Within humanized mice (hu-mice), employing MTSRG and NSG-SGM3 strains, we focused on testing the capacity of endogenously-generated human NK cells to display tolerance towards HLA-edited iPSC-derived cells. The use of human interleukin-15 (hIL-15) and IL-15 receptor alpha (hIL-15R), in conjunction with the engraftment of cord blood-derived human hematopoietic stem cells (hHSCs), led to high NK cell reconstitution. HLA class I-null hiPSC-derived hematopoietic progenitor cells (HPCs), megakaryocytes, and T cells were rejected by hu-NK mice, while HLA-A/B-knockout, HLA-C expressing HPCs were not. To the best of our understanding, this investigation represents the initial instance of recapitulating the powerful inherent NK cell reaction to non-cancerous HLA class I-reduced cells within a living organism. Hu-NK mouse models are well-suited for the preclinical evaluation of HLA-altered cells, and promise to aid in the development of universal, readily available regenerative therapies.
The process of autophagy, induced by thyroid hormone (T3), and its profound biological implications have been intensely examined over the last few years. Despite this, only a limited quantity of studies have addressed the critical role that lysosomes play in the process of autophagy. This research meticulously investigated the impact of T3 on lysosomal protein expression and transport mechanisms. T3's action on the lysosomal system was characterized by a rapid enhancement of lysosomal turnover alongside an increased expression of several lysosomal genes, including TFEB, LAMP2, ARSB, GBA, PSAP, ATP6V0B, ATP6V0D1, ATP6V1E1, CTSB, CTSH, CTSL, and CTSS, a process controlled by thyroid hormone receptors. The specific induction of the LAMP2 protein was observed in mice with hyperthyroidism, utilizing a murine model. T3-mediated microtubule assembly was markedly disrupted by vinblastine, resulting in an accumulation of the lipid droplet protein, PLIN2. Significant accumulation of LAMP2 protein, but not LAMP1, was evident in our study when exposed to the lysosomal autophagy inhibitors, bafilomycin A1, chloroquine, and ammonium chloride. A subsequent enhancement of the protein levels of both ectopically expressed LAMP1 and LAMP2 was triggered by T3. In the case of LAMP2 knockdown, cavities within lysosomes and lipid droplets increased in the presence of T3, but variations in the expression of LAMP1 and PLIN2 were less conspicuous. To be more specific, the protective mechanism of T3 from ER stress-caused cell death was nullified upon downregulating LAMP2. Our collective research demonstrates that T3 induces lysosomal gene expression, accompanied by improvements in LAMP protein stability and microtubule structure, thereby leading to heightened lysosomal capacity in handling any accrued autophagosomal load.
The serotonin transporter (SERT) facilitates the reuptake of the neurotransmitter serotonin (5-HT) into serotonergic neurons. Antidepressants often target SERT, leading to a considerable amount of research exploring the diverse relationship between SERT and depression. Still, how SERT is regulated at the cellular level is not fully known. B022 We present the post-translational modulation of SERT by S-palmitoylation, a process that involves the covalent attachment of palmitate to cysteine residues on proteins. We noted S-palmitoylation of immature SERT molecules within AD293 cells, a human embryonic kidney 293-derived cell line with superior adhesion, following transient transfection with FLAG-tagged human SERT. These immature SERT proteins, bearing high-mannose N-glycans or no N-glycans, are presumed to be localized within the endoplasmic reticulum, a component of the early secretory pathway. Analysis of mutations using alanine substitutions reveals that S-palmitoylation of immature serotonin transporter (SERT) occurs at least at cysteine residues 147 and 155, which are juxtamembrane cysteines located within the first intracellular loop. Concomitantly, modifying Cys-147 reduced the cell's uptake of a fluorescent SERT substrate that mimics 5-HT, with no concurrent decrease in surface-bound SERT. In contrast, the combined modification of cysteine residues 147 and 155 resulted in decreased SERT localization on the cell surface and a decline in the uptake of the 5-hydroxytryptamine analog. Therefore, the palmitoylation of cysteine residues 147 and 155 within SERT is essential for both its presence on the cell membrane and its ability to absorb 5-hydroxytryptamine. B022 The significance of S-palmitoylation in brain stability underscores the potential of further examining SERT S-palmitoylation in discovering innovative solutions for depression.
Tumor-associated macrophages (TAMs) play a critical role in facilitating the progression of tumor formation. A growing body of research suggests a possible link between miR-210 and the progression of tumor virulence, but the pro-carcinogenic effect of miR-210 in primary hepatocellular carcinoma (HCC) and its potential relationship with M2 macrophages has not been explored.
