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The purpose of this paper was to study the kinetics of calcium uptake of blanched and not blanched apple tissue during impregnation treatments under vacuum or/and at atmospheric pressure. The relative absorption of calcium in rats, regarding calcium carbonate as reference substance, was also analyzed as a first approach. Apple impregnation is conducted (with and without previous blanching in saturated vapor) in isotonic glucose aqueous solutions containing calcium gluconate and lactate. A 50mm Hg vacuum pressure is applied for a time t1 (0-180mm) or atmospheric pressure is used for a process time t2 (0-7.5h). Calcium uptake by apple tissue is influenced by system pressure, blanching, and impregnation time, reaching 500-4600 μg Ca/g according to process variables. Preliminary data of true calcium absorption ranges between 74 and 86% regarding calcium carbonate. [ABSTRACT FROM AUTHOR]

The development of human induced pluripotent stem cell (iPSC)-based regenerative therapies is challenged by the lack of specific cell markers to isolate differentiated cell types and improve differentiation protocols. This issue is particularly critical for notochordal-like cells and chondrocytes, which are crucial in treating back pain and osteoarthritis, respectively. Both cell types produce abundant proteoglycan aggrecan (ACAN), crucial for the extracellular matrix. We generated two human iPSC lines containing an ACAN-2A-mScarlet reporter. The reporter cell lines were validated using CRISPR-mediated transactivation and functionally validated during notochord and cartilage differentiation. The ability to isolate differentiated cell populations producing ACAN enables their enrichment even in the absence of specific cell markers and allows for comprehensive studies and protocol refinement. ACAN's prevalence in various tissues (e.g., cardiac and cerebral) underscores the reporter's versatility as a valuable tool for tracking matrix protein production in diverse cell types, benefiting developmental biology, matrix pathophysiology, and regenerative medicine.

An intricate meshwork of trabeculations lines the luminal side of cardiac ventricles. Compaction, a developmental process, is thought to reduce trabeculations by adding them to the neighboring compact wall which is then enlarged. When pig, a plausible cardiac donor for xenotransplantation, is compared to human, the ventricular walls appear to have fewer trabeculations. We hypothesized the trabecular volume is proportionally smaller in pig than in human. Macroscopically, we observed in 16 pig hearts that the ventricular walls harbor few but large trabeculations. Close inspection revealed a high number of tiny trabeculations, a few hundred, within the recesses of the large trabeculations. While tiny, these were still larger than embryonic trabeculations and even when considering their number, the total tally of trabeculations in pig was much fewer than in human. Volumetrics based on high-resolution MRI of additional six pig hearts compared to six human hearts, revealed the left ventricles were not significantly differently trabeculated (21.5 versus 22.8%, respectively), and the porcine right ventricles were only slightly less trabeculated (42.1 vs 49.3%, respectively). We then analyzed volumetrically 10 pig embryonic hearts from gestational day 14-35. The trabecular and compact layer always grew, as did the intertrabecular recesses, in contrast to what compaction predicts. The proportions of the trabecular and compact layers changed substantially, nonetheless, due to differences in their growth rate rather than compaction. In conclusion, processes that affect the trabecular morphology do not necessarily affect the proportion of trabecular-to-compact myocardium and they are then distinct from compaction., (© 2024 The Authors. Clinical Anatomy published by Wiley Periodicals LLC on behalf of American Association of Clinical Anatomists and British Association of Clinical Anatomists.)

Hip dysplasia causes major disability in dogs. Treatment options are limited to palliative treatment (e.g., pain relief, physical exercise, lifestyle changes, and weight control) or invasive surgeries such as pelvic osteotomies and total hip arthroplasty. Hence, a strong unmet need exists for an effective and dog-friendly solution that enhances the quality of life of man's best friend. We fill this treatment gap by offering a minimally traumatic and extraarticular, dog-specific, 3-dimensional-printed, hip implant (3DHIP) that restores hip joint stability. The surgical treatment using a 3DHIP implant is less invasive than osteotomies and can be performed bilaterally in one surgical session. The 3DHIP implant extends the dorsal acetabular rim of the dysplastic hip joint thereby increasing coverage of the femoral head and inhibiting joint subluxation with fast recovery. Sufficient access to the dorsal acetabular rim and ventral border of the iliac body together with optimal fitting and fixation of the implant are key steps for a successful 3DHIP implantation and imply the need for a specific approach. The present article aims to showcase this innovative surgical technique with tips and tricks as a surgical manual for implantation of the 3DHIP implant in dogs affected by hip dysplasia.

