Reduced adult stem cell fate specification led to eye reduction in cave planarians.

Eye loss occurs convergently in numerous animal phyla as an adaptation to dark environments. We investigate the cave planarian Girardia multidiverticulata (Gm), a representative species of the Spiralian clade, to study mechanisms of eye loss. We found that Gm, which was previously described as an eyeless species, retains rudimentary and functional eyes. Eyes are maintained in homeostasis and regenerated in adult planarians by stem cells, called neoblasts, through their fate specification to eye progenitors. The reduced number of eye cells in cave planarians is associated with a decreased rate of stem cell fate specification to eye progenitors during homeostasis and regeneration. Conversely, the homeostatic formation of new cells from stem cell-derived progenitors for other tissues, including for neurons, pharynx, and epidermis, is comparable between cave and surface species. These findings reveal a mode of evolutionary trait loss, with change in rate of fate specification in adult stem cells leading to tissue size reduction.

Isolating Synaptic Vesicles from Neurospheres for Proteomics.

This chapter presents an optimized method for isolating synaptic vesicles (SVs) from neurospheres derived from human induced pluripotent stem cells (hiPSCs). The protocol begins with neurosphere cultivation to achieve mature neurons, which is essential for the functional studies of neuronal activity. Following this, neurosphere-derived synaptosomes are isolated, and SVs are enriched through differential centrifugation. The method culminates in the proteomic analysis of SVs using nano-liquid chromatography coupled with high-resolution tandem mass spectrometry (nanoLC-MS/MS), providing a detailed proteome profile of the isolated vesicles. This protocol can contribute to the understanding of SV molecular heterogeneity and the mechanisms of neurotransmitter uptake and release and be applied to the field of research in neurological and neuropsychiatric disorders.

Comprehensive analysis of secretome and transcriptome stability of Wharton jelly mesenchymal stromal cells during good manufacturing practice-compliant production.

BACKGROUND: Mesenchymal stromal cells (MSCs) hold promise for cell-based therapies due to their ability to stimulate tissue repair and modulate immune responses. Umbilical cord-derived MSCs from Wharton jelly (WJ) offer advantages such as low immunogenicity and potent immune modulatory effects. However, ensuring consistent quality and safety throughout their manufacturing process remains critical. RNA sequencing (RNA-seq) emerges as a crucial tool for assessing genetic stability and expression dynamics in cell-based therapeutic products. METHODS: We examined the secretome and transcriptome of WJ-MSC signatures throughout Good Manufacturing Practice (GMP) production, focusing on the performance of total RNA or Massive Analysis of cDNA Ends (MACE) sequencing. RESULTS: Through extensive transcriptomic analysis, we demonstrated consistent stability of WJ-MSC expression signatures across different manufacturing stages. Notably, MACE-seq showed improved identification of key expression patterns related to senescence and immunomodulation. CONCLUSIONS: These findings highlight the potential of MACE-seq as a quality assessment tool for WJ-MSC-based therapies, ensuring their efficacy and safety in clinical applications. Importantly, MACE-seq demonstrated its value in characterizing WJ-MSC-derived products, offering insights that traditional assays cannot provide.

Bone Tissue Engineering With Chitosan, Carbon Nanotubes, and Hydroxyapatite Biomaterials Enriched With Mesenchymal Stem Cells: A Radiographic and Histological Evaluation in a Sheep Model Undergoing Ostectomy (Bone Tissue Engineering in a Sheep Model).

