

Nowadays, special concrete-like self-compacting concrete (SCC) requires sustainability by introducing recycled aggregates as a partial replacement for natural aggregate. Technological development initiatives in the construction sector estimate the 28 days' concrete compressive strength before casting due to faster requirement; one method selected is an artificial neural network. From works of literature, 515 mixed design are collected and utilized for training, validation, and testing data to prepare models. Different applications of SCC require different strengths of concrete. Based on control mix compressive strength, the mix designs are grouped into three families as low, medium, and high strength, apart from a common family. The correlation between input and output variables for three different families is analyzed. ANOVA analyses are done for input parameters. Coefficient of relation (R2) is used for sensitive assessment and results for family I (R2 = 0.9299), family II (R2 = 0.824), family III (R2 = 0.8775), and family IV (R2 = 0.7991). Two further sensitivity analyses indicate that input parameters' influence varies for different families.

Este trabalho teve como objetivo caracterizar o perfil do profissional de saúde de Unidades Básicas de Saúde de Olinda-PE. Trata-se de uma pesquisa quantitativa, observacional do tipo transversal. Os participantes voluntários foram 33 profissionais de saúde das equipes de Estratégias de Saúde da Família do município de Olinda-PE. Os dados foram coletados pelo Google formulário para os participantes da pesquisa, após os mesmos aceitarem participar da pesquisa e terem assinado o Termo de Consentimento Livre e Esclarecido (TCLE). As variáveis foram expressas como frequência absoluta. Este projeto foi analisado e aprovado pelo CEP da FMO. A maioria dos profissionais eram os Agentes Comunitário de Saúde (ACS), os técnicos (enfermagem e farmácia) e os enfermeiros. Destes profissionais, 84,8% eram do sexo feminino, idade variando entre 31 a 50 anos, e o grau de escolaridade reflete os profissionais que eram técnicos ou com formação superior. Houve uma prevalência de 50% testarem positivo, destes 60% diagnosticados pelo PCR e apenas 14,3% não relataram afastamento do trabalho. Apesar que todos os entrevistados relataram utilizar EPI, o que não surtiu efeito na prevenção completa. Percebe-se que os profissionais da atenção primária à saúde também estiveram susceptíveis ao coronavírus, apesar de não serem das equipes de linha de frente no combate à pandemia. Isto refletiu também no afastamento destes profissionais nas suas atividades de trabalho.

Application of beneficial microorganisms as probiotics targets a broad range of intended uses, from maintaining health and supporting normal bodily functions to curing and preventing diseases. Currently, three main regulatory fields of probiotic products can be defined depending on their intended use: the more similar probiotic foods and probiotic dietary supplements, and live biotherapeutic products. However, it is not always straightforward to classify a probiotic product into one of these categories. The regulatory nuances of developing, manufacturing, investigating and applying each category of probiotic products are not universal, and not always apparent to those unfamiliar with the various global probiotic regulatory guidelines. Various global markets can be significantly different regarding legislation, possible claims, market value and quality requirements for the development and commercialization of probiotic products. Furthermore, different probiotic product categories are also linked with variable costs at different stages of product development. This review outlines the current landscape comparing probiotic foods, probiotic dietary supplements, and live biotherapeutics as probiotic products from a regulatory lens, focusing on product development, manufacturing and production, and clinical research agenda. The aim is to inform and promote a better understanding among stakeholders by outlining the expectations and performance for each probiotic product category, depending on their intended use and targeted geographical region.