Objectives: To investigate the effects of material type and thickness on force generation and distribution by aligners., Materials and Methods: Sixty aligners were divided into six groups (n = 10): one group with a thickness of 0.89 mm using Zendura Viva (Multi-layer), four groups with a thickness of 0.75 mm using Zendura FLX (Multi-layer), CA Pro (Multi-layer), Zendura (Single-layer), and Duran (Single-layer) sheets, and one group with a thickness of 0.50 mm using Duran sheets. Force measurements were conducted using Fuji® pressure-sensitive films., Results: The lowest force values, both active and passive, were recorded for the multi-layered sheets: CA Pro (83.1 N, 50.5 N), Zendura FLX (88.9 N, 60.7 N), and Zendura Viva (92.5 N, 68.5 N). Conversely, the highest values were recorded for the single-layered sheets: Duran (131.9 N, 71.8 N) and Zendura (149.7 N, 89.8 N). The highest force was recorded at the middle third of the aligner, followed by the incisal third, and then the cervical third. The net force between the incisal and cervical thirds (F I -F C ) showed insignificant difference across different materials. However, when comparing the incisal and middle thirds, the net force (F I -F M ) was higher with single-layered materials. Both overall force and net force (F I -F M ) were significantly higher with 0.75 mm compared to those with a thickness of 0.50 mm., Conclusions: Multi-layered aligner materials exert lower forces compared to their single-layered counterparts. Additionally, increased thickness in aligners results in enhanced retention and greater force generation. For effective bodily tooth movement, thicker and single-layered rigid materials are preferred., Clinical Relevance: This research provides valuable insights into the biomechanics of orthodontic aligners, which could have significant clinical implications for orthodontists. Orthodontists might use this information to more effectively tailor aligner treatments, considering the specific tooth movement required for each individual patient. In light of these findings, an exchangeable protocol for aligner treatment is suggested, which however needs to be proven clinically. This protocol proposes alternating between multi-layered and single-layered materials within the same treatment phase. This strategy is suggested to optimize treatment outcomes, particularly when planning for a bodily tooth movement., (© 2024. The Author(s).)

Objectives: To use the finite element method (FEM) to investigate the effect of various attachment configurations and trimming line designs of orthodontic aligners on their biomechanical performance., Method: A 3D upper jaw model was imported into 3D design software. The upper right central incisor tooth (Tooth 11) was made mobile, and its periodontal ligament (PDL) and bone structures were designed. Aligners were modelled with three distinct attachment configurations: No attachment, rectangular horizontal, rectangular vertical, and two trimming line designs; scalloped and straight extended, with a homogeneous thickness of 0.6 mm. These models were then imported into an FE software. Simulations were conducted for three different movements, including facial translation, distalization, and extrusion., Results: Forces were recorded at 1.3-2.6 N during facial translation, 1.4-5.9 N in distalization, and 0.0-2.0 N in extrusion. The straight extended trimming line consistently generated higher forces than the scalloped design. Attachments had no significant impact on force components during facial translation but were more effective in distalization and extrusion. The combination of a straight extended trimming line with horizontal attachments exhibited the least stresses at the apical third during distalization, and the highest stresses during extrusion, suggesting superior retention., Conclusions: Rectangular attachments offer limited benefits in facial translation, but horizontal rectangular attachments can intensify load in distalization and are crucial for force generation in extrusion. Horizontal attachments are preferred over vertical options. Additionally, the straight extended trim line enhances control of tooth movement and can replace attachments in certain cases., Clinical Relevance: These findings provide biomechanical evidence and an optimal protocol to guide clinical practice in planning diverse teeth movements. The emphasis is on the influence of attachment utilization and the specific design of aligner trimming lines to enhance control over tooth movement., (© 2024 The Authors. Orthodontics & Craniofacial Research published by John Wiley & Sons Ltd.)

Here we describe the acute myocardial effects of an elapid (red spitting cobra, Naja pallida) and a viper (western diamondback rattlesnake, Crotalus atrox) venom using an ex vivo heart model. Our results reveal two different pathophysiological trajectories that influence heart function and morphology. While cobra venom causes a drop in contractile force, rattlesnake venom causes enhanced contractility and frequency that coincides with differences in myocellular morphology. This highlights the medical complexity of snake venom-induced cardiotoxicity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

A fruit leather (apple and acáchul berry) oriented toward women of reproductive age was developed. The snack was supplemented with an ingredient composed of folic acid (FA) and whey proteins (WPI) to ensure the required vitamin intake to prevent fetal neural tube defects. In order to generate a low-calorie snack, alternative sweeteners were used (stevia and maltitol). The fruit leather composition was determined. Also, an in vitro digestion process was carried out to evaluate the bioaccessibility of compounds with antioxidant capacity (AC), total polyphenols (TPCs), total monomeric anthocyanins (ACY), and FA. The quantification of FA was conducted by a microbiological method and by HPLC. The leather contained carbohydrates (70%) and antioxidant compounds, mainly from fruits. Bioaccessibility was high for AC (50%) and TPCs (90%), and low for ACY (17%). Regarding FA, bioaccessibility was higher for WPI-FA (50%) than for FA alone (37%), suggesting that WPI effectively protected the vitamin from processing and digestion. Furthermore, the product was shown to be non-cytotoxic in a Caco-2 cell model. The developed snack is an interesting option due to its low energy intake, no added sugar, and high content of bioactive compounds. Also, the supplementation with WPI-FA improved the conservation and bioaccessibility of FA.