Comminuted fractures associated with tissue loss can adversely affect bone regeneration. Biomaterials enriched with mesenchymal stem cells (MSCs) employed for supporting osteosynthesis and potentiating osteoconduction are necessary to fill these bone defects. Natural compound biomaterials, similar to bone tissue, have been extensively tested in animal models for clinical use. Bone tissue engineering studies have used critical-size defects in ovine tibia monitored by imaging and histological examinations to evaluate the regenerative process. This study aimed to monitor the regenerative process in ovine tibial defects with or without chitosan, carbon nanotubes, or hydroxyapatite biomaterials, enriched or not enriched with MSCs. A 3-cm ostectomy was performed in 18 female Suffolk sheep. A 10-hole 4.5 mm narrow locking compression plate was used for osteosynthesis. The animals were randomly divided into three groups (n = 6): control (CON); defects filled with chitosan, carbon nanotubes, and hydroxyapatite biomaterial (BIO); and the same biomaterial enriched with bone marrow MSCs (BIO + CELL). The animals were evaluated monthly using radiographic examinations until 90 postoperative days, when they were euthanized. The limbs were subjected to micro-computed tomography (micro-CT), and bone specimens were subjected to histological evaluations. The radiographic examinations revealed construction stability without plate deviation, fracture, or bone lysis. Micro-CT evaluation demonstrated a difference in bone microarchitecture between the CON and biomaterial treatment groups (BIO and BIO + CELL). In the histological evaluations, the CON group did not demonstrate bone formation, and in the treatment groups (BIO and BIO + CELL), biocompatibility with sheep tissue was noted, and bone formation with trabeculae interspersed with remnants of the biomaterial was observed, with no differences between the groups. In conclusion, biomaterials present osteoconduction with beneficial characteristics for filling bone-lost fractures, and MSCs did not interfere with bone formation.

Combined additive effects of neuronal membrane glycoprotein GPM6a and the intercellular cell adhesion molecule ICAM5 on neuronal morphogenesis.

The mechanisms underlying neuronal development and synaptic formation in the brain depend on intricate cellular and molecular processes. The neuronal membrane glycoprotein GPM6a promotes neurite elongation, filopodia/spine formation, and synapse development, yet its molecular mechanisms remain unknown. Since the extracellular domains of GPM6a (ECs) command its function, we investigated the interaction between ICAM5, the neuronal member of the intercellular adhesion molecule (ICAM) family, and GPM6a's ECs. Our study aimed to explore the functional relationship between GPM6a and ICAM5 in hippocampal culture neurons and cell lines. Immunostaining of 15 days in vitro (DIV) neurons revealed significant co-localization between endogenous GPM6a clusters and ICAM5 clusters in the dendritic shaft. These results were further corroborated by overexpressing GPM6a and ICAM5 in N2a cells and hippocampal neurons at 5 DIV. Moreover, results from the co-immunoprecipitations and cell aggregation assays prove the cis and trans interaction between both proteins in GPM6a/ICAM5 overexpressing HEK293 cells. Additionally, GPM6a and ICAM5 overexpression additively enhanced neurite length, the number of neurites in N2a cells, and filopodia formation in 5 DIV neurons, indicating their cooperative role. These findings highlight the dynamic association between GPM6a and ICAM5 during neuronal development, offering insights into their contributions to neurite outgrowth, filopodia formation, and cell-cell interactions.

Further biological characterization of small molecules UM171 and SR1: In vitro effects on three hematopoietic cell populations from human cord blood.

Small molecules UM171 and SR1 have already been taken into clinically-oriented protocols for the ex vivo expansion of hematopoietic stem (HSCs) and progenitor (HPCs) cells. In order to gain further insight into their biology, in the present study we have assessed their effects, both individually and in combination, on the in vitro long-term proliferation and expansion of HSCs and HPCs contained within three different cord blood-derived cell populations: MNCs (CD34+ cells = 0.8 %), LIN- cells (CD34+ cells = 41 %), and CD34+ cells (CD34+ cells >98 %). Our results show that when added to cultures in the absence of recombinant stimulatory cytokines, neither molecule had any effect. In contrast, when added in the presence of hematopoietic cytokines, UM171 and SR1 had significant stimulatory effects on cell proliferation and expansion in cultures of LIN- and CD34+ cells. No significant effects were observed in cultures of MNCs. The effects of both molecules were more pronounced in cultures with the highest proportion of CD34+ cells, and the greatest effects were observed when both molecules were added in combination. In the absence of small molecules, cell numbers reached a peak by days 25-30, and then declined; whereas in the presence of UM171 or/and SR1 cell numbers were sustained up to day 45 of culture. Our results indicate that besides CD34+ cells, LIN- cells could also be used as input cells in clinically-oriented expansion protocols, and that using both molecules simultaneously would be a better approach than using only one of them.

