Number Of Proliferating Cells Research Articles

SoxB family genes delay regeneration and cause abnormal movement in Dugesia japonica.

SoxB subfamily is an important branch of Sox family and plays a key role in animal physiological process, but little is known about their function in planarian regeneration. This study aims to evaluate the function of DjSoxB family genes in intact and regenerating planarians Dugesia japonica. Here, we amplify the full-length cDNA of DjSoxB1 and DjSoxB2 in D. japonica by rapid amplification of the cDNA ends (RACE), detect the expression of DjSoxB family genes in planarian. The results show that DjSoxBs are expressed in parenchymal tissue and the hybridization signals partially disappear after irradiation indicates DjSoxB family genes are expressed in neoblasts. After the RNA interference (RNAi) of DjSoxB1, DjSoxB2 and DjSoxB3 separately, the numbers of proliferative cells are all reduced that causes planarians show slower growth of blastema in the early stage of regeneration, and nerves of planarians are affected that the movement speed of planarians decreases in varying degrees. Specially, planarians in the DjSoxB3 RNAi group show shrinkage and twisting. Overall, this study reveals that DjSoxB family genes play a role in cell proliferation during regeneration. They also play an important role in the maintenance of normal nerve function and nerve regeneration. These results provide directions for the functional study of SoxB family genes and provide an important foundation for planarian regeneration.

Neonatal epithelial reparative capacity is enhanced by acellular placental extract in porcine models

Necrotizing enterocolitis (NEC) is a devastating condition with a ~30% mortality rate in pre-term, very low birth-weight infants. Premature infants lack vital in utero ingestion of amniotic fluid which accelerates intestinal maturation and reduces inflammation. Therefore, an acellular placental extract (PE) akin to amniotic fluid may be therapeutic for NEC. The objective of this study was to evaluate PE’s capacity to accelerate repair following or prevent the induction of NEC-like injury. We hypothesized that PE would enhance neonatal porcine intestinal epithelial cells’ capacity to repair in vitro and in vivo. NEC was induced in 12 piglets via pre-term cesarean delivery and hyperosmolar formula feeding. Prior to formula feeding, piglets were nil per os or enterally supplemented with PE. Tissue, collected at euthanasia, up to 72H after formula initiation, was evaluated grossly and histologically and with immunohistochemical staining for proliferation (Ki67), stem cell identity (Sox9), and apoptosis (CC3). Numerical outputs were assessed with Kolmogorov-Smirnov testing. PE’s effect on epithelial restitution was assessed with scratch assays performed on confluent monolayers derived from ~1-day old piglet ileum. Monolayers were pre-treated with either actin polymerization inhibitor, Latrunculin A, or a cell cycle inhibitor, hydroxyurea. After scratch, either media or PE in media was applied. Scratch-closure was monitored for 24H; percentage-closure was calculated and analyzed using 2-way ANOVA and Tukey multiple comparison. Scratch-wound leading-edge cells were examined for EdU and Ki67 immunofluorescent positivity which was evaluated using Kolmogorov-Smirnov analysis. Tight junction recovery was assessed using identically derived confluent monolayers grown on Transwell inserts and exposed to hypoxia (18H, 1%O2, 5%CO2). Following hypoxia, PE or media alone was added to the apical chamber. Transepithelial electrical resistance (TEER) was measured every 6-12H for 48H using a symmetrical disc electrode chamber. TEER rate of change was analyzed using a two-way ANOVA. For all analyses, significance was set at p<0.05. In vivo PE administration resulted in reduced gross and histological NEC damage in treated compared to untreated piglets (p=0.02). Ki67 and Sox9 expression increased (p=0.71; p=0.93) and CC3 expression decreased with PE treatment (p=0.65). Evaluation of these markers in additional animals is pending. In vitro, PE expedited scratch wound closure (p<0.05) and expanded proliferative cell number along scratch edges (EdU/DAPI, p<0.05; Ki67+EdU/DAPI, p<0.01). Other analyses are pending. PE application prevented gross NEC development in vivo and accelerated recovery in vitro. NIH #R44HD100243; #T32562967-39806. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Cardiac manifestations of human ACTA2 variants recapitulated in a zebrafish model