Background: Mild Cognitive Impairment (MCI) is a heterogeneous condition characterised by cognitive changes that do not affect everyday functioning and may represent a predementia phase. Research on the neuroanatomical correlates of cognitive tests used to diagnose MCI is heterogeneous and has mainly focused on elderly populations of patients with MCI, usually well above the age of 65. However, the effect of ageing on brain structure is known to be substantial and to affect brain-behaviour associations in older people. We explored the brain correlates of different cognitive tests in a group of young-onset MCI (i.e., with symptoms onset before the age of 65) to minimise the effect of ageing on brain-behaviour associations., Methods: Patients with a clinical diagnosis of young-onset MCI underwent extensive cognitive assessment and multimodal Magnetic Resonance Imaging (MRI) including high-resolution T1-weighted and Diffusion Tensor Imaging (DTI) sequences. Their scores on cognitive tests were related to measures of grey matter (GM) density and white matter (WM) integrity using, respectively, Voxel Based Morphometry (VBM) and Tract-Based Spatial Statistics (TBSS)., Results: 104 young-onset MCI were recruited. VBM and TBSS whole-brain correlational analyses showed that between-subject variability in cognitive performance was significantly associated with regional variability in GM density and WM integrity. While associations between cognitive scores and focal GM density in our young-onset MCI group reflected the well-known lateralization of verbal and visuo-spatial abilities on the left and right hemispheres respectively, the associations between cognitive scores and WM microstructural integrity were widespread and diffusely involved most of the WM tracts in both hemispheres., Conclusions: We investigated the structural neuroanatomical correlates of cognitive tests in young-onset MCI in order to minimise the effect of ageing on brain-behaviour associations., Competing Interests: The authors declare no conflict of interest. Giovanna Zamboni is the member of Editorial Board of this journal. Also, Giovanna Zamboni is serving as one of the Guest editors of this journal. We declare that Giovanna Zamboni had no involvement in the peer review of this article and has no access to information regarding its peer review. Full responsibility for the editorial process for this article was delegated to Gernot Riedel., (© 2023 The Author(s). Published by IMR Press.)

The textural changes during storage of two freeze-dried candies developed from blackcurrant fruits, unflavored yogurt, and different alternative sweeteners, one sweetened with honey/isomalt (HI) and another sweetened with isomalt/stevia (IS), were analyzed using three different methods (instrumental, sensory, and image analysis). Fresh candies were in the supercooled state and presented different structural and textural characteristics (HI: compact and homogeneous, and IS: porous and crunchy), with Fmax values of 139 ± 14 and 174 ± 16 N for HI and IS, respectively. After storage, the instrumental analysis showed approximately 60% average drop in F max and W values, in agreement with the decrease observed by sensory analysis in hardness, fracturability, and crispness. Image analysis showed an increase in parameters related to the homogeneity and the uniformity/smoothness for HI. Pearson's correlation coefficients analysis showed that there was a good correlation between the three techniques used, suggesting that the joint use of these methods could be performed for a better understanding of complex food texture., (© 2023 Wiley Periodicals LLC.)

Muscle-specific kinase (MuSK) is crucial for acetylcholine receptor (AChR) clustering and thereby neuromuscular junction (NMJ) function. NMJ dysfunction is a hallmark of several neuromuscular diseases, including MuSK myasthenia gravis. Aiming to restore NMJ function, we generated several agonist monoclonal antibodies targeting the MuSK Ig-like 1 domain. These activated MuSK and induced AChR clustering in cultured myotubes. The most potent agonists partially rescued myasthenic effects of MuSK myasthenia gravis patient IgG autoantibodies in vitro. In an IgG4 passive transfer MuSK myasthenia model in NOD/SCID mice, MuSK agonists caused accelerated weight loss and no rescue of myasthenic features. The MuSK Ig-like 1 domain agonists unexpectedly caused sudden death in a large proportion of male C57BL/6 mice (but not female or NOD/SCID mice), likely caused by a urologic syndrome. In conclusion, these agonists rescued pathogenic effects in myasthenia models in vitro, but not in vivo. The sudden death in male mice of one of the tested mouse strains revealed an unexpected and unexplained role for MuSK outside skeletal muscle, thereby hampering further (pre-) clinical development of these clones. Future research should investigate whether other Ig-like 1 domain MuSK antibodies, binding different epitopes, do hold a safe therapeutic promise., (© 2023. The Author(s).)

In previous studies, the Argentinean native plants called Zuccagnia punctata (jarilla, pus pus, lata) and Solanum betaceum (chilto, tree tomato) were reported as new natural sources of antioxidant compounds, mainly chalcones, anthocyanins and rosmarinic acid derivates. The present study deals with the production of antioxidant beverages of Z. punctata (Zp) extract and chilto juice with honey as sweetener. A Zp extract and red chilto juice were obtained according to Food Code and characterized. The beverages were formulated by using maltodextrin (MD) with two dextrose equivalents (DE), 10 and 15, and then spray-dried at an inlet air temperature of 130 °C. The physicochemical, microscopical, phytochemical and functional characteristics of the powders were surveyed. The experiments carried out showed good physical properties for both formulations showing high water solubility with adequate features for handling, transport and storage. The chromatic parameters of both powdered beverages indicate orange-pink tones regardless of the wall material used. The total polyphenol and flavonoid content in the beverages were kept after spray-drying (92 and 100%, respectively). The anthocyanins were less stable under drying conditions (yield 58%). Both powdered beverages showed high scavenger capacity on ABTS •+ , HO • and H 2 O 2 (SC 50 between 3.29 to 41.05 µg GAE/mL) and were able to inhibit xanthine oxidase (XOD) activity (CI 50 between 91.35 and 114.43 µg GAE/mL). The beverages were neither toxic nor mutagenic in the concentration range with biological activity. The results obtained in the present work scientifically support the use of the powdered beverages of Argentinean native plants as antioxidant.

