Mouse Cells Research Articles

C3G improves lipid droplet accumulation in the proximal tubules of high-fat diet-induced ORG mice.

Obesity-related glomerulopathy (ORG) represents an escalating public health with no effective treatments currently available. Abnormal lipid metabolism and lipid droplet deposition in the kidneys are key contributors to ORG. Cyanidin-3-glucoside (C3G) has shown potential in regulating lipid metabolism and may offer reno-protective effects; however, its therapeutic efficacy and underlying mechanisms in ORG remain unclear. An ORG mouse model was established, followed by an 8-week C3G intervention. The mice were divided into three groups: normal control (CT) group, ORG group, and C3G treatment group. Fecal 16S rRNA sequencing, metabolomics of feces-serum-kidney, and kidney single-cell RNA sequencing (scRNA-seq) were performed to investigate the effects and mechanisms of C3G. Compared to CT mice, ORG mice exhibited elevated serum CHO, TG, Cys-C, UACR, urinary Kim-1, and NAG levels, along with glomerular hypertrophy and tubular injury. These biochemical and pathological indicators improved following C3G treatment. Fecal 16S analysis revealed reduced gut microbiota diversity in ORG mice compared to CT mice, while C3G intervention increased gut microbiota diversity. Metabolic profiling of feces, serum, and kidney indicated reprogramming of glycerophospholipid metabolism in ORG mice, ameliorated by C3G treatment. Further analysis demonstrated that abnormal glycerophospholipid metabolites correlated with blood lipids, urinary protein, urinary tubular injury markers, and gut microbiota, specifically Lachnospiraceae and Blautia. Additionally, scRNA-seq analysis identified activation of the PPARγ/CD36 pathway in proximal tubule cells (PTCs) of ORG mice. C3G improved abnormal glycerophospholipid metabolism and alleviated injury in PTCs by inhibiting the PPARγ/CD36 pathway. C3G reduces lipid droplet accumulation in the PTCs of ORG mice by modulating the gut microbiota and inhibiting the PPARγ/CD36 pathway. These findings offer new insights and therapeutic targets for ORG.

Early postnatal GABAB antagonist treatment normalizes inhibitory/excitatory balance in neonatal Ts65Dn mice, a genetic model of down syndrome.

Brain abnormalities in Down syndrome (DS) most rapidly accumulate during the third trimester, a critical period for the formation of neural circuits in the hippocampus and neocortex. In mice, this stage roughly corresponds to the first 2.5weeks after birth. We hypothesized that enhanced Girk2 channel signaling during this critical period profoundly contributes to the formation of faulty neural circuits in mouse genetic models of DS, with a key feature being an imbalance of excitatory and inhibitory neurotransmission favoring inhibition. Major predictions of this hypothesis were tested. We observed that hippocampal Girk2 levels are enhanced, GABAB/Girk2 signaling efficiency is increased, and intrinsic neuronal excitability of dentate gyrus (DG) granule cells is reduced in neonatal Ts65Dn mice. Given this, we tested if suppressing the enhanced GABAB/Girk2 signaling in the early postnatal period would affect the inhibitory/excitatory (I/E) balance in Ts65Dn mice. Remarkably, GABAB antagonist treatment from postnatal day 2 (P2) to P17 normalized the exaggerated IPSC/EPSC ratio in DG granule cells in Ts65Dn mice. Our findings show that GABAB/Girk2 signaling is increased in neonatal Ts65Dn mice, and that pharmacological suppression of GABAB receptors during the early postnatal period normalizes the I/E balance. These results suggest that early intervention targeting GABAB/Girk2 signaling could be a promising therapeutic approach to mitigate cognitive impairment in DS.