Summary: Chromosome segregation in female meiosis in many metazoans is mediated by acentrosomal spindles. The analysis of the dynamics of self-assembled spindles is a challenge due to the low availability of oocytes. Here, we present a protocol for analyzing self-assembled spindle dynamics in fission yeast meiosis using in vivo fluorescence imaging. We describe steps for starter culture preparation, meiosis induction, and sample preparation. We then detail procedures for acquisition and analysis of images of self-assembled spindles.For complete details on the use and execution of this protocol, please refer to Pineda-Santaella and Fernández-Álvarez (2019)1 and Pineda-Santaella et al. (2021).2 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

AbstractPolymeric microparticles are widely used as drug delivery platforms either alone or embedded in more complex structures for regenerative medicine. Emulsion-solvent evaporation is the most extensively used technique for microparticles preparation. Despite the apparent simplicity of this method, there is no general procedure for producing microparticles of predictable characteristics (particle size, size distribution, encapsulation efficiency, and drug loading). Hybrid systems such as neurofuzzy logic allow identifying relationships between inputs and outputs, expressing the generated mathematical models through rules in linguistic format. In this work, the relationships between the variables involved in the emulsion-solvent evaporation process and the quality parameters of PLGA microparticles as drug delivery systems were established. Neurofuzzy logic software was able to generate models of high predictability (> 85%) for the microspheres properties namely particle size, size distribution, encapsulation efficiency and drug loading. Moreover, the generated sets of IF-THEN rules allowed to dictate general guidelines to better select the PLGA microparticles formulation parameters. This approach would be of great interest as a starting point to set-up protocols for the development of PLGA microparticles obtained by emulsion-solvent evaporation for many applications.

Presently, the rate for the development of ultra-high-performance concrete (UHPC) is increasing due to the requirement of long-span bridges, challenging architectural plans, and high-rise buildings. UHPC is still weak in tension and consumes a lot of cement, making it non-eco-friendly. This research is aimed at modifying the cement's matrix by incorporating two sustainable solid waste mineral admixtures, eggshell powder (ESP) and nano-silica (NS), to enhance the bonding between fibers and the binder's matrix and improve the strength characteristics of ultra-high-performance fiber-reinforced concrete (UHPFRC). The improving impacts of the ESP and NS on the double-hooked end (DHE) steel fibers were studied and analyzed by evaluating various strength parameters, i.e., compressive, splitting tensile and flexural strength and the load-deflection performance. To characterize the durability properties of UHPFRC, chloride ion coefficient, ultra-sonic pulse velocity (UPV), and water absorption tests were also carried out. Optimization of added materials was also done using a statistical tool called a design of experiments. Multi-variable statistical analysis was performed using response surface methodology (RSM) and analysis of variance (ANOVA). Both the experimental and statistical test outcomes depicted that introducing 5% ESP and 5% NS significantly improved the effectiveness of DHE steel fibers when the concrete was subjected to splitting tensile and flexural strength. UHPFRC with the 5% ESP and NS depicted 81% and 94% higher flexural strength than the reference mixture. While UHPFRC had almost 35 times higher flexural toughness than its counterparts. The inclusion of 5% and 10% eggshell powder depicted a favorable impact on the resistance of UHPFRC against the infiltration of water and chloride ions. The test outcomes of the present research confirmed the suitability and effectiveness of using ESP and NS as a partial substitute for OPC and significantly improved the engineering properties of UHPFRC, which was verified by the analytical results.