Objectives: To investigate in a numerical study the effect of the geometry and the extension of orthodontic aligner edges and the aligner thickness on force transmission to upper right central incisor tooth (Tooth 11)., Methods: A three-dimensional (3D) digital model, obtained from a 3D data set of a complete dentulous maxilla, was imported into 3-matic software. Aligners with four different trimming line designs (scalloped, straight, scalloped extended, straight extended) were designed, each with four different thicknesses (0.3, 0.4, 0.5, and 0.6 mm). The models were exported to a finite element (FE) software (Marc/Mentat). A facial 0.2 mm bodily malposition of tooth 11 was simulated., Results: The maximum resultant force was in the range of (7.5 - 55.2) N. The straight trimming designs had higher resultant force than the scalloped designs. The resultant force increases with increasing the edge extension of the aligner. The normal contact forces were unevenly distributed over the entire surface and were concentrated in six areas: Incisal, Mesio-Incisal, Disto-Incisal, Middle, Mesio-Cervical, and Disto-Cervical. The resultant force increases super linearly with increasing thickness., Conclusions: The design of the trimming line, the edge extension, and the thickness of the aligner affect significantly the magnitude of the resultant force and the distribution of normal contact force. The straight extended trimming design exhibited better force distribution that may favor a bodily tooth movement., Clinical Relevance: A straight extended trimming design of an orthodontic aligner may improve the clinical outcomes. In addition, the manufacturing procedures of the straight design are much simpler compared to the scalloped design., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

In 2016, the Dutch government declared its commitment to phasing out animal experiments by 2025. Although a high number of animal experiments are still performed and the 2025 target will not be met, the commitment remains. Efforts are being made to identify levers that might foster the transition to animal-free science. Education has been found to play a key role in the future of animal-free science and young generations are increasingly seen as key stakeholders. However, their attitudes toward the transition to animal-free innovations have not been investigated. The present article focuses on the values and beliefs held by students, who in 2022, participated in the course 'Replacing Animal Testing' (RAT) Challenge, organized by a consortium of Dutch universities. Contextually, students' motivations to follow the course were investigated. The research was based on a qualitative study, including semi-structured interviews and a literature review. Our analysis of the findings revealed that students feel aligned with the social, ethical, and scientific reasons that support the transition to animal-free innovations. Moreover, the participants identified a series of regulatory, educational, cultural, and political obstacles to the transition that align with those identified in recent literature. From the discussion of these findings, we extrapolated six fundamental challenges that need to be addressed to foster the transition to animal-free science in an acceptable and responsible way., Competing Interests: The authors declare no conflict of interest.

Background: Progranulin protein (GRN) is a growth factor, encoded by the GRN (Granulin precursor) gene, involved in several functions including inflammation, wound repair, signal transduction, proliferation, and tumorigenesis. Mutations in GRN gene are usually the genetic etiology of frontotemporal dementia (FTD), but different studies reported GRN mutations in Alzheimer 's disease (AD) patients., Objective: Here, we analyzed FTD linked gene GRN in 23 patients with a clinical diagnosis of AD and a family history of AD (FAD), not carrying mutations in AD candidate genes (PSEN 1, PSEN 2, and APP). In addition, Microtubule-associated protein tau (MAPT) gene was studied too. All patients underwent an extensive neuropsychological battery., Methods: Genetic analyses were performed thought PCR assay and sequencing. Variants were annotated with ANNOVAR and allele frequency was checked on population databases. In silico prediction tools were consulted to check nonsynonymous variants and their effect on protein function and structure. The clinical data were retrospectively collected from medical records., Results: Genetic screening of MAPT and GRN in 23 FAD patients highlighted two rare different variants in two probands (2/23 = 8,7%) located in GRN gene: R433W (p.Arg433Trp) and C521Y (p.Cys521Tyr). The R433W and C521Y are variants with uncertain significant, that are predicted to affect GRN protein structure and function, with a possible damaging effect., Conclusions: Our data provide evidence of the importance of GRN genetic analysis also in the study of familial AD.

Lumbar interbody fusion (IF) is a common procedure to obtain fusion of the spine by replacement of the intervertebral disc with a cage. Optimization of spinal cages is ongoing to reduce complications such as a pseudoarthrosis and subsidence of the cage. IF animal models (primate, dog, pig, goat and sheep) remain important to assess implant effectivity. But currently the available literature is dispersed and not IF model specific. Therefore unwanted inconsistencies between studies occur that limit generalizability. Based on our experience, anatomical preparation and literature research, we present a rationale for species selection and a practical guide for the surgical procedure in the goat animal model., The Translational Potential of This Article: Rigorous methodologic design is an important means to improve translational value and generalizability of large animal IF efficacy studies. This paper provides a rationale and practical guide for animal selection and surgical decision making that can help reduce unnecessary variation between models and improve methodologic rigor and documentation for future experiments., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Authors.)

Objectives: To investigate how the stress distribution and forces transmitted from orthodontic aligners to the tooth surface are affected by the geometry and extension of the trimming line., Materials and Methods: Thirty-six aligners were thermoformed from Zendura FLX sheets (0.75 mm thick) and divided into four groups based on the design of the trimming line: Scalloped, Scalloped extended, Straight and Straight extended. Fuji pressure-sensitive films were used for pressure measurement. The pressurized films were scanned and evaluated. Pressures and forces were measured over the entire facial surface of an upper right central incisor (Tooth 11) and at 7 different locations [cervical, middle, incisal, mesio-incisal, mesio-cervical, disto-incisal, and disto-cervical]. In addition, the thickness of the aligners at these 7 sites was measured with a digital caliper., Results: The active force ranged from (2.2 to 6.9) N, and the average pressure was (1.6-2.7) MPa. The highest values were recorded for the (straight extended) design, while the lowest values were recorded for the scalloped design. The forces and stresses were not uniformly distributed over the surface. When the values in each area were compared separately, significant differences were found between the different designs in the cervical area, with the scalloped design transmitting the lowest cervical forces. Aligner thickness was drastically reduced (60-75% thinning) over the entire tooth surface after thermoforming., Conclusions: The straight extended design of aligner's trimming line exhibited more uniform force transfer and stress distribution across the surface than the other designs., Clinical Relevance: The trimming line design could have a significant impact on the clinical outcome of orthodontic aligner treatment., Competing Interests: Conflicts of interest The authors declare that they have no conflict of interest., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

