-
Molecular Neurobiology Aug 2023The study of psychiatric and neurological diseases requires the substrate in which the disorders occur, that is, the nervous tissue. Currently, several types of human...
The study of psychiatric and neurological diseases requires the substrate in which the disorders occur, that is, the nervous tissue. Currently, several types of human bio-specimens are being used for research, including postmortem brains, cerebrospinal fluid, induced pluripotent stem (iPS) cells, and induced neuronal (iN) cells. However, these samples are far from providing a useful predictive, diagnostic, or prognostic biomarker. The olfactory epithelium is a region close to the brain that has received increased interest as a research tool for the study of brain mechanisms in complex neuropsychiatric and neurological diseases. The olfactory sensory neurons are replaced by neurogenesis throughout adult life from stem cells on the basement membrane. These stem cells are multipotent and can be propagated in neurospheres, proliferated in vitro and differentiated into multiple cell types including neurons and glia. For all these reasons, olfactory epithelium provides a unique resource for investigating neuronal molecular markers of neuropsychiatric and neurological diseases. Here, we describe the isolation and culture of human differentiated neurons and glial cells from olfactory epithelium of living subjects by an easy and non-invasive exfoliation method that may serve as a useful tool for the research in brain diseases.
Topics: Humans; Basement Membrane; Biomarkers; Cell Adhesion; Cell Culture Techniques; Cell Differentiation; Cell Proliferation; Cell Separation; Cells, Cultured; Culture Media; Flow Cytometry; Immunohistochemistry; Magnetics; Neural Stem Cells; Neurogenesis; Neuroglia; Neurons; Olfactory Mucosa; Organ Specificity
PubMed: 37118325
DOI: 10.1007/s12035-023-03363-2 -
Animal Bioscience Nov 2023The number of bovine mammary epithelial cells (BMECs) is closely associated with the quantity of milk production in dairy cows; however, the optimal levels and the...
OBJECTIVE
The number of bovine mammary epithelial cells (BMECs) is closely associated with the quantity of milk production in dairy cows; however, the optimal levels and the combined effects of hormones and essential amino acids (EAAs) on cell proliferation are not completely understood. Thus, the purpose of this study was to determine the optimal combination of individual hormones and EAAs for cell proliferation and related signaling pathways in BMECs.
METHODS
Immortalized BMECs (MAC-T) were treated with six hormones (insulin, cortisol, progesterone, estrone, 17β-estradiol, and epidermal growth factor) and ten EAAs (arginine, histidine, leucine, isoleucine, threonine, tryptophan, lysine, methionine, phenylalanine, and valine) for 24 h.
RESULTS
Cells were cultured in a medium containing 10% fetal bovine serum (FBS) as FBS supplemented at a concentration of 10% to 50% showed a comparable increase in cell proliferation rate. The optimized combination of four hormones (insulin, cortisol, progesterone, and 17β-estradiol) and 20% of a mixture of ten EAAs led to the highest cell proliferation rate, which led to a significant increase in cell cycle progression at the S and G2/M phases, in the protein levels of proliferating cell nuclear antigen and cyclin B1, cell nucleus staining, and in cell numbers.
CONCLUSION
The optimal combination of hormones and EAAs increased BMEC proliferation by enhancing cell cycle progression in the S and G/2M phases. Our findings indicate that optimizing hormone and amino acid levels has the potential to enhance milk production, both in cell culture settings by promoting increased cell numbers, and in dairy cows by regulating feed intake.
PubMed: 37641829
DOI: 10.5713/ab.23.0199 -
Current Issues in Molecular Biology Dec 2023Limbal epithelial stem/progenitor cells (LESCs) proliferate, migrate and differentiate into mature corneal epithelium cells (CECs) that cover the ocular surface. LESCs...
Limbal epithelial stem/progenitor cells (LESCs) proliferate, migrate and differentiate into mature corneal epithelium cells (CECs) that cover the ocular surface. LESCs play a crucial role in the maintenance and regeneration of the corneal epithelium, and their dysfunction can lead to various corneal diseases. Neuregulin 1 (NRG1) is a member of the epidermal growth factor family that regulates the growth and differentiation of epithelial tissues. Here, we depicted the dynamic transcriptomic profiles during human CEC differentiation, identifying six gene co-expression modules that were specific to different differentiation stages. We found that the expression of was high in human LESCs and decreased dramatically upon differentiation. Knockdown of significantly inhibited LESC proliferation and upregulated the expression of the terminal differentiation marker genes , and . In addition, the scratch wound closure assay showed that knockdown of attenuated wound closure of LESCs over 24 h. Together, we dissected the transcriptional regulatory dynamics during CEC differentiation and identified NRG1 as a key regulator that promoted LESC proliferation and migration and maintained the undifferentiated state.