The ACTA2 gene encodes actin α2, a major smooth muscle protein in vascular smooth muscle cells. Missense variants in the ACTA2 gene can cause inherited thoracic aortic diseases with characteristic symptoms, such as dysfunction of smooth muscle cells in the lungs, brain vessels, intestines, pupils, bladder, or heart. We identified a heterozygous missense variant of Gly148Arg (G148R) in a patient with a thoracic aortic aneurysm, dissection, and left ventricular non-compaction. We used zebrafish as an in vivo model to investigate whether or not the variants might cause functional or histopathological abnormalities in the heart. Following the fertilization of one-cell stage embryos, we injected in vitro synthesized ACTA2 mRNA of wild-type, novel variant G148R, or the previously known pathogenic variant Arg179His (R179H). The embryos were maintained and raised for 72 h post-fertilization for a heart analysis. Shortening fractions of heart were significantly reduced in both pathogenic variants. A histopathological evaluation showed that the myocardial wall of ACTA2 pathogenic variants was thinner than that of the wild type, and the total cell number within the myocardium was markedly decreased in all zebrafish with pathogenic variants mRNAs. Proliferating cell numbers were also significantly decreased in the endothelial and myocardial regions of zebrafish with ACTA2 variants compared to the wild type. These results demonstrate the effects of ACTA2 G148R and R179H on the development of left ventricle non-compaction and cardiac morphological abnormalities. Our study highlights the previously unknown significance of the ACTA2 gene in several aspects of cardiovascular development.

Abstract TMP112: Von Willebrand Factor (VWF) Modulates Smooth Muscle Cell Proliferation During the Outward Remodeling of Leptomeningeal Collateral Arterioles Following Stroke

Introduction: The leptomeningeal collaterals (LMCs) undergo outward remodeling after stroke to increase rescue flow toward the occluded region. We previously showed increased VWF in remodeled LMCs and others have shown that VWF can act as a smooth muscle cell (SMC) mitogen. Here we test the hypothesis that VWF modulates vascular remodeling of LMCs in the post-stroke brain. Methods: Permanent distal middle cerebral artery occlusion (pdMCAO) was performed in C57BL6 WT mice (cohort 1; 4 mos), aged (cohort 2; 24 mos) or VWF KO and WT male mice (cohort 3; 4 mos). At 3, 7, and 14 days after pdMCAO, the pial vessels were processed for VWF/Ki67/SMA immunofluorescence and morphometric analysis. Lastly, distribution of VWF was evaluated in LMCs from human and pig brains. Results: During the 3-14 days post-stroke, WT mice of cohort 1 demonstrated significantly increased VWF in ipsilateral LMCs as well as increased Ki67 positive cells. In ECs, Ki67-positive cells were 10.68 ± 1.48 and 10.73 ± 1.26 per mm of vessel at 3 and 7 days, respectively. In SMCs, Ki67-positive cells were 0.049 ± 0.22 and 4.546 ± 0.95 per mm vessel at 3 and 7 days, respectively. By 14 days, LMC no longer showed Ki67-positive ECs or SMCs. In aged cohort 2, along with attenuated VWF expression and remodeling, the Ki67-positive ECs and SMCs were significantly decreased in the aged mice compared to young mice (Ki67-positive ECs were reduced by 40% and 50% at 3 and 7 days, respectively; Ki67-positive SMCs were reduced by 75% at 7 days). In cohort 3, Ki67-positive SMCs were absent in the VWF-KOs at 3 days post-stroke. However, by 7 days, Ki67-positive SMCs were equivalent between WT and VWF-KOs (4.8-5.0 per mm vessel). Lastly, the presence of VWF was detected in the smooth muscle layer of LMCs of human and pig brains. Conclusions: Our study revealed increased VWF in the mural layer of remodeled LMCs and both EC and SMC proliferation. The remodeling response, VWF expression, and number of proliferative cells were attenuated in LMCs from aged brains. Knockout of VWF resulted in reduced LMC remodeling and delayed SMC proliferation. Our findings also provide evidence for VWF within the smooth muscle layer of pig and human brain arterioles, supporting the broader potential role of VWF as a molecular player in vascular remodeling.

Mechanisms of γδ T cell accumulation in visceral adipose tissue with aging.

γδ T cells are resident in visceral adipose tissue (VAT) where they show an age-associated increase in numbers and contribute to local and systemic chronic inflammation. However, regulation of this population and mechanisms for the age-dependent accumulation are not known. In this study, we identified a progressive trend of γδ T cell accumulation in VAT over the lifespan in mice and explored physiological mechanisms contributing to accumulation. Using isochronic parabiotic pairs of wild-type (WT) and T cell receptor delta knockout (TCRδ KO) mice at young and old age, we confirmed that VAT γδ T cells are predominately a tissue-resident population which is sustained in aging. Migration of peripheral γδ T cells into VAT was observed at less than 10%, with a decreasing trend by aging, suggesting a minor contribution of recruitment to γδ T cell accumulation with aging. Since tissue-resident T cell numbers are tightly regulated by a balance between proliferation and programmed cell death, we further explored these processes. Using in vivo EdU incorporation and the proliferation marker Ki67, we found that the absolute number of proliferating γδ T cells in VAT is significantly higher in the aged compared to young and middle-aged mice, despite a decline in the proportion of proliferating to non-proliferating cells by age. Analysis of apoptosis via caspase 3/7 activation revealed that VAT γδ T cells show reduced apoptosis starting at middle age and continuing into old age. Further, induction of apoptosis using pharmacological inhibitors of Bcl2 family proteins revealed that VAT γδ T cells at middle age are uniquely protected from apoptosis via a mechanism independent of traditional anti-apoptotic Bcl2-family proteins. Collectively, these data indicate that protection from apoptosis at middle age increases survival of tissue-resident γδ T cells resulting in an increased number of proliferative cells from middle age onward, and leading to the age-associated accumulation of γδ T cells in VAT. These findings are important to better understand how adipose tissue dysfunction and related changes in the immune profile contribute to inflammaging among the elderly.