In Gaucher disease (GD), the deficiency of glucocerebrosidase causes lysosomal accumulation of glucosylceramide (GlcCer), which is partly converted by acid ceramidase to glucosylsphingosine (GlcSph) in the lysosome. Chronically elevated blood and tissue GlcSph is thought to contribute to symptoms in GD patients as well as to increased risk for Parkinson's disease. On the other hand, formation of GlcSph may be beneficial since the water soluble sphingoid base is excreted via urine and bile. To study the role of excessive GlcSph formation during glucocerebrosidase deficiency, we studied zebrafish that have two orthologs of acid ceramidase, Asah1a and Asah1b. Only the latter is involved in the formation of GlcSph in glucocerebrosidase-deficient zebrafish as revealed by knockouts of Asah1a or Asah1b with glucocerebrosidase deficiency (either pharmacologically induced or genetic). Comparison of zebrafish with excessive GlcSph (gba1 -/- fish) and without GlcSph (gba1 -/- :asah1b -/- fish) allowed us to study the consequences of chronic high levels of GlcSph. Prevention of excessive GlcSph in gba1 -/- :asah1b -/- fish did not restrict storage cells, GlcCer accumulation, or neuroinflammation. However, GD fish lacking excessive GlcSph show an ameliorated course of disease reflected by significantly increased lifespan, delayed locomotor abnormality, and delayed development of an abnormal curved back posture. The loss of tyrosine hydroxylase 1 (th1) mRNA, a marker of dopaminergic neurons, is slowed down in brain of GD fish lacking excessive GlcSph. In conclusion, in the zebrafish GD model, excess GlcSph has little impact on (neuro)inflammation or the presence of GlcCer-laden macrophages but rather seems harmful to th1-positive dopaminergic neurons., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Animal models continue to be used to investigate cartilage repair strategies. Adequate anaesthesia and pain management are essential in order to guarantee acceptable animal welfare as well as reproducible experimental results. This systematic review evaluates reporting of anaesthesia and pain management in surgical large animal models (horse, pig, dog, goat and sheep) of (osteo)chondral repair. Manuscripts published between 2015 and 2020 were included after a comprehensive search strategy. Data were evaluated using descriptive statistics and qualitative review. Out of 223 eligible studies, 220 studies contained incomplete information on anaesthetic and pain management. Pre-, intra- and post-operative analgesia were not mentioned in 68%, 94%, and 64% of manuscripts respectively. A total of 176 studies reported that animals underwent general anaesthesia during surgery. Surprisingly, 30% of these articles did not provide any detail on anaesthetic management, while 37% reported using inhalant, hypnotic or sedative drugs only, without mention of analgesics. Pain monitoring was not reported in 87% of manuscripts. The vast majority of preclinical large animal studies on cartilage repair did not meet veterinary clinical standards for anaesthesia and analgesia, and failed to report according to the ARRIVE international guidelines. In light of serious welfare, ethical and translational validity concerns, improvement is urgently needed., Competing Interests: The authors have no competing interest to declare., (© 2022 The Authors.)

This work aimed at developing powders rich in antioxidants and pigments from two wild berries: maqui ( Aristotelia chilensis ) and murra ( Rubus ulmifolius ). Fruits were subjected to successive ultrasound-assisted extractions (UAE) and then freeze-dried. Physical properties, anthocyanin stability of powders, and their performance as natural colorants in yogurts were evaluated. The optimum extraction methods were: UAE for 10 min in murra, and without UAE (control) in maqui, with juice extraction yields ranging between 80 and 82%. Maqui powder exhibited ≈ 2.8 times more polyphenol and anthocyanin content than murra. However, murra powder showed better stability characteristics as powder colorant since it exhibited greater protection of anthocyanins by means of copigmentation phenomena. Regarding consumer's perception of colored yogurt, samples with 4% and 8% maqui powder could be considered as future prototypes to be launched into the market. The obtained powders may be used in different industrial food applications., Competing Interests: Conflict of interestThe authors declare that they have no conflict of interest., (© Association of Food Scientists & Technologists (India) 2021.)

Recombinase-activating gene-1 (RAG1)-deficient severe combined immunodeficiency (SCID) patients lack B and T lymphocytes due to the inability to rearrange immunoglobulin and T cell receptor genes. Gene therapy is an alternative for those RAG1-SCID patients who lack a suitable bone marrow donor. We designed lentiviral vectors with different internal promoters driving codon-optimized RAG1 to ensure optimal expression. We used Rag1 -/- mice as a preclinical model for RAG1-SCID to assess the efficacy of the various vectors. We observed that B and T cell reconstitution directly correlated with RAG1 expression. Mice with low RAG1 expression showed poor immune reconstitution; however, higher expression resulted in phenotypic and functional lymphocyte reconstitution comparable to mice receiving wild-type stem cells. No signs of genotoxicity were found. Additionally, RAG1-SCID patient CD34 + cells transduced with our clinical RAG1 vector and transplanted into NSG mice led to improved human B and T cell development. Considering this efficacy outcome, together with favorable safety data, these results substantiate the need for a clinical trial for RAG1-SCID., (© 2020 The Authors.)