Phenotypic Characterization of B-Lymphocyte Subpopulations in Human Peripheral Blood: A Cost-Effective Seven-Color One-Tube Protocol.

B cells are crucial components of the immune system, responsible for producing specific antibodies in response to infections and vaccines. Despite their uniform appearance, B cells display diverse surface molecules and functional properties, which are not yet fully understood. Apart from antibody production, B cells also play roles in antigen presentation and cytokine secretion, essential for initiating T-cell immune responses. Their significance as disease biomarkers and therapeutic targets has led to increased research focus. However, the lack of standardized protocols for B-cell identification and the variability in defining B-lymphocyte subpopulations pose some challenges. This paper proposes a B-cell identification panel throughout the evaluation of previous cytometry panels and nomenclature heterogeneity for B-cell subpopulations. Major subpopulations recognized in human peripheral blood include transitional, naive, switched memory, unswitched memory, double negative, and plasmablasts, characterized based on their functional and phenotypic features. We present a standardized flow cytometry protocol utilizing surface phenotypic markers (CD3, CD19, IgD, CD27, CD38, and CD24) to differentiate and analyze B-cell subpopulations. This practical and cost-effective panel can be used in various research and laboratory settings. The challenges of standardizing names and markers for classifying B-lymphocyte subpopulations are discussed, along with protocols utilizing multiple markers and gating strategies, allied with the importance of considering viability markers. In summary, this standardized protocol and panel provide a comprehensive approach to identifying B-cell subpopulations to enhance the reproducibility and comparability of B-cell subpopulation studies.

Establishment and characterization of fibroblast lines from the northern tiger cat (Leopardus tigrinus, Schreber, 1775) during extended passage and cryopreservation.

The establishment of fibroblast lines enables several applications from the formation of biobanks for the conservation of biodiversity to the use of these cells in physiological and toxicological assays. Considered a species vulnerable to extinction, the characterization of fibroblastic lines of northern tiger cat would contribute to its conservation. Therefore, we established and characterized fibroblasts derived from northern tiger cat during extended passage (third, seventh, and eleventh passages) and cryopreservation with regard to the morphology, viability, apoptotic classification, metabolism, proliferative activity, and oxidative stress by reactive oxygen species (ROS) levels and mitochondrial membrane potential (ΔΨm). Initially, we identified four dermal fibroblast lines by morphology, immunophenotyping, and karyotyping assays. In vitro culture after the third, seventh, and eleventh passages did not affect the viability, apoptotic classification, and ROS levels. Nevertheless, cells at seventh and eleventh passages featured a reduction in metabolism and an alteration in ΔΨm when compared to third passage cells. Additionally, cells at eleventh passage showed changes in the proliferative activity and morphology when compared to other passages. Regarding cryopreservation, no effect was observed on cryopreserved cells for morphology, viability, apoptotic classification, metabolism, and proliferative activity. Nevertheless, cryopreserved cells had alteration for ROS levels and ΔΨm. In summary, fibroblasts from northern tiger cat were affected by extended passage (seventh and eleventh passages) and cryopreservation. Adjustments to the in vitro culture and cryopreservation are necessary to reduce cellular oxidative stress caused by in vitro conditions.

Repopulation of a 3D simulated periapical lesion cavity with dental pulp stem cell spheroids with triggered osteoblastic differentiation.