Developing ultra-high fiber-reinforced concrete (UHPFRC) demands an immense quantity of ordinary Portland cement (OPC). Using a high percentage of cement in UHPFRC leads to uneconomic concrete and significant release of CO2 into the atmosphere. Therefore, it is critical to address this issue by substituting the part of cement with a sustainable material. The current research has tried to tackle this issue by assessing the impact of adding wheat straw ash (WSA) and silica fume (SF) as a partial substitute of cement on the properties of UHPFRC on the static and dynamic compressive strength test, among other strength and durability characteristics. Two different batches of mixes were developed. The OPC was substituted for up to 40% in the first batch, with combined WSA plus SF at 10% intervals. In the second batch, OPC was replaced up to 40% with only WSA at an interval of 10%. Polypropylene fibers (PPFs) at 2% (by vol.) were added to modified samples to improve the ductility of concrete. The dynamic compression strength was evaluated utilizing a Split Hopkinson pressure bar, and quasi-static compression strength was determined using the compression machine. Moreover, splitting and flexural strength were also assessed, and water permeability and sorptivity tests were carried out for durability. The test outcome showed that the sample with 15% WSA plus 15% SF had higher compressive strength (174.8 MPa at 90 days) and improved durability than the sample with only 30% WSA (170.2 MPa at 90 days) and the control sample. The assessed properties tend to decline at 40% of WSA and 20% WSA with 20% SF. In dynamic compression strength, it was observed that the WSA and SF, and fibers samples performed similarly to the control sample, and these modified samples were not sensitive to strain rates. Adding 15% plus 15% SF had an optimal effect in improving the durability properties of UHPFRC. Also, the thermogravimetric analysis (TGA) confirms the role of WSA and SF in densifying the matrix of UHPFRC, decreasing the porous pores and amount of calcium-hydroxide and developing additional gels of calcium-silicate-hydrate, which enhances the strength and durability of UHPFRC. The current study revealed that the modified samples (with WSA and SF) had a low embedded CO2 for the sustainability features compared to the control sample.

The requirement of the construction sector pushes researchers and academicians to determine the 28-day concrete compressive strength due to less consumption of natural products and reduced cost. One recommended method to reduce the cost and simultaneously adopt sustainability is introducing recycled aggregates in concrete. Most typical structures require concrete, which is self-flowable and compactable; specific structures require concrete, which is self-flowable and compactable; one such concrete is self-compacting concrete (SCC). 515 mix design samples for SCC with recycled aggregates are collected from the literature and used for training, validation, and testing to create the model using machine learning techniques (Extra Gradient (XG) Boosting, Ada Boosting, Gradient Boosting, Light Gradient Boosting, Category Boosting, K Nearest Neighbors, Extra Trees, Decision Trees, Random Forest, and Support Vector Machine). The correlation between input and output variables is analyzed using ANOVA and is indicated that data can be used to develop machine learning models successfully. Sensitive analysis and error assessment are performed to choose the best machine learning methods, and it found that CB, KNN, and ERT have the highest R2 value and lowest MSE.

The effect of embryo reinsertion immediately after embryo flushing was studied. In Experiment 1, eight mares were used during 32 cycles (8 cycles in each group). For the first two groups, inseminated mares were flushed 8 days after ovulation and prostaglandin F2α was not administered: in group EF-ET (embryo flushing and embryo transfer) the embryo was reinserted in the same donor mare, while in the EF group, no further procedure was performed. In the third group (ET), non-inseminated mares (recipients) received a Day 8 embryo. Progesterone concentration was measured before EF/ET and 72 h after in the three groups. In Experiment 2, twelve mares were used during 17 cycles in two groups, EF-ET (n = 11) and ET (n = 6), as in Experiment 1, except that every mare was flushed 24 h after embryo transfer to retrieve the embryo. Fewer pregnancies resulted after transfer in EF-ET cycles (0/8, 0%) than in the ET group (6/8, 75%). Progesterone concentration decreased significantly (p = 0.05) 72 h after EF-ET but not in EF or ET cycles (p > 0.1). Three mares from the EF-ET showed full luteolysis and signs of endometritis. In Experiment 2, more (5/6; p = 0.08) grade 1 embryos were recovered in the ET compared to the EF-ET group (3/7); 4 embryos were graded 3–4 (were broken or had signs of degeneration) in the EF-ET group but none in the ET group. In both groups, capsule fragments were obtained as indicative of the presence of a recently destroyed embryo in the EF-ET (n = 3) and ET (n = 1) groups. Positive bacterial cultures were obtained in 2/11 and 1/6 embryo flushes from the EF-ET and ET groups, respectively.