Liquid biopsy-based biomarkers, such as microRNAs, represent valuable tools for patient management, but often do not make it to integration in the clinic. We aim to explore issues impeding this transition, in the setting of germ cell tumors, for which novel biomarkers are needed. We describe a model for identifying and validating clinically relevant microRNAs for germ cell tumor patients, using both in vitro, in vivo (mouse model) and patient-derived data. Initial wide screening of candidate microRNAs is performed, followed by targeted profiling of potentially relevant biomarkers. We demonstrate the relevance of appropriate (negative) controls, experimental conditions (proliferation), and issues related to sample origin (serum, plasma, cerebral spinal fluid) and pre-analytical variables (hemolysis, contaminants, temperature), all of which could interfere with liquid biopsy-based studies and their conclusions. Finally, we show the value of our identification model in a specific scenario, contradicting the presumed role of miR-375 as marker of teratoma histology in liquid biopsy setting. Our findings indicate other putative microRNAs (miR-885-5p, miR-448 and miR-197-3p) fulfilling this clinical need. The identification model is informative to identify the best candidate microRNAs to pursue in a clinical setting.

The aim of this work was to obtain powders rich in bioactive compounds from maqui berry aqueous extracts by spray drying. First, the process parameters of the maqui aqueous extraction were optimized. The optimal operating conditions were found using an experimental Box-Behnken design with three factors: solvent/fruit ratio (2:1, 3.5:1 and 5:1), extraction temperature (25, 50 and 75 °C) and extraction time (30, 75 and 120 min). Soluble solids content, monomeric anthocyanin content (ACY), total polyphenol content (TPC) and antioxidant capacity in the liquid extracts were analyzed as key responses to find the optimal extraction conditions. Secondly, the best aqueous extract (solvent/fruit ratio = 2:1; extraction temperature = 75 °C and extraction time = 75 min) was subjected to spray drying. The effects of different drying adjuvants (maltodextrin, colloidal silicon dioxide, arabic gum, and microcrystalline cellulose) on the powders flow properties, the process yield (PY), the bioactive compounds content and the superficial color were studied. The product based on colloidal silicon dioxide presented the best powder properties: excellent flowability (α: 30.4 ± 0.7°, CI: 8.0 ± 1.7%), adequate moisture content (4.9 ± 0.3%), very good PY (70 ± 1%), high ACY (1528 ± 41 mg cy-3glu/100 g of powder) and TPC (3936 ± 132 mg GAE/100 g of powder), and a purple hue. This maqui powder offers valuable properties that allow its use, among other applications, as a functional ingredient, natural colorant and nutraceutical product.

Tissue factor, coagulation factor XII, platelets, and neutrophils are implicated as important players in the pathophysiology of (experimental) venous thrombosis (VT). Their role became evident in mouse models in which surgical handlings were required to provoke VT. Combined inhibition of the natural anticoagulants antithrombin ( Serpinc1 ) and protein C ( Proc ) using small interfering RNA without additional triggers also results in a venous thrombotic phenotype in mice, most notably with vessel occlusion in large veins of the head. VT is fatal but is fully rescued by thrombin inhibition. In the present study, we used this VT mouse model to investigate the involvement of tissue factor, coagulation factor XII, platelets, and neutrophils. Antibody-mediated inhibition of tissue factor reduced the clinical features of VT, the coagulopathy in the head, and fibrin deposition in the liver. In contrast, genetic deficiency in, and small interfering RNA-mediated depletion of, coagulation factor XII did not alter VT onset, severity, or thrombus morphology. Antibody-mediated depletion of platelets fully abrogated coagulopathy in the head and liver fibrin deposition. Although neutrophils were abundant in thrombotic lesions, depletion of circulating Ly6G-positive neutrophils did not affect onset, severity, thrombus morphology, or liver fibrin deposition. In conclusion, VT after inhibition of antithrombin and protein C is dependent on the presence of tissue factor and platelets but not on coagulation factor XII and circulating neutrophils. This study shows that distinct procoagulant pathways operate in mouse VT, dependent on the triggering stimulus., (© 2019 by The American Society of Hematology.)

Background: Sweet cherries are an excellent source of phenolic compounds, which may contribute to a healthy diet. The objective of this work was to generate dehydrated ingredients from postharvest discard of sweet cherries., Results: Four dried ingredients were obtained from fresh sweet cherry discard (Lapins var.) using an osmotic dehydration pretreatment and freeze drying or air drying. The ingredients showed an important phenolic contribution (2.8-6.6 g gallic acid kg -1 of product) and preserved the natural color of the fruit to a great extent. Freeze-dried ingredients were less hygroscopic than air-dried ones, and presented with a softer texture. All the ingredients were in a supercooled state at room temperature (T g range: -23.0 to -18.8 °C). Sugar infusion pretreatment caused a decrease in water sorption capacity and molecular mobility; it also reduced the initial rehydration rate., Conclusion: Relevant differences in nutritional and structural characteristics of the ingredients were observed depending on the processing method used. These ingredients could be incorporated into different processed foods, such as snacks, cereal mixtures, cereal bars, and bakery and confectionery products. Air-dried control ingredients presented better nutritional qualities and air-dried sweet cherries with sugar infusion pretreatment could be appropriate ingredients for applications where sweet flavor and slow rehydration rate are required. © 2018 Society of Chemical Industry., (© 2018 Society of Chemical Industry.)