We established a proof-of-concept model system for the biological healing of periapical lesions using stem cell spheroids. Mesenchymal stem cells from human exfoliated deciduous teeth (SHED) were cultured in a 2D monolayer and then as 3D multicellular spheroids. An image of a periapical lesion of an upper lateral incisor tooth was obtained by computed tomography and was used as a model for photopolymer resin 3D printing to generate a negative frame of the lesion. The negative model served to prepare a positive model of the periapical lesion cavity in an agarose gel. SHED that were cultured in monolayers or as spheroids were seeded in the positive lesion mold before or after osteoblastic differentiation. The results showed that compared to cells cultured in monolayers, spheroids exhibited uniform cellularity and a greater viability within the lesion cavity, which was accompanied by a temporal reduction in the expression of CD13, CD29, CD44, CD73, and CD90 mRNAs that are typically expressed by stem cells. Concomitantly, the expression of markers that characterize osteoblastic differentiation (RUNX2, ALP, and BGLAP) increased. These results provide a new perspective for regenerative endodontics with the use of SHED-derived spheroids to repair periapical lesions.

Dimethyl sulfoxide affects the viability and mineralization activity of apical papilla cells in vitro.

Dimethyl sulfoxide (DMSO) is widely used as an adjuvant in dissolving insoluble compounds in an aqueous medium; however, it can induce significant molecular changes in cells. The possible damages may occur obeying a tissue-specific profile, and the effect on human apical papilla cells (hAPC) remains unknown. Therefore, this study aimed to evaluate DMSO effects on the viability and mineralization activity in hAPC cultures in vitro and to establish standards of maximum concentrations for its use in laboratory routines. hAPCs were cultured, plated, and maintained in media containing increasing concentrations of Dimethyl sulfoxide (0.1%, 0.5%, 1%, 5%, and 10%) for 24 h, 48 h, 72 h, and 7 days. At each time point, the cells were subjected to the MTT assay. The Alizarin red S staining assay was performed to evaluate the osteo/odontogenic mineralization potential of hAPC DMSO-exposed (0.1%, 0.5%, and 1%) in the 21-day time-point. Statistical analysis was performed using one-way analysis of variance followed by Tukey's post hoc test (p<0.05). In general, the 5% and 10% DMSO concentrations were shown to be cytotoxic for hAPC at all analyzed time points, and the hAPC DMSO-stimulated presented higher osteo/odontogenic mineralization potential. Therefore, the 5% and 10% DMSO concentrations should be avoided, and the mineralization activity assay should be carefully designed in order to avoid biases at in vitro assays using hAPC cultures.

Chemical transdifferentiation of somatic cells to neural cells: a systematic review.

INTRODUCTION: Transdifferentiation is the conversion of a specific somatic cell into another cell type, bypassing a transient pluripotent state. This implies a faster method to generate cells of interest with the additional benefit of reduced tumorigenic risk for clinical use. OBJECTIVE: We describe protocols that use small molecules as direct conversion inducers, without the need for exogenous factors, to evaluate the potential of cell transdifferentiation for pharmacological and clinical applications. METHODS: In this systematic review, using PRISMA guidelines, we conducted a personalized search strategy in four databases (PubMed, Scopus, Embase, and Web Of Science), looking for experimental works that used exclusively small molecules for transdifferentiation of non-neural cell types into neural lineage cells. RESULTS: We explored the main biological mechanisms involved in direct cell conversion induced by different small molecules used in 33 experimental in vitro and in vitro transdifferentiation protocols. We also summarize the main characteristics of these protocols, such as the chemical cocktails used, time for transdifferentiation, and conversion efficiency. CONCLUSION: Small molecules-based protocols for neuronal transdifferentiation are reasonably safe, economical, accessible, and are a promising alternative for future use in regenerative medicine and pharmacology.

Amniotic fluid-derived stem cells: potential factories of natural and mimetic strategies for congenital malformations.