PubMed: 38132478
DOI: 10.3390/cimb45120632 -
Circulation Jun 2024Endothelial cell (EC) apoptosis and proliferation of apoptosis-resistant cells is a hallmark of pulmonary hypertension (PH). Yet, why some ECs die and others proliferate...
BACKGROUND
Endothelial cell (EC) apoptosis and proliferation of apoptosis-resistant cells is a hallmark of pulmonary hypertension (PH). Yet, why some ECs die and others proliferate and how this contributes to vascular remodeling is unclear. We hypothesized that this differential response may: (1) relate to different EC subsets, namely pulmonary artery (PAECs) versus microvascular ECs (MVECs); (2) be attributable to autophagic activation in both EC subtypes; and (3) cause replacement of MVECs by PAECs with subsequent distal vessel muscularization.
METHODS
EC subset responses to chronic hypoxia were assessed by single-cell RNA sequencing of murine lungs. Proliferative versus apoptotic responses, activation, and role of autophagy were assessed in human and rat PAECs and MVECs, and in precision-cut lung slices of wild-type mice or mice with endothelial deficiency in the autophagy gene (). Abundance of PAECs versus MVECs in precapillary microvessels was assessed in lung tissue from patients with PH and animal models on the basis of structural or surface markers.
RESULTS
In vitro and in vivo, PAECs proliferated in response to hypoxia, whereas MVECs underwent apoptosis. Single-cell RNA sequencing analyses support these findings in that hypoxia induced an antiapoptotic, proliferative phenotype in arterial ECs, whereas capillary ECs showed a propensity for cell death. These distinct responses were prevented in hypoxic mice or after silencing, yet replicated by autophagy stimulation. In lung tissue from mice, rats, or patients with PH, the abundance of PAECs in precapillary arterioles was increased, and that of MVECs reduced relative to controls, indicating replacement of microvascular by macrovascular ECs. EC replacement was prevented by genetic or pharmacological inhibition of autophagy in vivo. Conditioned medium from hypoxic PAECs yet not MVECs promoted pulmonary artery smooth muscle cell proliferation and migration in a platelet-derived growth factor-dependent manner. Autophagy inhibition attenuated PH development and distal vessel muscularization in preclinical models.
CONCLUSIONS
Autophagic activation by hypoxia induces in parallel PAEC proliferation and MVEC apoptosis. These differential responses cause a progressive replacement of MVECs by PAECs in precapillary pulmonary arterioles, thus providing a macrovascular context that in turn promotes pulmonary artery smooth muscle cell proliferation and migration, ultimately driving distal vessel muscularization and the development of PH.
PubMed: 38873770
DOI: 10.1161/CIRCULATIONAHA.124.068726 -
Trends in Cell Biology Apr 2024The retinoblastoma protein (RB)-mediated regulation of E2F is a component of a highly conserved cell cycle machine. However, RB's tumor suppressor activity, like RB's... (Review)
Review
The retinoblastoma protein (RB)-mediated regulation of E2F is a component of a highly conserved cell cycle machine. However, RB's tumor suppressor activity, like RB's requirement in animal development, is tissue-specific, context-specific, and sometimes appears uncoupled from cell proliferation. Detailed new information about RB's genomic distribution provides a new perspective on the complexity of RB function, suggesting that some of its functional specificity results from context-specific RB association with chromatin. Here we summarize recent evidence showing that RB targets different types of chromatin regulatory elements at different cell cycle stages. RB controls traditional RB/E2F targets prior to S-phase, but, when cells proliferate, RB redistributes to cell type-specific chromatin loci. We discuss the broad implications of the new data for RB research.