Aire attenuate collagen-induced arthritis by suppressing T follicular helper cells through ICOSL

ObjectiveTo assess the expression levels of autoimmune regulator (Aire) and inducible costimulator molecule ligand (ICOSL), as well as T follicular helper (Tfh) cell numbers in rheumatoid arthritis (RA) patients, and to explore their relationship with RA severity. We also aimed to investigate the effect of Aire on arthritis and its underlying mechanisms. MethodsThe expression levels of Aire, ICOSL, and Tfh cell numbers were measured in RA patients. The relationship between these factors and the levels of anticyclic citrullinated peptide antibodies (Anti-CCP) as well as the Disease Activity Score in 28 joints (DAS28) was analyzed. To investigate the effect of Aire on arthritis, Aire-overexpressing bone marrow-derived dendritic cells (Aire-BMDCs) and recombinant ICOSL were transferred into collagen-induced arthritis (CIA) mice, the symptoms, clinical scores, anti-collagen type II antibodies (anti-CII Abs), rheumatoid factor (RF), proportions of Tfh cells, and percentages of germinal center (GC) B cells in CIA mice were examined. ResultsThe results showed that Aire levels were significantly decreased in CD14+ PBMCs from patients with RA, and there were negative correlations between ICOSL expression levels, Tfh cell numbers, and Aire expression. Additionally, these factors were correlated with Anti-CCP levels and DAS28. Aire-BMDCs could influence Tfh differentiation, GC B cell development, as well as the levels of anti-CII Abs and RF, which further alleviate the symptoms and reduce clinical scores partly through ICOSL in CIA mice. ConclusionsThe findings suggest that Aire may alleviate rheumatoid arthritis by inhibiting ICOSL, which further inhibits Tfh cell differentiation and autoantibody production.

Sakuranetin Prevents Acetaminophen-Induced Liver Injury via Nrf2-Induced Inhibition of Hepatocyte Ferroptosis.

Oxidative stress is an important cause of acetaminophen (APAP)-induced liver injury (AILI). Sakuranetin (Sak) is an antitoxin from the cherry flavonoid plant with good antioxidant effects. However, whether sakuranetine has a protective effect on APAP-induced liver injury is not clear. Mouse and HepG2 cell models of APAP injury were used to investigate the effect of sakuranetin on AILI and its mechanism. Serum transaminase levels, histological changes, inflammatory mediators, oxidative stress, ferroptosis-related markers and Nrf2 signaling pathway proteins were analyzed. Sakuranetin significantly reduced serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST), as well as inflammatory factor; increased HepG2 activity and decreased cell death; inhibited ROS production, increased glutathione (GSH) content, expression of Glutathione Peroxidase 4 (GPX4) and Solute Carrier Family 7 Member 11 (SLC7A11), and decreased malondialdehyde and Acyl-CoA Synthetase Long Chain Family Member 4 (ACSL4) expression in mice and HepG2 cells after APAP treatment. Further analysis showed that sakuranetin induced the activation of the NFE2 Like BZIP Transcription Factor 2 (Nrf2) signaling pathway in liver tissue and HepG2 cells and promoted the nuclear translocation of Nrf2. Moreover, the hepatoprotective effect of sakuranetin and its inhibitory effect on ferroptosis were significantly attenuated by the Nrf2 inhibitor ML385. Sakuranetin alleviates AILI by activating the Nrf2 signaling pathway and inhibiting ferroptosis, and sakuranetin may be a potential therapeutic agent for the treatment of AILI.

Integration of ipsc‐derived human photoreceptors into rodent models of retinal degeneration

A main cause for vision impairment and blindness is the loss of light‐sensing photoreceptors in the retina. However, photoreceptors do not regenerate in mammals including humans, thus cell transplantation has emerged as a potential treatment approach for blinding diseases. Therefore, a pipeline for assessing the transplantation of human stem cell‐derived photoreceptors into mouse models of retinal generation was developed. Human photoreceptor‐specific reporter iPSC‐derived retinal organoids were used for isolation of donor cells via flow‐cytometry. After transplantation we observed extensive incorporation of human photoreceptors in mice with cone dysfunction/loss. This incorporative phenotype was validated in both cone‐only as well as pan‐photoreceptor transplantations. Rather than forming a glial barrier, Müller cells extend throughout the graft, and additionally form a series of adherens junctions between mouse and human cells, reminiscent of an outer limiting membrane. Donor‐host interaction appears to promote transplant polarization as well as development of morphological features critical for light detection, namely formation of inner‐ and outer‐segments mainly oriented towards the host retinal pigment epithelium. Putative synapse formation and graft function was observed both at the structural level and by electrophysiological analysis using micro‐electrode recordings. Transcriptomic analysis reveals increased expression of cone photoreceptor maturation genes with time after transplantation, in comparison to cones of age‐matched retinal organoids in vitro. While highly integrative donor cell clusters were observed in partially degenerated recipients, transplantation into hosts with late‐stage degeneration, i.e. with complete photoreceptor loss, lead to more wide‐spread distribution of donor cells within the sub‐retinal space and reduced signs for integration and maturation. Thus, interaction with the host retina appears to be important for graft integration, maturation, polarization and function. Overall, these results show that human photoreceptors interact readily with a partially degenerated mouse retina.Funding:BMBF – ReSight (01EK1613A)DFG – AD375/7‐1 (SPP2127), SEQ2146FFB – TA‐RM‐0522‐0824‐TUD‐TRAP