BACKGROUND: Mesenchymal stem cells (MSCs) derived from gestational tissues offer a promising avenue for prenatal intervention in congenital malformations although their application is hampered by concerns related to cellular plasticity and the need for invasive, high-risk surgical procedures. Here, we present naturally occurring exosomes (EXOs) isolated from amniotic fluid-derived MSCs (AF-MSCs) and their mimetic analogs (MIMs) as viable, reproducible, and stable alternatives. These nanovesicles present a minimally invasive therapeutic option, addressing the limitations of MSC-based treatments while retaining therapeutic efficacy. METHODS: MIMs were generated from AF-MSCs by combining sequential filtration steps through filter membranes with different porosity and size exclusion chromatography columns. A physicochemical, structural, and molecular comparison was conducted with exosomes (EXOs) released from the same batch of cells. Additionally, their distribution patterns in female mice were evaluated following in vivo administration, along with an assessment of their safety profile throughout gestation in a mouse strain predisposed to neural tube defects (NTDs). The possibility to exploit both formulations as mRNA-therapeutics was explored by evaluating cell uptake in two different cell types(fibroblasts, and macrophages) and mRNA functionality overtime in an in vitro experimental setting as well as in an ex vivo, whole embryo culture using pregnant C57BL6 dams. RESULTS: Molecular and physiochemical characterization showed no differences between EXOs and MIMs, with MIMs determining a threefold greater yield. Biodistribution patterns following intraperitoneal administration were comparable between the two particle types, with the uterus being among targeted organs. No toxic effects were observed in the dams during gestation, nor were there any malformations or significant differences in the number of viable versus dead fetuses detected. MIMs delivered a more intense and prolonged expression of mRNA encoding for green fluorescent protein in macrophages and fibroblasts. An ex-vivo whole embryo culture demonstrated that MIMs mainly accumulate at the level of the yolk sac, while EXOs reach the embryo. CONCLUSIONS: The present data confirms the potential application of EXOs and MIMs as suitable tools for prevention and treatment of NTDs and proposes MIMs as prospective vehicles to prevent congenital malformations caused by in utero exposure to drugs.

Fibroblast Growth Factor 8 enhances the chondrogenesis of trunk neural crest cells: a possible gene regulatory network.

The neural crest (NC) is an embryonic cell population with high migratory capacity. It contributes to forming several organs and tissues, such as the craniofacial skeleton and the peripheral nervous system of vertebrates. Both pre-migratory and post-migratory NC cells are plastic, adopting multiple differentiation paths by responding to different inductive environmental signals. Cephalic neural crest cells (CNCCs) give rise to most of the cartilage and bone tissues in the head. On the other hand, the mesenchymal potential of trunk neural crest cells (TNCCs) is sparsely detected in some animal groups. The mesenchymal potential of TNCCs can be unveiled through specific environmental conditions of NC cultures. In this study, we present evidence that FGF8 treatment can foster increased chondrogenic differentiation of TNCCs, particularly during treatment at the migratory stage. Additionally, we conducted a transcriptomic analysis of TNCCs in the post-migratory stage, noting that exogenous FGF8 signaling can sustain multipotent status and, possibly, at the same time, a pro-cartilage regulatory gene network. Our results provide a more comprehensive understanding of the mechanisms underlying chondrogenic differentiation from TNCCs.

[Association between alterations in the expanded lymphoid profileby flow cytometry and inborn errors of immunity]. / Asociación entre alteraciones del perfil linfoide ampliado por citometría de flujo y errores innatos de la inmunidad.

OBJECTIVE: To evaluate the association between the expanded lymphoid profile and inborn errors of immunity using flow cytometry. METHODS: Observational and cross-sectional, case-control study, carried out in patients with a diagnosis or clinical suspicion of inborn errors of immunity, treated at the Santísima Trinidad Children's Hospital in Córdoba, Argentina, from August 2021 to November 2022. Clinical data were collected, and peripheral blood samples were obtained for flow cytometry analysis, using the PIDOT tube, to identify lymphocyte subpopulations. For statistical analysis, Fisher's exact test, odds ratio and binary logistic regression model were used. RESULTS: 40 cases and 20 controls were analyzed. The most frequently altered lymphocyte subpopulations were: CD4+ n (63%), Mem c/s (60%) and Mem s/s (55%). A statistically significant association was found between several lymphocyte subpopulations and health-disease status. Binary logistic regression reported Mem s/s and CD4+n as altered lymphocyte subpopulations with a greater probability to have inborn errors of immunity. CONCLUSIONS: This study contributes to improving the understanding of inborn errors of immunity and demonstrates a strong association with altered lymphocyte subpopulation profiles. Mem s/s and CD4+n emerge as relevant biomarkers for diagnosis. Heterogeneity in different diseases and in flow cytometry underlines the importance of evaluating each patient individually, to improve diagnosis and treatment.