Allicin inhibits the biological activities of cervical cancer cells by suppressing circEIF4G2.

Allicin is a safe herbal extract believed to have antitumor effects, which, however, remain unclear. The aim of the present work was to discuss Allicin antitumor effects on cervical cancer using cell experiments. Using Hela and Siha to our research objectives in our study, first step, difference concentration of Allicin (20, 40, and 80 μM) treated Hela and Siha cell lines, and next step, discuss circEIF4G2 effects in Allicin antitumor effects in Hela and Siha cell lines; the cell proliferation and EdU-positive cell number by CCK-8 and EdU staining; cell apoptosis rate by flow cytometry; invasion cell number by transwell assay; wound healing rate by wound healing assay; and relative mRNA and protein levels using qRT-PCR and WB assay. With Allicin supplement, the cell proliferation and EdU-positive cell number were significantly depressed with cell apoptosis rate significantly increasing; invasion cell number and wound healing rate significantly suppressed with circEIF4G2 mRNA expression significantly down-regulation (p < .05, respectively). However, there was no significant difference among Allicin, si-circEIF4G2, and Allicin+si-circEIF4G2 in cell biological activities including cell proliferation, apoptosis, invasion and migration, and relative gene and protein expression. Allicin depresses biological activities of cervical cancer cells through down-regulating circEIF4G2/HOXA1/AKT/mTOR.

The Construction of Foot and Ankle Joint by Autologous Oxygen-Releasing Nano-Bionic Scaffold Combined with Bone Marrow Mesenchymal Stem Cells

This study assesses the role of a scaffold constructed by co-cultivating autologous oxygen-releasing nano-bionic materials and bone marrow stromal cells (BMSCs) in joint repairing. A scaffold constructed of autologous oxygen-releasing nano-bionic materials and BMSCs was transplanted into SD rats. The immunofluorescence detected the expression of nuclear antigen (PCNA) and analyzed the proliferation of BMSCs. Reverse transcription polymerase chain reaction (RT-PCR) examined the expression of osteogenic markers and TUNEL staining analyzed BMSCs apoptosis. There was a significant difference in the apoptosis and proliferation cell number of BMSCs in ankle joint between solely autologous oxygen-releasing nano-bionic material scaffold group and composite BMSCs nano-bionic scaffold (P &lt;0.05). There was a statistical difference in PCNA and TUNEL expression between two groups at 4 weeks and 8 weeks (P &lt;0.05). The expression of osteogenesis markers in ankle joint at 1 week, 4 weeks, and 8 weeks were continuously reduced, and the composite of autologous oxygen-releasing nanomaterials and BMSCs increased the expression of osteogenic markers (P &lt;0.05). The composite scaffold constructed by autologous oxygen-releasing nano-bionic materials and BMSCs has a good two-way immune regulation function and is able to carry lipids, proteins, nutritional factors, and growth factors, which can effectively promote tissue engineering repairing and delay the scaffold degradation. Combined with the nano-release system to repair bone tissue, composite material can effectively promote the proliferation of joint osteoblasts and osteogenic differentiation, thus help repairing the ankle joint.

An Astyanax mexicanus mao knockout line uncovers the developmental roles of monoamine homeostasis in fish brain.