Akkermansia muciniphila inhibits jejunal lipid absorption and regulates jejunal core bacteria.

Insufficiency of Akkermansia muciniphila (Akk) has been implicated in the pathogenesis of metabolic diseases, and administration or restoration of Akk has ameliorated these disorders. Recently, Pasteurized Akk (PA-Akk) has been approved as a functional food. However, the impact of Akk on lipid absorption in the proximal intestine, which is directly exposed to orally administered Akk, remains largely unexplored. In this study, we orally administered Akk and PA-Akk to mice and investigated the subsequent lipid absorption. Long-term administration of Akk resulted in reduced lipid deposits in the liver and adipocytes, along with improved glucose metabolism. This was primarily attributed to a reduction in lipid absorption by epithelial cells in the proximal jejunum. Mechanistically, Akk activated AMP-activated protein kinase (AMPK) and directly inhibit lipids absorption in both mouse and human jejunal epithelial cells. Furthermore, we demonstrated that Akk treatment, but not PA-Akk treatment, promotes the abundance of genera that are highly abundant in the normal jejunum and belong to the phylum Firmicutes. Thus, our study concludes that oral administration of Akk provides beneficial effects on metabolism, partially through inhibiting jejunal lipid absorption and promoting the abundance of core jejunal microbes.

Hypoxia inducible factor-dependent upregulation of Agrp in glomus type I cells of the carotid body.

Agouti-related peptide (AgRP) is a well-established potent orexigenic peptide primarily expressed in hypothalamic neurons. Nevertheless, the expression and functional significance of extrahypothalamic AgRP remain poorly understood. In this study, utilizing histological and molecular biology techniques, we have identified a significant expression of Agrp mRNA and AgRP peptide production in glomus type I cells within the mouse carotid body (CB). Furthermore, we have uncovered evidence supporting the expression of the AgRP receptor melanocortin receptor 3 (Mc3r) in adjacent sympathetic neurons, suggesting a potential local paracrine role for AgRP within the CB. Importantly, AgRP immunoreactivity was also identified in glomus type I cells of the human CB. Given the unexpected abundance of AgRP in glomus type I cells, a chemoreceptor cell specialized i.a. for oxygen sensing, we proceeded to investigate whether Agrp expression in the CB is regulated by hypoxemia and associated oxygen-sensing molecular mechanisms. Invitro luciferase assays reveal that hypoxia stimulates the human and mouse Agrp promoters in a Hypoxia Inducible Factor (HIF1/2)-dependent manner. Our invivo experiments further demonstrate that exposure to environmental hypoxia (10%) robustly induces Agrp expression in type I glomus cells of mice. Furthermore, these findings collectively highlight the hitherto unknown source of AgRP in murine and human type I glomus cells and underscore the direct control of Agrp transcription by HIF signaling.

Epigenetics in Learning and Memory.