OBJETIVO: Evaluar la asociación entre el perfil linfoide ampliado y los errores innatos de la inmunidad mediante citometría de flujo. MÉTODOS: Estudio observacional y transversal, de casos y controles, llevado a cabo en pacientes con diagnóstico o sospecha clínica de errores innatos de la inmunidad, atendidos en el Hospital de Niños de la Santísima Trinidad de Córdoba, Argentina, de agosto de 2021 a noviembre de 2022. Se recolectaron los datos clínicos y se obtuvieron muestras de sangre periférica para el análisis por citometría de flujo, mediante el tubo PIDOT, para identificar subpoblaciones de linfocitos alteradas. Para el análisis estadístico se utilizó la prueba exacta de Fisher, razón de momios y modelo de regresión logística binaria. RESULTADOS: Se analizaron 40 casos y 20 controles. Las subpoblaciones de linfocitos más frecuentemente alteradas fueron: CD4+ n (63%), Mem c/s (60%) y Mem s/s (55%). Se encontró asociación estadísticamente significativa entre varias subpoblaciones de linfocitos y el estado de salud-enfermedad. La regresión logística binaria reportó Mem s/s y CD4+n como las subpoblaciones de linfocitos alteradas con mayor probabilidad de padecer errores innatos de la inmunidad. CONCLUSIONES: Este estudio contribuye a mejorar la comprensión de los errores innatos de la inmunidad, y demuestra una sólida asociación con los perfiles de subpoblaciones de linfocitos alteradas. Los Mem s/s y CD4+n emergen como biomarcadores relevantes para su diagnóstico. La heterogeneidad en diferentes enfermedades y en la citometría de flujo subraya la importancia de evaluar a cada paciente individualmente, para mejorar el diagnóstico y tratamiento.

Cotton cellulose nanofiber/chitosan scaffolds for skin tissue engineering and wound healing applications.

Chitosan (CS) is a promising polymeric biomaterial for use in scaffolds forin vitroskin models and wound dressings, owing to its non-antigenic and antimicrobial properties. However, CS often exhibits insufficient physicochemical properties, mechanical strength, and bioactivity, limiting its efficacy in demanding applications. To address these challenges, cotton cellulose nanofibers (CNFs) represent a promising nanomaterial for enhancing CS-based scaffolds in tissue engineering. CNF offers superior stiffness, and mechanical properties that enhance cellular adhesion and proliferation, both crucial for effective tissue regeneration and healing. This study aimed to develop and characterize a scaffold combining cotton CNF and CS, focusing on its cytocompatibility with human fibroblasts and keratinocytes. The cotton CNF/CS scaffold was fabricated using the casting technique, and its physicochemical properties and cellular compatibility were assessedin vitro. The results demonstrated that incorporating cotton CNF significantly enhanced the stability of the CS matrix. The CS scaffold with 1000 µg ml-1of cotton CNF exhibited increased roughness and reduced rupture strain compared to the pure CS scaffold. The cotton CNF/CS scaffold effectively promoted the adhesion, viability, proliferation, migration, and collagen synthesis of skin cells. Notably, increased cell viability was observed in human fibroblasts cultured on scaffolds with higher concentrations of cotton CNF (100 and 1000 µg ml-1). Based on the findings, the cotton CNF/CS scaffold demonstrates enhanced physicochemical properties and bioactivity, making it a promising candidate for the development ofin vitrohuman skin models and wound healing dressings.