M2-polarized macrophages, differentiated from THP-1 monocytes, were cultivated using phorbol myristate acetate (PMA) and IL-4, IL-13. The transfection of M2 macrophages involved the addition of miR-210 mimics or the addition of miR-210 inhibitors. Flow cytometry served as the method to identify macrophage markers and apoptosis. To determine the levels of autophagy within M2 macrophages and the expression of mRNAs and proteins associated with the PI3K/AKT/mTOR signaling pathway, quantitative real-time PCR and Western blotting were used. To investigate the impact of miR-210, secreted by M2 macrophages, on HepG2 and MHCC-97H HCC cell proliferation, migration, invasion, and apoptosis, M2 macrophage-conditioned medium was utilized for cell culture.
qRT-PCR measurements indicated a heightened expression of miR-210 specifically in M2 macrophages. Enhanced autophagy-related gene and protein expression was observed in M2 macrophages transfected with miR-210 mimics, while apoptosis-related proteins were downregulated. In the miR-210 mimic group, M2 macrophages exhibited an accumulation of MDC-labeled vesicles and autophagosomes, as visualized by MDC staining and transmission electron microscopy. The expression of the PI3K/AKT/mTOR signaling pathway in M2 macrophages was lowered by miR-210 mimic treatment. Enhanced proliferation and invasive capacity were observed in HCC cells co-cultured with M2 macrophages transfected with miR-210 mimics, contrasted with the control group, where apoptosis levels were decreased. Beyond this, the stimulation or inhibition of autophagy could respectively intensify or diminish the previously observed biological effects.
miR-210 triggers autophagy within M2 macrophages by way of the PI3K/AKT/mTOR signaling cascade. Autophagy, a process driven by M2 macrophage-derived miR-210, contributes to the progression of hepatocellular carcinoma (HCC), implying that macrophage autophagy could be a novel therapeutic target in HCC, and interventions aimed at miR-210 could potentially reverse the influence of M2 macrophages on HCC.
Through its involvement in the PI3K/AKT/mTOR signaling pathway, miR-210 encourages autophagy in M2 macrophages. M2 macrophage-derived miR-210 contributes to the malignant transformation of hepatocellular carcinoma (HCC) via autophagy. This implies that targeting macrophage autophagy could be a novel therapeutic strategy for HCC, and manipulating miR-210 might counteract the detrimental effects of M2 macrophages on HCC.
Chronic liver disease invariably leads to liver fibrosis, a condition characterized by an excessive buildup of extracellular matrix components, primarily due to the activation of hepatic stellate cells (HSCs). It has been demonstrated that HOXC8 participates in governing cell reproduction and fibrosis progression within the context of tumors. Despite this, the role of HOXC8 in liver fibrosis and the associated molecular underpinnings are currently unknown. In this study, we discovered that HOXC8 mRNA and protein expression were elevated in a carbon tetrachloride (CCl4)-induced liver fibrosis mouse model and in transforming growth factor- (TGF-) treated human (LX-2) hepatic stellate cells. Our in vivo studies highlighted a crucial connection between downregulating HOXC8 and the relief of liver fibrosis, alongside the suppression of fibrogenic gene expression provoked by CCl4. Besides, inhibiting HOXC8 reduced HSC activation and the expression of fibrosis-related genes (-SMA and COL1a1) triggered by TGF-β1 in vitro LX-2 cells, conversely, increasing HOXC8 levels fostered these effects. Our mechanistic study revealed that HOXC8 stimulates TGF1 transcription and increases the levels of phosphorylated Smad2/Smad3, implying a positive feedback mechanism between HOXC8 and TGF-1, thus boosting TGF- signaling and activating HSCs. Our research findings unequivocally demonstrate that a positive feedback loop between HOXC8 and TGF-β1 is essential for regulating HSC activation and driving the liver fibrosis process, suggesting that targeting HOXC8 could be a beneficial therapeutic strategy for such diseases.
The intricate regulation of chromatin plays a crucial role in gene expression, yet the precise impact of this system on nitrogen metabolism within Saccharomyces cerevisiae remains largely unexplored. B022 Prior studies indicated a regulatory function of the chromatin protein Ahc1p in controlling multiple key genes related to nitrogen metabolism in the yeast S. cerevisiae, but the precise regulatory pathway is not understood. This research highlighted multiple key genes involved in nitrogen metabolism, directly controlled by Ahc1p, and investigated the transcription factors interacting with Ahc1p. After thorough investigation, it was discovered that Ahc1p might modulate specific key nitrogen metabolism genes by employing two different strategies. Transcription complex binding to the core promoter regions of target genes is a consequence of the recruitment of Ahc1p, a co-factor, in partnership with Rtg3p or Gcr1p transcription factors, initiating transcription. The second mechanism involves Ahc1p binding enhancer elements to stimulate the transcription of its target genes, alongside the action of transcription factors.