In animals, memory formation and recall are essential for their survival and for adaptations to a complex and often dynamically changing environment. During memory formation, experiences prompt the activation of a selected and sparse population of cells (engram cells) that undergo persistent physical and/or chemical changes allowing long-term memory formation, which can last for decades. Over the past few decades, important progress has been made on elucidating signaling mechanisms by which synaptic transmission leads to the induction of activity-dependent gene regulation programs during the different phases of learning (acquisition, consolidation, and recall). But what are the molecular mechanisms that govern the expression of immediate-early genes (IEGs; c-fos, Npas4) and plasticity-related genes (PRGs; Dlg4/PSD95 and Grin2b/NR2B) in memory ensemble? Studies in relatively simple in vitro and in vivo neuronal model systems have demonstrated that synaptic activity during development, or when induced by chemical stimuli (i.e., cLTP, KCl, picrotoxin), activates the NMDAR-Ca2+-CREB signaling pathway that upregulates gene expression through changes in the epigenetic landscape (i.e., histone marks and DNA methylation) and/or 3D chromatin organization. The data support a model in which epigenetic modifications in promoters and enhancers facilitate the priming and activation of these regulatory regions, hence leading to the formation of enhancer-promoter interactions (EPIs) through chromatin looping. The exploration of whether similar molecular mechanisms drive gene expression in learning and memory has presented notable challenges due to the distinct phases of learning and the activation of only sparse population of cells (the engram). Consequently, such studies demand precise temporal and spatial control. By combining activity-dependent engram tagging strategies (i.e., TRAP mice) with multi-omics analyses (i.e., RNA-seq, ChiP-seq, ATAC-seq, and Hi-C), it has been recently possible to associate changes in the epigenomic landscape and/or 3D genome architecture with transcriptional waves in engram cells of mice subjected to contextual fear conditioning (CFC), a relevant one-shot Pavlovian learning task. These studies support the role of specific epigenetic mechanisms and of the 3D chromatin organization during the control of gene transcription waves in engram cells. Advancements in our comprehension of the molecular mechanisms driving memory ensemble will undoubtedly play a crucial role in the development of better-targeted strategies to tackle cognitive diseases, including Alzheimer's disease and frontotemporal dementia, among other information-processing disorders.

Vitamin D and Tinospora cordifolia modulate TLR3 and TLR4 pathways, reduce inflammation, and maintain antimicrobial peptide levels in infected mice

The activation of Toll-Like Receptor-3 (TLR3) and Toll-Like Receptor-4 (TLR4) signalling pathways is a regular pathway for immune system activation during infection. This study aimed to investigate the effects of vitamin D (VD) and Tinospora cordifolia ethanol extract (TC) on TLR3 and TLR4 receptor protein expression, proinflammatory cytokine (IL1 and IL-6) production, and antimicrobial peptide cathelicidin (CAP) production in CD11b+ cells of mice infected with Escherichia coli. The treatments consisted of administration of VD (0.325 µg/kg bw), TC (100 mg/kg bw), and a combination of both in the same dose for 28 days, followed by induction of E. coli infection on day 29. The flow cytometry method was analyzed of TLR3, TLR4, IL-1, IL-6, and CAP expression in CD11b+ cells of experimental animals. The following measurement results were compared with healthy controls and infected animals with the significance of differences between treatments analyzed by One-way ANOVA with p < 0.05. The results showed that administering VD, TC, and a combination of both reduced the expression of TLR3, TLR4, and IL-1 compared to treating infected animals. The combination treatment of VD + TC increased CAP production more than all other treatments. This significant finding suggests that the combination of VD + TC has the potential to control inflammation without disrupting the body’s defence mechanisms against infection, providing valuable insights for the field of immunology.

In vitro NIH3T3 mouse embryonic fibroblast cell model does not predict AAV2 or AAVdj-mediated cell transformation.

One of the potential risk factors of recombinant adeno-associated virus (rAAV)-based gene therapy is insertional mutagenesis, which has been associated with the development of hepatocellular carcinoma (HCC) in rAAV-treated neonatal mice. The objective of this study was to investigate if well-established in vitro cell transformation assays (CTA) in mouse cell lines can detect AAV2 or AAVdj-mediated cell transformation. Since AAV integration at the Rian locus in neonatal mice has been implicated in AAV-mediated HCC, an rAAV vector specifically targeting the mouse Rian locus and an additional rAAV vector previously shown to cause HCC in neonatal mice were both tested for the induction of cell transformation in NIH3T3 cells. To increase the frequency of AAV DNA integration at the Rian locus in the genome of NIH3T3 cells, double-strand breaks in Rian locus of NIH3T3 cells were created by CRISPR-Cas9 to increase the homologous crossover between viral DNA and the cell genome. When transduced cells were assayed in CTA, the transformation frequency observed in AAV-transduced NIH3T3 cells was not significantly different from that of untreated vehicle cells. The finding that rAAV is unable to transform the NIH3T3 in vitro indicates that either the transformation rate is less than the spontaneous rate of NIH3T3 cellular transformation, or in vitro CTA are not predictive of rAAV-induced HCC in mice.