Modeled grid cells aligned by a flexible attractor.

Entorhinal grid cells implement a spatial code with hexagonal periodicity, signaling the position of the animal within an environment. Grid maps of cells belonging to the same module share spacing and orientation, only differing in relative two-dimensional spatial phase, which could result from being part of a two-dimensional attractor guided by path integration. However, this architecture has the drawbacks of being complex to construct and rigid, path integration allowing for no deviations from the hexagonal pattern such as the ones observed under a variety of experimental manipulations. Here, we show that a simpler one-dimensional attractor is enough to align grid cells equally well. Using topological data analysis, we show that the resulting population activity is a sample of a torus, while the ensemble of maps preserves features of the network architecture. The flexibility of this low dimensional attractor allows it to negotiate the geometry of the representation manifold with the feedforward inputs, rather than imposing it. More generally, our results represent a proof of principle against the intuition that the architecture and the representation manifold of an attractor are topological objects of the same dimensionality, with implications to the study of attractor networks across the brain.

Follistatin like-1 (Fstl1) regulates adipose tissue development in zebrafish.

Obesity is a highly prevalent disorder with complex aetiology. Therefore, studying its associated cellular and molecular pathways may be aided by analysing genetic tractable diseases. In this context, the study of ciliopathies such as Bardet-Biedl syndrome has highlighted the relevance of primary cilia in obesity, both in the central nervous system and peripheral tissues. Based on our previous in vitro results supporting the role of a novel Bbs4-cilia-Fstl1 axis in adipocyte differentiation, we evaluated the in vivo relevance of the zebrafish orthologous genes fstl1a and fstl1b in primary cilia and adipose tissue development. Using a combination of knockdowns and a new fstl1a mutant line, we show that fstl1a promotes primary cilia formation in early embryos and participates in adipose tissue formation in larvae. We also show that fstl1b partially compensates for the loss of fstl1a. Moreover, in high fat diet, fstl1a depletion affects the expression of differentiation and mature adipocyte markers. These results agree with our previous in vitro data and provide further support for the role of FSTL1 as a regulator of adipose tissue formation. Dissecting the exact biological role of proteins such as FSTL1 will likely contribute to understand obesity onset and presentation.

GLUT1 and prorenin receptor mediate differential regulation of TGF-ß and CTGF in renal inner medullary collecting duct cells during high glucose conditions.

BACKGROUND: During diabetes, prorenin is highly produced by the renal collecting ducts. The binding of prorenin to (pro)renin receptor (PRR) on the apical plasma membrane triggers intracellular profibrotic genes, including TGF-ß and CTGF. However, the underlying mechanisms contributing to the stimulation of these pathways remain unclear. Hence, we hypothesize that the glucose transporter-1 (GLUT1) favors the PRR-dependent stimulation of TGF-ß and CTGF in the distal nephron segments during high glucose (HG) conditions. METHODS: To test this hypothesis, primary cultured renal inner medullary collecting duct (IMCD) cells were treated with normal glucose (NG, 5 mM) or high glucose (HG, 25 mM) for 48 h in the presence or absence of the GLUT1-specific inhibitor BAY 876 (2 nM). Additionally, IMCD cells were treated with the PRR antagonist PRO20. The expression of TGF-ß and CTGF was quantified by immunoblot and qRT-PCR. RESULTS: HG increased GLUT1 mRNA and protein abundance, while BAY 876 inhibited these responses. HG treatment upregulated PRR, but the concomitant treatment with BAY 876 partially prevented this effect. TGF-ß and CTGF expressions were augmented in IMCD cells treated with HG. However, PRO20 prevented the increases in TGF-ß but not those of CTGF. GLUT1 inhibition partially prevented the increases in reactive oxygen species (ROS) during HG while PRO20 did not. ROS scavenging impaired CTGF upregulation during HG conditions. Additionally, long-term exposure to HG increases lipid peroxidation and reduced cell viability. CONCLUSIONS: The data indicate that glucose transportation via GLUT1 is implicated in the PRR-dependent upregulation of TGF-ß while CTGF is mediated mainly via a mechanism depending on ROS formation in renal medullary collecting duct cells.