Monoaminergic systems are conserved in vertebrates, yet they present variations in neuroanatomy, genetic components and functions across species. MonoAmine Oxidase, or MAO, is the enzyme responsible for monoamine degradation. While mammals possess two genes, MAO-A and MAO-B, fish possess one single mao gene. To study the function of MAO and monoamine homeostasis on fish brain development and physiology, here we have generated a mao knockout line in Astyanax mexicanus (surface fish), by CRISPR/Cas9 technology. Homozygote mao knockout larvae died at 13 days post-fertilization. Through a time-course analysis, we report that hypothalamic serotonergic neurons undergo fine and dynamic regulation of serotonin level upon loss of mao function, in contrast to those in the raphe, which showed continuously increased serotonin levels - as expected. Dopaminergic neurons were not affected by mao loss-of-function. At behavioral level, knockout fry showed a transient decrease in locomotion that followed the variations in the hypothalamus serotonin neuronal levels. Finally, we discovered a drastic effect of mao knockout on brain progenitors proliferation in the telencephalon and hypothalamus, including a reduction in the number of proliferative cells and an increase of the cell cycle length. Altogether, our results show that MAO has multiple and varied effects on Astyanax mexicanus brain development. Mostly, they bring novel support to the idea that serotonergic neurons in the hypothalamus and raphe of the fish brain are different in nature and identity, and they unravel a link between monoaminergic homeostasis and brain growth.

Platelet-Rich Fibrin-Conditioned Medium as an Alternative to Fetal Bovine Serum Promotes Osteogenesis of Human Dental Pulp Stem Cells.

Human dental pulp stem cells (DPSCs) exhibit multilineage differentiation capabilities and superior clonogenic and proliferative properties. However, the use of animal-derived components such as FBS raises concerns regarding the clinical application of stem-cell-based therapies. Platelet-rich fibrin (PRF) derived from human blood is rich in fibrin, platelets, and growth factors and acts as a bioactive scaffold for grafting with biomaterials. In this study, we assessed the efficacy of PRF-conditioned medium (CM) in promoting DPSCs proliferation and osteogenic differentiation compared with the standard culture medium supplemented with FBS. A comparison of DPSCs cultured in FBS and PRF-CM revealed no differences in characteristics or morphology. However, cells cultured with PRF-CM exhibited inferior proliferation rates and cell numbers during passage in comparison with those cultured with FBS. In contrast, DPSCs cultured in PRF-CM showed significantly higher levels of calcification, and RT-PCR confirmed that the gene expression levels of markers associated with osteoblast differentiation were significantly increased. The PRF-CM approach offers a convenient, straightforward, and advantageous method for culturing DPSCs, without relying on animal-derived components. In summary, this study introduces a novel application of PRF-CM for enhancing the osteogenesis of DPSCs, which provides an alternative to FBS culture medium and addresses concerns associated with the use of animal-derived components in clinical settings.

TrpA1 is a shear stress mechanosensing channel regulating intestinal stem cell proliferation in Drosophila

Adult stem cells are essential for tissue maintenance and repair. Although genetic pathways for controlling adult stem cells are extensively investigated in various tissues, much less is known about how mechanosensing could regulate adult stem cells and tissue growth. Here, we demonstrate that shear stress sensing regulates intestine stem cell proliferation and epithelial cell number in adult Drosophila. Ca2+ imaging in ex vivo midguts shows that shear stress, but not other mechanical forces, specifically activates enteroendocrine cells among all epithelial cell types. This activation is mediated by transient receptor potential A1 (TrpA1), a Ca2+-permeable channel expressed in enteroendocrine cells. Furthermore, specific disruption of shear stress, but not chemical, sensitivity of TrpA1 markedly reduces proliferation of intestinal stem cells and midgut cell number. Therefore, we propose that shear stress may act as a natural mechanical stimulation to activate TrpA1 in enteroendocrine cells, which, in turn, regulates intestine stem cell behavior.