HDAC and MEK inhibition synergistically suppresses HOXC6 and enhances PD-1 blockade efficacy in BRAFV600E-mutant microsatellite stable colorectal cancer

BackgroundB-Raf proto-oncogene, serine/threonine kinase (BRAF)V600E-mutant microsatellite stable (MSS) colorectal cancer (CRC) constitutes a distinct CRC subgroup, traditionally perceived as minimally responsive to standard therapies. Recent clinical attempts, such as BRAF...

Endothelial Dysfunction in the Tubule Area Accelerates the Progression of Early Diabetic Kidney Disease

Diabetic kidney disease (DKD) is the leading cause of end-stage renal disease. Therefore, understanding the molecular regulatory mechanisms underlying the pathogenesis of DKD is imperative. In this study, we aimed to explore the molecular mechanisms of tubule region endothelial dysfunction in early DKD. Early-stage DKD model was established in 16-week-old female db/db mice for 16 weeks. Body weight, glucose level, and urine albumin-to-creatinine ratio (UACR) were measured. Hematoxylin and eosin (H&E) and periodic acid-Schiff (PAS) staining were performed to evaluate pathological lesions. RNA sequencing data of the kidneys and integrated publicly available single-cell and spatial transcriptome datasets were used to investigate the mechanism of endothelial dysfunction. There was a significant increase in body weight (p = 0.001), glucose levels (p=0.0008), and UACR (p=0.006) in db/db mice compared with db/m mice. H&E and PAS staining showed that vacuolar lesions and protein casts of tubules were the major histopathological changes observed in early-stage DKD mice. The apoptotic pathway in endothelial cells was notably activated in DKD, and Thbs1 was identified as the central gene involved in this apoptotic process. Deconvolution of the cell composition in the RNA sequencing data showed a decrease in the proportion of endothelial cells in the DKD mice. Further analysis of the activity and regulatory network of transcription factors showed that Creb1 was activated in both mouse and human early-stage DKD, suggesting that Creb1 activation may be involved in early kidney injury. The endothelial cell apoptotic pathway is activated in DKD, and the proportion of endothelial cells was reduced in the DKD mice, which is significantly associated with Thbs1.

CCN3: lactational bone booster

Mammalian reproduction requires that nursing mothers transfer large amounts of calcium to their offspring through milk. Meeting this demand requires the activation of a brain-breast-bone circuit during lactation that coordinates changes in systemic hormones, dietary calcium intake, skeletal turnover, and calcium transport into milk. Classically, increased bone resorption via increased parathyroid hormone-related protein and low estrogen levels is the main source of calcium for milk production during lactation. Over the past few decades, investigators have described many aspects of this brain-breast-bone axis during lactation, yet many unanswered questions remain. Using a comprehensive set of parabiosis coupled with in vivo µCT, bone transplant studies, cell culturing and differentiation assays, mouse genetic models, pharmacologic interventions, hepatic viral transduction, and sequencing analysis, a recent study discovered that cellular communication network factor 3 (CCN3), derived from ARHERα/Kiss1 neurons, functions as an osteogenic hormone to sustain bone formation and progeny survival during lactation. Compelling evidence has been presented to show that (1) CCN3 expression in ARHERα/Kiss1 neurons fluctuates, almost exclusively appearing during lactation; (2) CCN3 stimulates mouse and human skeletal stem cell activity, increases bone remodeling and fracture repair in young and old mice of both sexes; (3) knockdown Ccn3 transcripts in the ARHKiss1 neurons in lactating dams causes devastating bone loss and failure to sustain progeny survival. These findings suggested that the stage-specific expression of CCN3 in female ARHERα/Kiss1 neurons during lactation is a newly identified brain-bone axis evolved to sustain the skeleton in mammalian mothers and offspring.