Topics: Animals; Chromatin; E2F Transcription Factors; Cell Cycle; Retinoblastoma Protein; Cell Division
PubMed: 37648594
DOI: 10.1016/j.tcb.2023.07.012 -
Cells Feb 2024proliferates by budding, which includes the formation of a cytoplasmic protrusion called the 'bud', into which DNA, RNA, proteins, organelles, and other materials are... (Review)
Review
proliferates by budding, which includes the formation of a cytoplasmic protrusion called the 'bud', into which DNA, RNA, proteins, organelles, and other materials are transported. The transport of organelles into the growing bud must be strictly regulated for the proper inheritance of organelles by daughter cells. In yeast, the RING-type E3 ubiquitin ligases, Dma1 and Dma2, are involved in the proper inheritance of mitochondria, vacuoles, and presumably peroxisomes. These organelles are transported along actin filaments toward the tip of the growing bud by the myosin motor protein, Myo2. During organelle transport, organelle-specific adaptor proteins, namely Mmr1, Vac17, and Inp2 for mitochondria, vacuoles, and peroxisomes, respectively, bridge the organelles and myosin. After reaching the bud, the adaptor proteins are ubiquitinated by the E3 ubiquitin ligases and degraded by the proteasome. Targeted degradation of the adaptor proteins is necessary to unload vacuoles, mitochondria, and peroxisomes from the actin-myosin machinery. Impairment of the ubiquitination of adaptor proteins results in the failure of organelle release from myosin, which, in turn, leads to abnormal dynamics, morphology, and function of the inherited organelles, indicating the significance of proper organelle unloading from myosin. Herein, we summarize the role and regulation of E3 ubiquitin ligases during organelle inheritance in yeast.
Topics: Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Ubiquitin-Protein Ligases; Peroxisomes; Myosins; Ubiquitins; Cell Cycle Proteins; Mitochondrial Proteins
PubMed: 38391905
DOI: 10.3390/cells13040292 -
Cellular and Molecular Life Sciences :... Jul 2023Insulin deficiency may be due to the reduced proliferation capacity of islet β-cell, contributing to the onset of diabetes. It is therefore imperative to investigate...
Insulin deficiency may be due to the reduced proliferation capacity of islet β-cell, contributing to the onset of diabetes. It is therefore imperative to investigate the mechanism of the β-cell regeneration in the islets. NKX6.1, one of the critical β-cell transcription factors, is a pivotal element in β-cell proliferation. The ubiquitin-binding enzyme 2C (UBE2C) was previously reported as one of the downstream molecules of NKX6.1 though the exact function and mechanism of UBE2C in β-cell remain to be elucidated. Here, we determined a subpopulation of islet β-cells highly expressing UBE2C, which proliferate actively. We also discovered that β-cell compensatory proliferation was induced by UBE2C via the cell cycle renewal pathway in weaning and high-fat diet (HFD)-fed mice. Moreover, the reduction of β-cell proliferation led to insulin deficiency in βUbe2cKO mice and, therefore, developed type 2 diabetes. UBE2C was found to regulate PER1 degradation through the ubiquitin-proteasome pathway via its association with a ubiquitin ligase, CUL1. PER1 inhibition rescues UBE2C knockout-induced β-cell growth inhibition both in vivo and in vitro. Notably, overexpression of UBE2C via lentiviral transduction in pancreatic islets was able to relaunch β-cell proliferation in STZ-induced diabetic mice and therefore partially alleviated hyperglycaemia and glucose intolerance. This study indicates that UBE2C positively regulates β-cell proliferation by promoting ubiquitination and degradation of the biological clock suppressor PER1. The beneficial effect of UBE2C on islet β-cell regeneration suggests a promising application in treating diabetic patients with β-cell deficiency.
Topics: Animals; Mice; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 2; Insulin; Insulin-Secreting Cells; Islets of Langerhans; Ubiquitins
PubMed: 37486389
DOI: 10.1007/s00018-023-04868-8 -
Journal of Neuroinflammation Oct 2023Childhood represents a period of significant growth and maturation for the brain, and is also associated with a heightened risk for mild traumatic brain injuries (mTBI)....