Glutamate alliviate the intestinal damage caused by chemotherapeutic agents

Background &amp; Aim: 5-fluorouracil (5-FU), a type of anticancer drug, induces enteritis with diarrhea in high frequency and significantly reduces the quality of life of patients. Though inhibition of cell growth, induction of apoptosis, and changes in the intestinal microflora have been reported in the pathogenesis of 5-FU-induced enteritis, efficient treatment and prevention have not yet been established. In recently years, the usefulness of amino acids for various gastrointestinal disorders has been reported. Glutamate is the most abundant nonessential amino acid in the diet and is involved in the regulation and maintenance of function of gastrointestinal tract. The purpose of this study is to examine the protective effect of glutamate against 5-FU-induced small intestinal inflammation and to propose new targets for prevention and treatment. Materials &amp; Methods: Intestinal mucositis was induced in male C57BL/6 mice by repeated administration of 5-FU (50 mg/kg, i.p.) for 6 days. glutamate was administered twice daily for 7 days prior to the start of 5-FU administration, and body weight and fecal status were measured daily during 5-FU administration. Twenty-four hours after the last dose of 5-FU, ileal tissue was removed for histological and immunohistological evaluation. The epithelial barrier function was also examined by using 4kDa FITC-labeled dextran (FD-4). Rat intestinal cell line (IEC-6) was also used to measure the epithelial barrier function. To analyze the factors that were involved in mucositis by 5-FU, Western blotting were performed. Results: Continuous administration of 5-FU was observed to cause weight loss and exacerbation of diarrhea. Administration of 5-FU shortened the villi, decreased the number of proliferative cells, induced apoptotic cells around the crypts, and reduced intestinal barrier function. Glutamate showed a protective effect against 5-FU-induced diarrhea, shortening of small intestinal villi, inhibition of proliferating cells and induction of apoptosis. The translocation of FD-4 into the blood was significantly increased by 5-FU, while glutamate had no significant effect. However, it clearly reduced FD-4 infiltration into ileal tissue. The effect of glutamate was associated with epithelial barrier function and the pretreatment of glutamate significantly recovered the decrease of epithelial electrical resistance by 5-FU. Moreover, glutamate inhibited the decrease of the expression of excitatory amino acid transporter 3 (EAAT3) by 5-FU. Conclusion: We conclude that glutamate prevents 5-FU-induced intestinal mucositis. It is suggested that glutamate improves epithelial damages caused by 5-FU through enhancing glutamate uptake and improves intestinal morphology and functions. Thus, glutamate may be helpful for preventing and treating intestinal mucositis during cancer chemotherapy. University This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

TMEM59 ablation leads to loss of olfactory sensory neurons and impairs olfactory functions via interaction with inflammation

The olfactory epithelium undergoes constant neurogenesis throughout life in mammals. Several factors including key signaling pathways and inflammatory microenvironment regulate the maintenance and regeneration of the olfactory epithelium. In this study, we identify TMEM59 (also known as DCF1) as a critical regulator to the epithelial maintenance and regeneration. Single-cell RNA-Seq data show downregulation of TMEM59 in multiple epithelial cell lineages with aging. Ablation of TMEM59 leads to apparent alteration at the transcriptional level, including genes associated with olfactory transduction and inflammatory/immune response. These differentially expressed genes are key components belonging to several signaling pathways, such as NF-κB, chemokine, etc. TMEM59 deletion impairs olfactory functions, attenuates proliferation, causes loss of both mature and immature olfactory sensory neurons, and promotes infiltration of inflammatory cells, macrophages, microglia cells and neutrophils into the olfactory epithelium and lamina propria. TMEM59 deletion deteriorates regeneration of the olfactory epithelium after injury, with significant reduction in the number of proliferative cells, immature and mature sensory neurons, accompanied by the increasing number of inflammatory cells and macrophages. Anti-inflammation by dexamethasone recovers neuronal generation and olfactory functions in the TMEM59-KO animals, suggesting the correlation between TMEM59 and inflammation in regulating the epithelial maintenance. Collectively, TMEM59 regulates olfactory functions, as well as neuronal generation in the olfactory epithelium via interaction with inflammation, suggesting a potential role in therapy against olfactory dysfunction associated with inflamm-aging.

Good Manufacturing Practice–compliant change of raw material in the manufacturing process of a clinically used advanced therapy medicinal product–a comparability study

The development of medicinal products often continues throughout the different phases of a clinical study and may require challenging changes in raw and starting materials at later stages. Comparability between the product properties pre- and post-change thus needs to be ensured. Here, we describe and validate the regulatory compliant change of a raw material using the example of a nasal chondrocyte tissue-engineered cartilage (N-TEC) product, initially developed for treatment of confined knee cartilage lesions. Scaling up the size of N-TEC as required for the treatment of larger osteoarthritis defects required the substitution of autologous serum with a clinical-grade human platelet lysate (hPL) to achieve greater cell numbers necessary for the manufacturing of larger size grafts. A risk-based approach was performed to fulfill regulatory requirements and demonstrate comparability of the products manufactured with the standard process (autologous serum) already applied in clinical indications and the modified process (hPL). Critical attributes with regard to quality, purity, efficacy, safety and stability of the product as well as associated test methods and acceptance criteria were defined. Results showed that hPL added during the expansion phase of nasal chondrocytes enhances proliferation rate, population doublings and cell numbers at passage 2 without promoting the overgrowth of potentially contaminant perichondrial cells. N-TEC generated with the modified versus standard process contained similar content of DNA and cartilaginous matrix proteins with even greater expression levels of chondrogenic genes. The increased risk for tumorigenicity potentially associated with the use of hPL was assessed through karyotyping of chondrocytes at passage 4, revealing no chromosomal changes. Moreover, the shelf-life of N-TEC established for the standard process could be confirmed with the modified process. In conclusion, we demonstrated the introduction of hPL in the manufacturing process of a tissue engineered product, already used in a late-stage clinical trial. Based on this study, the national competent authorities in Switzerland and Germany accepted the modified process which is now applied for ongoing clinical tests of N-TEC. The described activities can thus be taken as a paradigm for successful and regulatory compliant demonstration of comparability in advanced therapy medicinal products manufacturing.