Possible immunomodulatory role of Filifactor alocis through beta-defensin 2 in gingival keratinocytes.

OBJECTIVES: The present study aimed to investigate a possible immunomodulatory role of the periodontopathogen Filifactor alocis through the antimicrobial peptide hBD-2 on the expression of chemokines in human gingival keratinocytes. MATERIALS AND METHODS: Cells were cultured in the presence or absence of periodontopathogenic bacteria, such as F. alocis, Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, and Treponema denticola, to evaluate the regulation of hBD-2, CXCL8 and CCL20. Furthermore, the cells were exposed or not to hBD-2 and the expression of CXCL8 and CCL20 and their receptors was evaluated. RESULTS: All bacteria induced a significant upregulation of hBD-2, CXCL8, and CCL20 gene expressions. In addition, F. alocis significantly increased their protein levels, as detected by ELISA. Pre-incubation of the cells with the TLR2 inhibitor resulted in a significant downregulation of hBD-2 expression in F. alocis-treated cells. Gingival keratinocytes exposed to hBD-2 resulted in a significant and dose-dependent increase of all chemokines and their receptors. CONCLUSIONS: F. alocis increased the production of chemotactic cytokines, suggesting an increase in the recruitment of immunoinflammatory cells in periodontal diseases. The chemotaxis-promoting effect is partly direct, but is also mediated via hBD-2. F. alocis stimulates the synthesis of hBD-2, which in turn could promote the expression and synthesis of these chemokines and their receptors. In addition, hBD-2 has an autostimulatory effect and stimulates the synthesis of these chemokines, so that the chemotaxis triggered by F. alocis is further fueled. CLINICAL RELEVANCE: F. alocis and hBD-2 have a significant role in periodontitis, showing their importance for diagnostic and treatment approaches.

Combined catalytic strategies applied to in-office tooth bleaching: whitening efficacy, cytotoxicity, and gene expression of human dental pulp cells in a 3D culture model.

OBJECTIVES: To evaluate the feasibility of using a 3D model with human dental pulp cells (HDPCs) to compare bleaching therapies and assess whether coating enamel with a nanofiber scaffold (NS) and polymeric catalyst primer (PCP), combined with violet LED (LEDv) irradiation, enhances bleaching efficacy (BE) and reduces cytotoxicity (CT). MATERIALS AND METHODS: After using NS + PCP to cover enamel of enamel/dentin discs adapted to artificial pulp chambers containing 3D culture with HDPCs, a bleaching gel with 35%H2O2 was applied and then irradiated with LEDv. The following groups were established (n = 8): NC - no treatment; PC- 35%H2O2 for 45 min, and EXP: NS + PCP + 35%H2O2 + LEDv for 15 min. The study evaluated BE (ΔE00 and ΔWID), CT (alamarBlue), and HDPCs gene modulation (TNF, IL1B, PTGS2, IL8, IL6, PPRAG, HMOX1, DSPP, DMP1, SPP1, BGLAP, and ALPL; RTqPCR). RESULTS: BE showed no significant difference between PC and EXP (p > 0.05). EXP had lower oxidative stress, higher cell viability, reduced inflammatory marker expression, and increased mineralization marker expression compared to PC (p < 0.05). CONCLUSION: The 3D model using HDPCs effectively compared bleaching protocols. Coating enamel with NS + PCP and applying violet LED (LEDv) reduced bleaching time from 45 to 15 min while lowering cytotoxicity compared to conventional in-office treatments. CLINICAL RELEVANCE: This study shows that a 3D model with human dental pulp cells (HDPCs) effectively compares tooth bleaching protocols. The combination of a nanofiber scaffold with violet LED (LEDv) reduces bleaching time, as well as minimizes cytotoxicity and inflammation, offering a safer alternative to conventional treatments.