By their interaction with IgG immune complexes, FcγR and complement link innate and adaptive immunity, showing functional redundancy. In complement-deficient mice, IgG downstream effector functions are often impaired, as well as adaptive immunity. Based on a variety of model systems using FcγR-knockout mice, it has been concluded that FcγRs are also key regulators of innate and adaptive immunity; however, several of the model systems underpinning these conclusions suffer from flawed experimental design. To address this issue, we generated a novel mouse model deficient for all FcγRs (FcγRI/II/III/IV -/- mice). These mice displayed normal development and lymphoid and myeloid ontogeny. Although IgG effector pathways were impaired, adaptive immune responses to a variety of challenges, including bacterial infection and IgG immune complexes, were not. Like FcγRIIb-deficient mice, FcγRI/II/III/IV -/- mice developed higher Ab titers but no autoantibodies. These observations indicate a redundant role for activating FcγRs in the modulation of the adaptive immune response in vivo. We conclude that FcγRs are downstream IgG effector molecules with a restricted role in the ontogeny and maintenance of the immune system, as well as the regulation of adaptive immunity., (Copyright © 2018 by The American Association of Immunologists, Inc.)

Blackcurrants contain high levels of polyphenolics, particularly flavonols and anthocyanins, which contribute to their high antioxidant activity. The aims of this work were the recovery of bioactive compounds from the remaining solid (waste) after processing blackcurrant juice and to obtain spray-dried powders from the blackcurrant juice and extracts. The extraction of bioactive compounds from the fruit pulp was performed by ultrasound-assisted extraction. Experiments were conducted to select the more suitable solvent, and citric acid was chosen. Then, to optimize the extraction conditions (time, solvent concentration, and amplitude) an experimental design using a Box-Behnken Design was done. Comparing the optimized extract with the fruit, 31% total monomeric anthocyanins, 19% total phenolic compounds, and 10% antioxidant capacity were obtained. The optimized extract and the juice were mixed and spray dried, using maltodextrin as carrier matrix. A blackcurrant powder with low hygroscopicity 14.46 ± 0.13 (g a.w./100 g d.m) and high solubility 94.25 ± 4% was obtained. High concentration of bioactive compounds and antioxidant capacity was recorded: Total monomeric anthocyanins 63.01 ± 1 (mg cyn-3-glu/100 g.d.m), total phenolic content 116.87 ± 5 (mg gallic acid/100 g d.m.), and antioxidant capacity 144.40 ± 0.11 (mg eq Trolox/100 g.d.m.).

Aims: The Z-disc is a crucial structure of the sarcomere and is implicated in mechanosensation/transduction. Dysregulation of Z-disc proteins often result in cardiomyopathy. We have previously shown that the Z-disc protein Cytoskeletal Heart-enriched Actin-associated Protein (CHAP) is essential for cardiac and skeletal muscle development. Furthermore, the CHAP gene has been associated with atrial fibrillation in humans. Here, we studied the misregulated expression of CHAP isoforms in heart disease., Methods and Results: Mice that underwent transverse aortic constriction and calcineurin transgenic (Tg) mice, both models of experimental heart failure, displayed a significant increase in cardiac expression of fetal isoform CHAPb. To investigate whether increased expression of CHAPb postnatally is sufficient to induce cardiomyopathy, we generated CHAPb Tg mice under the control of the cardiac-specific αMHC promoter. CHAPb Tg mice displayed cardiac hypertrophy, interstitial fibrosis and enlargement of the left atrium at three months, which was more pronounced at the age of six months. Hypertrophy and fibrosis were confirmed by evidence of activation of the hypertrophic gene program (Nppa, Nppb, Myh7) and increased collagen expression, respectively. Connexin40 and 43 were downregulated in the left atrium, which was associated with delayed atrioventricular conduction. Tg hearts displayed both systolic and diastolic dysfunction partly caused by impaired sarcomere function evident from a reduced force generating capacity of single cardiomyocytes. This co-incided with activation of the actin signalling pathway leading to the formation of stress fibers., Conclusion: This study demonstrated that the fetal isoform CHAPb initiates progression towards cardiac hypertrophy, which is accompanied by delayed atrioventricular conduction and diastolic dysfunction. Moreover, CHAP may be a novel therapeutic target or candidate gene for screening in cardiomyopathies and atrial fibrillation.

The canonical Wnt signalling pathway has been implicated in organogenesis and self-renewal of essentially all stem cell systems. In vivo reporter systems are crucial to assess the role of Wnt signalling in the biology and pathology of stem cell systems. We set out to develop a Turquoise (TQ) fluorescent protein based Wnt reporter. We used a CRISPR-Cas9 approach to insert a TQ fluorescent protein encoding gene into the general Wnt target gene Axin2, thereby establishing a Wnt reporter mouse similar to previously generated Wnt reporter mice but with the mTurquoise2 gene instead of E. coli-β-galactosidase (LacZ). The use of mTurquoise2 is especially important in organ systems in which cells need to a be alive for further experimentation such as in vitro activation or transplantation studies. We here report successful generation of Axin2-TQ mice and show that cells from these mice faithfully respond to Wnt signals. High Wnt signals were detected in the intestinal crypts, a classical Wnt signalling site in vivo, and by flow cytometry in the thymus. These mice are an improved tool to further elucidate the role of Wnt signalling in vivo., (© 2017 Wiley Periodicals, Inc.)