Dose-Response Relationship between Head and Neck Radiation and Damages to Gustatory Cells in Mice.

To investigate the dose-response relationship between radiation to the head and neck regions and damage observed in mice gustatory cells. A total number of 45 mice (C57BL/6) (aged 8-12 weeks) were enrolled in this study. The head and neck regions of the mice were irradiated at doses of 8 Gy (low-dose group, n = 15), 16 Gy (moderate-dose group, n = 15), and 24 Gy (high-dose group, n = 15). Each time, 3 mice from each group were sacrificed before radiation and then 2-day, 4-day, 7-day, and 14-day post the irradiation, respectively. The immune-histochemical staining method was employed to obtain gustatory papilla tissues and mark gustatory cells. Careful calculation of the numbers of proliferative cells, taste buds, and type II gustatory cells was conducted. A decrease in the number of Ki-67-marked proliferative cells was noted at 2 days postirradiation (DPI), and the number of cells was recovered to the normal level at 4-DPI in each group. The number of Ki-67-marked proliferative cells was hypercompensation (significantly higher than normal) in the moderate-dose and high-dose groups at 7-DPI and insufficient compensation (significantly lower than normal) in the high-dose group at 14-DPI. There was a significant reduction of taste buds and type II gustatory cells at 2-DPI and is lowest at 4-DPI in the moderate-dose and high-dose groups, while little change was observed in the low-dose group. Damages to Gustatory Cells after head and neck radiation were dose-related and compensation occurred in 14-DPI and may be insufficient when overdosed.

Cisplatin attenuates taste cell homeostasis and induces inflammatory activation in the circumvallate papilla.

Rationale: Gustation is important to several biological functions in mammals. However, chemotherapy drugs often harm taste perception in cancer patients, while the underlying mechanism is still unclear for most drugs and there is no effective way to restore taste function. This study investigated the effects of cisplatin on the taste cell homeostasis and gustatory function. Methods: We used both mice and taste organoid models to study the effect of cisplatin on taste buds. Gustometer assay, gustatory nerve recording, RNA-Sequencing, quantitative PCR, and immunohistochemistry was performed to analyze the cisplatin-induced alteration in taste behavior and function, transcriptome, apoptosis, cell proliferation and taste cell generation. Results: Cisplatin inhibited proliferation and promoted apoptosis in the circumvallate papilla, leading to significant impairment in taste function and receptor cell generation. The transcriptional profile of genes associated with cell cycle, metabolic process and inflammatory response was significantly altered after cisplatin treatment. Cisplatin inhibited growth, promoted apoptosis, and deferred taste receptor cell differentiation in taste organoids. LY411575, a γ-secretase inhibitor, reduced the number of apoptotic cells and increased the number of proliferative cells and taste receptor cells, potentially suggesting as a taste tissue protective agent against chemotherapy. LY411575 treatment could offset the increased number of Pax1+ or Pycr1+ cells induced by cisplatin in the circumvallate papilla and taste organoids. Conclusion: This study highlights the inhibitory effects of cisplatin on taste cell homeostasis and function, identifies critical genes and biological processes regulated by chemotherapy, and proposes potential therapeutic targets and strategy for taste dysfunction in cancer patients.

Efficacy of probiotic Streptococcus thermophilus in counteracting TGF-β1-induced fibrotic response in normal human dermal fibroblasts

BackgroundAbnormal and deregulated skin wound healing associated with prolonged inflammation may result in dermal fibrosis. Since the current therapeutic strategies revealed unsatisfactory, the investigation of alternative approaches such as those based on the use of specific probiotic strains could provide promising therapeutic options. In this study, we aimed to evaluate whether the lysate from S. thermophilus could antagonize the fibrogenic effects of TGF-β1 in normal human dermal fibroblasts (NHDF).MethodsNHDF were exposed to TGF-β1 to establish a fibrotic phenotype. Proliferation rate and cell number were measured using the IncuCyte® Live Cell Imager system and the trypan blue dye exclusion test. Phenoconversion markers (α-SMA and fibronectin) and collagen I levels were assessed by western blot and immunofluorescence. The mRNA levels of TGF-β1 were evaluated by RT-PCR. The Smad2/3 phosphorylation level as well as β-catenin and PPARγ expression, were assessed by western blot. The cell contractility function and migration of NHDF were studied using collagen gel retraction assay, and scratch wound healing assay, respectively. The effects of S. thermophilus lysate, alone or combined with TGF-β1, were evaluated on all of the above-listed parameters and markers associated with TGF-β1-induced fibrotic phenotype.ResultsExposure to the S. thermophilus lysate significantly reduced the key mediators and events involved in the abnormal activation of myofibroblasts by TGF-β1 within the fibrotic profile. The S. thermophilus treatment significantly reduced cell proliferation, migration, and myo-differentiation. In addition, the treatment with probiotic lysate reduced the α-SMA, fibronectin, collagen-I expression levels, and affected the collagen contraction ability of activated dermal fibroblasts. Moreover, the probiotic targeted the TGF-β1 signaling, reducing Smad2/3 activation, TGF-β1 mRNA level, and β-catenin expression through the upregulation of PPARγ.ConclusionThis is the first report showing that S. thermophilus lysate had a remarkable anti-fibrotic effect in TGF-β1-activated NHDF by inhibiting Smad signaling. Notably, the probiotic was able to reduce β-catenin and increase PPARγ levels. The findings support our point that S. thermophilus may help prevent or treat hypertrophic scarring and keloids.