Abstract On‐going projects of the team are currently dealing with microbiota, xenobiotics, endocrine‐disrupting chemicals (EDCs), obesity, inflammation and probiotics. The combination of diet, lifestyle and the exposure to dietary xenobiotics categorised into microbiota‐disrupting chemicals (MDCs) could determine obesogenic‐related dysbiosis. This modification of the microbiota diversity impacts on individual health–disease balance, inducing altered phenotypes. Specific, complementary, and combined prevention and treatments are needed to face these altered microbial patterns and the specific misbalances triggered. In this sense, searching for next‐generation probiotics (NGP) by microbiota culturing, and focusing on their demonstrated, extensive scope and well‐defined functions could contribute to counteracting and repairing the effects of obesogens. Therefore, EU‐FORA project contributes to present a perspective through compiling information and key strategies for directed taxa searching and culturing of NGP that could be administered for preventing obesity and endocrine‐related dysbiosis by (i) observing the differential abundance of specific microbiota taxa in obesity‐related patients and analysing their functional roles, (ii) developing microbiota‐directed strategies for culturing these taxa groups, and (iii) design and applying the successful compiled criteria from recent NGP clinical studies. New isolated or cultivable microorganisms from healthy gut microbiota specifically related to xenobiotic obesogens' neutralisation effects might be used as an NGP single strain or in consortia, both presenting functions and the ability to palliate metabolic‐related disorders. Identification of holistic approaches for searching and using potential NGP, key aspects, the bias, gaps and proposals of solutions were also considered in this workplan.

Despite the potential of CAR-T therapies for hematological malignancies, their efficacy in patients with relapse and refractory Acute Myeloid Leukemia has been limited. The aim of our study has been to develop and manufacture a CAR-T cell product that addresses some of the current limitations. We initially compared the phenotype of T cells from AML patients and healthy young and elderly controls. This analysis showed that T cells from AML patients displayed a predominantly effector phenotype, with increased expression of activation (CD69 and HLA-DR) and exhaustion markers (PD1 and LAG3), in contrast to the enriched memory phenotype observed in healthy donors. This differentiated and more exhausted phenotype was also observed, and corroborated by transcriptomic analyses, in CAR-T cells from AML patients engineered with an optimized CAR construct targeting CD33, resulting in a decreased in vivo antitumoral efficacy evaluated in xenograft AML models. To overcome some of these limitations we have combined CRISPR-based genome editing technologies with virus-free gene-transfer strategies using Sleeping Beauty transposons, to generate CAR-T cells depleted of HLA-I and TCR complexes (HLA-IKO/TCRKO CAR-T cells) for allogeneic approaches. Our optimized protocol allows one-step generation of edited CAR-T cells that show a similar phenotypic profile to non-edited CAR-T cells, with equivalent in vitro and in vivo antitumoral efficacy. Moreover, genomic analysis of edited CAR-T cells revealed a safe integration profile of the vector, with no preferences for specific genomic regions, with highly specific editing of the HLA-I and TCR, without significant off-target sites. Finally, the production of edited CAR-T cells at a larger scale allowed the generation and selection of enough HLA-IKO/TCRKO CAR-T cells that would be compatible with clinical applications. In summary, our results demonstrate that CAR-T cells from AML patients, although functional, present phenotypic and functional features that could compromise their antitumoral efficacy, compared to CAR-T cells from healthy donors. The combination of CRISPR technologies with transposon-based delivery strategies allows the generation of HLA-IKO/TCRKO CAR-T cells, compatible with allogeneic approaches, that would represent a promising option for AML treatment.