Recently, platelets, neutrophils, and factor XII (FXII) have been implicated as important players in the pathophysiology of venous thrombosis. Their role became evident in mouse models in which surgical handling was used to provoke thrombosis. Inhibiting anticoagulation in mice by using small interfering RNA (siRNA) targeting Serpinc1 and Proc also results in a thrombotic phenotype, which is spontaneous (no additional triggers) and reproducibly results in clots in the large veins of the head and fibrin deposition in the liver. This thrombotic phenotype is fatal but can be fully rescued by thrombin inhibition. The mouse model was used in this study to investigate the role of platelets, neutrophils, and FXII. After administration of siRNAs targeting Serpinc1 and Proc, antibody-mediated depletion of platelets fully abrogated the clinical features as well as microscopic aspects in the head. This was corroborated by strongly reduced fibrin deposition in the liver. Whereas neutrophils were abundant in siRNA-triggered thrombotic lesions, antibody-mediated depletion of circulating Ly6G-positive neutrophils did not affect onset, severity, or thrombus morphology. In addition, absence of circulating neutrophils did not affect quantitative liver fibrin deposition. Remarkably, siRNA-mediated depletion of plasma FXII accelerated the onset of the clinical phenotype; mice were affected with more severe thrombotic lesions. To summarize, in this study, onset and severity of the thrombotic phenotype are dependent on the presence of platelets but not circulating neutrophils. Unexpectedly, FXII has a protective effect. This study challenges the proposed roles of neutrophils and FXII in venous thrombosis pathophysiology., (© 2016 by The American Society of Hematology.)

Naive mouse embryonic stem cells (mESCs) are in a metastable state and fluctuate between inner cell mass- and epiblast-like phenotypes. Here, we show transient activation of the BMP-SMAD signaling pathway in mESCs containing a BMP-SMAD responsive reporter transgene. Activation of the BMP-SMAD reporter transgene in naive mESCs correlated with lower levels of genomic DNA methylation, high expression of 5-methylcytosine hydroxylases Tet1/2 and low levels of DNA methyltransferases Dnmt3a/b. Moreover, naive mESCs, in which the BMP-SMAD reporter transgene was activated, showed higher resistance to differentiation. Using double Smad1;Smad5 knockout mESCs, we showed that BMP-SMAD signaling is dispensable for self-renewal in both naive and ground state. These mutant mESCs were still pluripotent, but they exhibited higher levels of DNA methylation than their wild-type counterparts and had a higher propensity to differentiate. We showed that BMP-SMAD signaling modulates lineage priming in mESCs, by transiently regulating the enzymatic machinery responsible for DNA methylation., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Gastrointestinal tumor growth is thought to be promoted by gastrointestinal bacteria and their inflammatory products. We observed that intestine-specific conditional Apc mutant mice (FabplCre;Apc (15lox/+)) developed many more colorectal tumors under conventional than under pathogen-low housing conditions. Shotgun metagenomic sequencing plus quantitative PCR analysis of feces DNA revealed the presence of two bacterial species in conventional mice, absent from pathogen-low mice. One, Helicobacter typhlonius, has not been associated with cancer in man, nor in immune-competent mice. The other species, mucin-degrading Akkermansia muciniphila, is abundantly present in healthy humans, but reduced in patients with inflammatory gastrointestinal diseases and in obese and type 2 diabetic mice. Eradication of H.typhlonius in young conventional mice by antibiotics decreased the number of intestinal tumors. Additional presence of A.muciniphila prior to the antibiotic treatment reduced the tumor number even further. Colonization of pathogen-low FabplCre;Apc (15lox/+) mice with H.typhlonius or A.muciniphila increased the number of intestinal tumors, the thickness of the intestinal mucus layer and A.muciniphila colonization without H.typhlonius increased the density of mucin-producing goblet cells. However, dual colonization with H.typhlonius and A.muciniphila significantly reduced the number of intestinal tumors, the mucus layer thickness and goblet cell density to that of control mice. By global microbiota composition analysis, we found a positive association of A.muciniphila, and of H.typhlonius, and a negative association of unclassified Clostridiales with increased tumor burden. We conclude that A.muciniphila and H.typhlonius can modulate gut microbiota composition and intestinal tumor development in mice., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Mortality from colorectal cancer increases with latitude and decreases with ambient UV radiation. We investigated whether moderate UV dosages could inhibit intestinal tumor development and whether this corresponded with UV-induced vitamin D. FabplCre;Apc(15lox/+) mice, which develop intestinal tumors, and their parents were put on a vitamin D-deficient diet. Next to a control group, one group was vitamin D supplemented and another one group was daily UV irradiated from 6 weeks of age. Vitamin D statuses after 6 weeks of treatment were markedly increased: mean ± SD from 7.7 ± 1.9 in controls to 75 ± 15 nmol/l with vitamin D supplementation (no gender difference), and to 31 ± 13 nmol/l in males and 85 ± 17 nmol/l in females upon UV irradiation. The tumor load (area covered by tumors) at 7.5 months of age was significantly reduced in both the vitamin D-supplemented group (130 ± 25 mm(2), p = 0.018) and the UV-exposed group (88 ± 9 mm(2), p < 0.0005; no gender differences) compared to the control group (202 ± 23 mm(2)). No reductions in tumor numbers were found. Only UV exposure appeared to reduce progression to malignancy (p = 0.014). Our experiments clearly demonstrate for the first time an inhibitory effect of moderate UV exposure on outgrowth and malignant progression of primary intestinal tumors, which at least in part can be attributed to vitamin D., (© 2014 UICC.)