Mother cells control daughter cell proliferation in intestinal organoids to minimize proliferation fluctuations.

During renewal of the intestine, cells are continuously generated by proliferation. Proliferation and differentiation must be tightly balanced, as any bias toward proliferation results in uncontrolled exponential growth. Yet, the inherently stochastic nature of cells raises the question how such fluctuations are limited. We used time-lapse microscopy to track all cells in crypts of growing mouse intestinal organoids for multiple generations, allowing full reconstruction of the underlying lineage dynamics in space and time. Proliferative behavior was highly symmetric between sister cells, with both sisters either jointly ceasing or continuing proliferation. Simulations revealed that such symmetric proliferative behavior minimizes cell number fluctuations, explaining our observation that proliferating cell number remained constant even as crypts increased in size considerably. Proliferative symmetry did not reflect positional symmetry but rather lineage control through the mother cell. Our results indicate a concrete mechanism to balance proliferation and differentiation with minimal fluctuations that may be broadly relevant for other tissues.

Identification of Molecular Mechanisms Governing CAR-T Cell Response in MM Patients Using Single Cell Transcriptomics

Background: CAR-T therapies have revolutionized cancer immunotherapy, especially for hematological malignancies, representing a promising advanced therapy for relapsed/refractory (R/R) multiple myeloma (MM) patients. Nevertheless, despite the high remission rates observed with BCMA CAR-T cells, not every patient achieves a complete response, and a significant number of patients still relapse. Application of single-cell technologies to study CAR-T cells has helped to shed light on important aspects of their expansion and evolution after administration. Methodology: CAR-T cells were FACS-isolated from infusion products (IP) as well as from bone marrow (BM) and peripheral blood (PB) from three MM patients (HCB08, IBS08 and HVA02), enrolled in the academic clinical trial CARTBCMA-HCB-01 (NCT04309981) assessing ARI0002h, an autologous CAR T-cell product targeting BCMA1, at different times after cell infusion (1 and 3 months). Single-cell analysis was performed using the Chromium Single-Cell Immune Profiling solution from 10X Genomics that allows simultaneous analysis of gene expression and paired T-cell receptors. Gene Regulatory Network (GRN) analysis was performed using SimiC, a novel computational method developed by our group, that infers regulatory dissimilarities2. Phenotypic analyses were performed by multidimensional flow cytometry. Results: scRNAseq analysis revealed that although CAR-T cells from IP presented similar phenotype, with CD4+ and CD8+ memory cells, and a high number of proliferative cells, CAR-T cells remaining after cell infusion were mainly resting CD8+ cells, with an effector/effector-memory phenotype. In addition, cell type distribution varied among patients, with a population of terminally differentiated effector cells in HCB08 patient, that presented a partial response and an early relapse, that increased from month 1 to 3 and presented a more exhausted phenotype. In contrast, IBS08 and HVA02 (complete responders) presented CD8+ cells in transition to central memory phenotype and a relatively constant pool of effector memory CD8+ CAR-T cells, respectively. Interestingly, CAR-T cells infiltrating BM presented increased expression of cytotoxic (GZMA, PRF1) and exhaustion (LAG3, HAVCR2, TIGIT) markers, compared to their PB counterparts. Functional characterization showed enrichment of T cell activation and cell-cell adhesion pathways in CAR-T cells from BM. GRN analysis at single-cell level with SimiC showed several regulons, such as IRF3 and PRDM1, presenting increased activity in BM. IRF3, a key transcriptional regulator of type I interferon response, was associated to DUSP2, IL32 and CCL5, genes associated to lymphocyte activation and inflammation. PRDM1, with crucial role in maintaining effector T cells homeostasis, was correlated with CST7, PRF1, MAP4K1 and CYTOR, genes associated with effector function and proliferation. All these data suggest that these regulons would be modulating the increased activation and inflammatory response of CAR-T cells observed in the BM. Further analysis using single-cell TCRseq allowed the identification of a hyperexpanded CAR-T cell clone in the BM of HCB08 patient (with an early relapse). Deeper characterization showed that this clone had higher expression of cytotoxicity (PRF1, GZMB) and activation markers (HLA-DRA, TNFRSF9). Strikingly, we also found increased expression of IL10, an immunosuppressor cytokine. Further analysis of this clone with SimiC showed increased activity of regulons from TFs related to exhausted CD8+ T cells (CREM, BHLHE40), meanwhile regulons from TFs associated to naïve and memory T cells (FOXP1, KLF2) had reduced activity. Interestingly, PRDM1, that is implicated in IL10 production in Treg, also presented increased activity in this clone. These data would suggest mechanistic insights regulating the phenotype of this hyperexpanded clone, that could represent a mechanism of therapeutic failure and/or early relapse. Conclusions: Overall, our data show that multiomic analysis are useful tools to characterize CAR-T cells and their dynamics after infusion, that presented different characteristics regarding their location (PB vs BM) and a heterogeneous behavior within the patients. Moreover, GRN analysis at single cell level offers an important tool to understand mechanisms modulating CAR-T cell response and to identify possible mechanisms of relapse. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Early life nociceptive stimulus and fentanyl exposure increase hippocampal neurogenesis and anxiety but do not affect spatial learning and memory