Recycled aggregate (RA) made from waste concrete is an environmentally friendly alternative to natural aggregate (NA) for concrete manufacturing. However, compared to NA concrete, concrete produced with recycled aggregates has poor characteristics. Supplementary cementitious materials (SCMs) can be used to enhance the poor properties of recycled aggregate concrete (RAC). Silica fume and fly ash are commonly used SCMs in the World, but their high usage led to a shortage of silica fume and fly ash. Still, the deficiency of these materials in large parts of the world is a challenge that requires exploring alternative feedstock materials for the construction industry in the coming years. Wheat straw ash (WSA) is an agricultural waste product that could be used as an alternative SCM due to its pozzolanic behavior to enhance the properties of RAC. In addition, concrete is brittle and needs reinforcement, for which polypropylene fibers (PPFs) can be used. The current research examines the mechanical characteristics of fiber-reinforced RAC, including compressive strength, splitting tensile strength, and ductility performance. Durability indicators, such as chloride diffusion, chloride penetration, acid resistance, and water absorption test, were also assessed. The results showed that concrete samples with 10% WSA, 50% RA and 1.5% PPFs had the highest compressive and splitting tensile strength, 60.2 MPa and 7.25 MPa, respectively, representing increases of 24.75% and 30.65%, as compared to plain samples at 56 days. In these samples, water absorption was reduced by 13% due to the finer WSA particles resulting in the lowest reduction in strength and mass recorded when exposing concrete samples to acidic media. The statistical analysis also validated that irrespective of WSA and PPFs, the concrete with 0% RA had the highest performance in strength and durability behavior. The study showed that WSA and PPFs might be employed in tandem to offset the poor behavior of RA, enhance the bond between fibers and concrete, and improve the mechanical strength and durability performance of RAC, thus demonstrating its suitability as a sustainable and economical construction material.

Concrete significantly impacts the atmosphere as its primary raw material, Portland cement, determines a considerable proportion of carbon dioxide in its production process. Improving the strength and durability properties of concrete buildings could decrease their influence on the atmosphere. Adding a small proportion of Nano-silica (NS) to concrete could change concrete’s configuration at the Nano level. Compared to other nano-materials, Nano-silica has riveted a lot of consideration due to its pozzolan reaction with calcium-hydroxide to make a gel of calcium-silicate-hydrate. Nano-silica can promote the adhesion between the concrete’s binder and aggregate by coupling and efficiently enhancing and densifying the concrete’s matrix. The present review article focuses on studying the influence of different types and proportions of nano-silica on the hydration, strength, durability, and microstructural characteristics of different cement-based concrete. This review article shows that around 2–4 % of Nano-silica in various types of concrete is suitable to improve the influence of NS on the physical, durability, and mechanical properties and densify concrete microstructure. Also, the present challenges that the concrete industry is facing in the commercial utilization of nano-silica in concrete are discussed in this review article. It is established that this review article will produce novel concepts to endorse the practical utilization of Nano-silica in the construction sector and offer some productive designs for similar research studies in the future.

Cracks are one of the worsening reasons for concrete failure, which permits the penetration of chemical solutions and could significantly impact the physical, mechanical, and durability characteristics of concrete buildings. To protect, heal and assimilate concrete structures, numerous coating materials, binding materials, and adhesives have been generally exercised. Though these methods are highly appropriate, because of their different essential procedure, critical issues, for instance, lack of effectiveness in cost and delamination, have caused the exploration of substitute procedures for sealing cracks and self-healing concrete. One of the newer self-healing methods is employing bacterial material modified with precipitation of calcite in concrete mixes to fill or heal cracking in concrete. In this method, the mineralization of bacteria is carried out via the decomposition of calcium and urea to form calcium carbonate, which could fill the cracking. To review the methods for this kind of precipitation, the present paper aims to offer an in-depth study of precipitation of calcium carbonate, physical, mechanical, and durability characteristics, and micro-structure performance of concrete. One hundred fifty articles were studied to perform the present study. Their results have been presented about the dose and type of bacteria and its impact on strength and durability characteristics. The present study shows that bio-mineralization largely relies on several factors, for instance, the preservation of bacterial cells and the application procedure. Furthermore, the impact of bacterial material on the environment is observed to be straight related to the proportion of urea in concrete mixes.