Childhood represents a period of significant growth and maturation for the brain, and is also associated with a heightened risk for mild traumatic brain injuries (mTBI). There is also concern that repeated-mTBI (r-mTBI) may have a long-term impact on developmental trajectories. Using an awake closed head injury (ACHI) model, that uses rapid head acceleration to induce a mTBI, we investigated the acute effects of repeated-mTBI (r-mTBI) on neurological function and cellular proliferation in juvenile male and female Long-Evans rats. We found that r-mTBI did not lead to cumulative neurological deficits with the model. R-mTBI animals exhibited an increase in BrdU + (bromodeoxyuridine positive) cells in the dentate gyrus (DG), and that this increase was more robust in male animals. This increase was not sustained, and cell proliferation returning to normal by PID3. A greater increase in BrdU + cells was observed in the dorsal DG in both male and female r-mTBI animals at PID1. Using Ki-67 expression as an endogenous marker of cellular proliferation, a robust proliferative response following r-mTBI was observed in male animals at PID1 that persisted until PID3, and was not constrained to the DG alone. Triple labeling experiments (Iba1+, GFAP+, Brdu+) revealed that a high proportion of these proliferating cells were microglia/macrophages, indicating there was a heightened inflammatory response. Overall, these findings suggest that rapid head acceleration with the ACHI model produces an mTBI, but that the acute neurological deficits do not increase in severity with repeated administration. R-mTBI transiently increases cellular proliferation in the hippocampus, particularly in male animals, and the pattern of cell proliferation suggests that this represents a neuroinflammatory response that is focused around the mid-brain rather than peripheral cortical regions. These results add to growing literature indicating sex differences in proliferative and inflammatory responses between females and males. Targeting proliferation as a therapeutic avenue may help reduce the short term impact of r-mTBI, but there may be sex-specific considerations.
Topics: Humans; Rats; Female; Male; Animals; Child; Brain Concussion; Bromodeoxyuridine; Rats, Long-Evans; Head Injuries, Closed; Cell Proliferation; Inflammation
PubMed: 37907981
DOI: 10.1186/s12974-023-02916-5 -
Frontiers in Immunology 2023Type 1 diabetes (T1D) affects three million Americans, with 80 new people diagnosed each day. T1D is currently uncurable and there is an urgent need to develop...
Type 1 diabetes (T1D) affects three million Americans, with 80 new people diagnosed each day. T1D is currently uncurable and there is an urgent need to develop additional drug candidates to achieve the prevention of T1D. We propose AZD6738 (ATRi), an orally available drug currently in phases I and II of clinical trials for various cancers, as a novel candidate to prevent T1D. Based on previously reported findings of ATRi inducing cell death in rapidly proliferating T cells, we hypothesized that this drug would specifically affect self-antigen activated diabetogenic T cells. These cells, if left unchecked, could otherwise lead to the destruction of pancreatic β cells, contributing to the development of T1D. This work demonstrates that increasing the duration of ATRi treatment provides extended protection against T1D onset. Remarkably, 5-week ATRi treatment prevented T1D in a robust adoptive transfer mouse model. Furthermore, the splenocytes of animals that received 5-week ATRi treatment did not transfer immune-mediated diabetes, while the splenocytes from control animal transferred the disease in 10 days. This work shows that ATRi prevents T1D by specifically inducing cell death in self-antigen activated, highly proliferative diabetogenic T cells through the induction of DNA damage, resulting in the inhibition of IFNγ production and proliferation. These findings support the consideration of repurposing ATRi for T1D prevention.
Topics: Animals; Mice; Humans; Diabetes Mellitus, Type 1; Indoles; Antineoplastic Agents; Autoantigens; Neoplasms
PubMed: 38164129
DOI: 10.3389/fimmu.2023.1290058 -
ELife Nov 2023An imbalance of the gut microbiota, termed dysbiosis, has a substantial impact on host physiology. However, the mechanism by which host deals with gut dysbiosis to...
An imbalance of the gut microbiota, termed dysbiosis, has a substantial impact on host physiology. However, the mechanism by which host deals with gut dysbiosis to maintain fitness remains largely unknown. In , , which is its bacterial diet, proliferates in its intestinal lumen during aging. Here, we demonstrate that progressive intestinal proliferation of activates the transcription factor DAF-16, which is required for maintenance of longevity and organismal fitness in worms with age. DAF-16 up-regulates two lysozymes and , thus limiting the bacterial accumulation in the gut of worms during aging. During dysbiosis, the levels of indole produced by are increased in worms. Indole is involved in the activation of DAF-16 by TRPA-1 in neurons of worms. Our finding demonstrates that indole functions as a microbial signal of gut dysbiosis to promote fitness of the host.
Topics: Animals; Caenorhabditis elegans Proteins; Escherichia coli; Dysbiosis; Caenorhabditis elegans; Longevity; Bacteria; Indoles
PubMed: 37987602
DOI: 10.7554/eLife.85362