This study aimed to determine whether preemptive fentanyl administration in neonatal rats reduces the impact of a nociceptive stimulus initiated during the first day of life (P1) on hippocampal neurogenesis, behavior, and learning. At P1, Wistar rat pups received either a subcutaneous injection of fentanyl (F) before intraplantar injection of complete Freund’s adjuvant (CFA) (CFA + F group), an isolated injection of CFA (CFA group), or subcutaneous injection of fentanyl without CFA injection (F). Control animals received saline injections using the same route and volume as the treatment groups. Hippocampal neurogenesis was evaluated by 5′ –bromo-2′-deoxyuridine (BrdU) staining on P10 and P39 to assess neuronal proliferation and survival, respectively. Anxiety behavior in adulthood was assessed using an open field test (OF) and an elevated plus maze test (EPM). Spatial memory was assessed on a Morris water maze test (MWM), where the animals were trained for seven days, beginning on P81, and the probe trial was performed to evaluate memory retention. Although the CFA + F group showed an increased number of proliferative cells on P10, this finding did not persist on P39. The CFA + F group spent more time in the closed arms in the EPM, revealing more anxious behavior, although the early noxious experience, both with and without fentanyl, did not alter neurogenesis in adolescence and learning in adulthood. This study highlights that the impact of pain in early life pain combined with fentanyl on hippocampal neurogenesis on P10 did not persist on P39. In addition, this combined intervention during the first week of life was associated with higher anxiety levels.

Pequi oil (Caryocar brasilense Cambess.) nanoemulsion alters cell proliferation and damages key organelles in triple-negative breast cancer cells in vitro

Pequi oil is extracted from the fruit of a Brazilian native plant (Caryocar brasiliense Camb) that contains some molecules with anticancer potential. Due to its hydrophobic property, the administration of pequi oil associated with nanoemulsion systems represents a successful strategy to improve oil bioavailability. Breast cancer is the most frequent type of cancer among women and conventional therapies used are frequently associated with several side effects. Thus, the aim of this study was to investigate the effects of pequi oil-based nanoemulsion (PeNE) on triple-negative breast cancer cells (4T1), in vitro. PeNE presented a dose- and time-dependent cytotoxic effect with lower IC50 than free pequi oil after 48 h of exposure (p < 0.001). At 180 µg/mL, PeNE demonstrated numerous cell alterations, when compared to free pequi oil, such as morphological alterations, reduction in cell proliferation and total cell number, damage to plasmatic membrane, induction of lysosomal membrane permeability and depolarization of mitochondrial membrane, alteration of intracellular ROS production and calcium level, and increase in phosphatidylserine exposure. Taken together, the results suggest an interesting induction of cell death mechanisms involving a combined action of factors that impair nucleus, mitochondria, lysosome, and ER function. In addition, more pronounced effects were observed in cells treated by PeNE at 180 µg/mL when compared to free pequi oil, thereby reinforcing the advantages of using nanometric platforms. These promising results highlight the use of PeNE as a potential complementary therapeutic approach to be employed along with conventional treatments against breast cancer in the future.