The characteristics of the whole PEDV genome that has circulated in Mexico from the first outbreak to the present are unknown. We chose samples obtained from 2013 to 2017 and sequenced them, which enabled us to identify the genetic variation and phylogeny in the virus during the first four years that it circulated in Mexico. A 99% identity was found among the analyzed pandemic strains; however, the 1% difference affected the structure of the S glycoprotein, which is essential for the binding of the virus to the cellular receptor. The S protein induces the most efficacious antibodies; hence, these changes in structure could be implicated in the clinical antecedents of the outbreaks. Antigenic changes could also help PEDV avoid neutralization, even in the presence of previous immunity. The characterization of the complete genome enabled the identification of three circulating strains that have a deletion in ORF1a, which is present in attenuated Asian vaccine strains. The phylogenetic analysis of the complete genome indicates that the first PEDV outbreaks in Mexico were caused by INDEL strains and pandemic strains related to USA strains; however, the possibility of the entry of European strains exists, which may have caused the 2015 and 2016 outbreaks.

We investigate the generation of ultrabroadband femtosecond ultraviolet (UV) radiation via third-order harmonic generation in highly confined gas media. A dual-stage differential-pumping scheme integrated into a glass microfluidic chip provides an exceptional gas confinement up to several bar and allows the apparatus to be operated under high-vacuum environment. UV pulses are generated both in argon and neon with up to ∼0.8 μ J energy and 0.2% conversion efficiency for spectra that cover the UVB and UVC regions between 200 and 325 nm. Numerical simulations based on the unidirectional pulse propagation equation reveal that ionization plays a critical role for extending the spectral bandwidth of the generated third-harmonic pulse beyond the tripled 800 nm driving laser pulse bandwidth. By delivering UV supercontinua supporting Fourier transform limits below 2 fs, as well as comparable pulse energies with respect to capillary-based techniques that typically provide high spectral tunability but produce narrower bandwidths, our compact device makes a step forward towards the production and application of sub-fs UV pulses for the investigation of electron dynamics in neutral molecules.

The protection of the neonate against pathogens depends largely on the antibodies transferred placentally from the mother; for this reason, maternal vaccination against emerging viruses, such as SARS-CoV-2, is of vital importance. Knowing some of the immunogenic factors that could alter the placental transfer of antibodies could aid in understanding the immune response and neonatal protection after maternal vaccination. In this study, we analyzed the efficiency of the placental transfer of binding and neutralizing antibodies, as well as some factors that could alter the passive immune response, such as the trimester of gestation at the time of immunization, the number of doses received by the mother and the type of vaccine. Binding IgG antibodies were detected by ELISA, and the detection of neutralizing antibodies was carried out using flow cytometry. Our results show efficient transfer rates (>1), which are higher when maternal vaccination occurs during the third trimester of gestation. Antibodies are detectable in mothers and their neonates after 12 months of maternal immunization, suggesting than the vaccination against COVID-19 before and during pregnancy in the Mexican population induces a lasting neutralizing response in mothers and their newborns.

Genome-editing strategies, especially CRISPR-Cas9 systems, have substantially increased the efficiency of innovative therapeutic approaches for monogenic diseases such as primary hyperoxalurias (PHs). We have previously demonstrated that inhibition of glycolate oxidase using CRISPR-Cas9 systems represents a promising therapeutic option for PH type I (PH1). Here, we extended our work evaluating the efficacy of liver-specific inhibition of lactate dehydrogenase (LDH), a key enzyme responsible for converting glyoxylate to oxalate; this strategy would not be limited to PH1, being applicable to other PH subtypes. In this work, we demonstrate a liver-specific inhibition of LDH that resulted in a drastic reduction of LDH levels in the liver of PH1 and PH3 mice after a single-dose delivery of AAV8 vectors expressing the CRISPR-Cas9 system, resulting in reduced urine oxalate levels and kidney damage without signs of toxicity. Deep sequencing analysis revealed that this approach was safe and specific, with no off-targets detected in the liver of treated animals and no on-target/off-tissue events. Altogether, our data provide evidence that in vivo genome editing using CRISPR-Cas9 systems would represent a valuable tool for improved therapeutic approaches for PH.