Enhancing antibody levels and T cell activity of quadrivalent influenza vaccine by combining it with CpG HP021

Influenza virus infections are a serious danger to people’s health worldwide as they are responsible for seasonal flu outbreaks. There is an urgent need to improve the effectiveness and durability longevity of the immune response to influenza vaccines. We synthesized the CpG HP021 and examined the impact of it on the immune response to an influenza vaccine. In BALB/c mice, hemagglutination inhibition (HI) titers to the vaccine were increased four- to eightfold against H1N1, H3N2, BV, and BY viruses by 3 μg IIV4 + 40 μg CpG HP021 compared with those of the non-adjuvanted IIV4 group, and the CpG HP021 group had a broader HI activity. Additionally, the immune response was directed towards Type 1 T helper (Th1) cells due to the CpG HP021 adjuvant. The CpG HP021-adjuvanted IIV4 induced a higher number of T cells secreting interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α), and increased the percentage of effector memory T cells in mice. In SD rats, the immune responses induced by IIV4 with CpG HP021 were similar to those in BALB/c mice. The development of CpG HP021 may expand the options for adjuvants in vaccines against infectious diseases.

Efficient radiolabeling of mesoporous silica nanoparticles for single-cell PET imaging.

Nanoparticles are highly efficient vectors for ferrying contrast agents across cell membranes, enabling ultra-sensitive in vivo tracking of single cells with positron emission tomography (PET). However, this approach must be fully characterized and understood before it can be reliably implemented for routine applications. We developed a Langmuir adsorption model that accurately describes the process of labeling mesoporous silica nanoparticles (MSNP) with 68Ga. We compared the binding efficiency of three different nanoparticle systems by fitting the model to experimental data. We then chose the MSNP with the highest affinity for 68Ga to study uptake and efflux kinetics in cancer cells. After intracardiac injection of 50-100 cells in mice, PET imaging was performed to test the effectiveness of cellular radiolabeling. We found that highly porous mesoporous nanoparticles (d = 100nm) with MCM-41 pore structures can achieve radiolabeling efficiency > 30 GBq/mg using 68Ga, without the need for any chelator. These 68Ga conjugated particles showed strong serum stability in vitro. In mice, the 68Ga-MSNPs predominantly accumulated in the liver with a high signal-to-background ratio and no bladder signal, indicating excellent stability of the labeled nanoparticles in vivo. Additionally, these MSNPs were efficiently taken up by B16F10 and MDA-MB-231 cancer cells, as confirmed by confocal imaging, flow cytometry analysis, and gamma counting. Finally, cardiac injection of < 100 68Ga-MSNP-labeled cells allowed PET/CT tracking of these cells in various organs in mice. We characterized the critical parameters of MSNP-mediated direct cellular radiolabeling to improve the use of these nanoparticles as cellular labels for highly sensitive preclinical PET imaging.

Opioid-induced respiratory depression: clinical aspects and pathophysiology of the respiratory network effects.

Important insights and consensus remain lacking for risk prediction of opioid-induced respiratory depression (OIRD), reversal of respiratory depression (RD), the pathophysiology of OIRD, and which sites make the most significant contribution to its induction. The ventilatory response to inhaled carbon dioxide is the most sensitive biomarker of OIRD. To accurately predict respiratory depression (RD), a multivariant RD prospective trial using continuous capnograph and oximetry examining 5 independent variables: age ≥60, sex, opioid naivety, sleep disorders, and chronic heart failure (PRODIGY trial), was undertaken. Intermittent oximetry alone substantially underestimates the incidence of RD. Naloxone, with an elimination half-life of ~33 min (c.f. morphine 2-3 h; fentanyl and congeners only 5-15 min) has limitations for the to rescue of patients with severe OIRD. Buprenorphine is potentially valuable in patients being treated long-term since its high μ-receptor (MOR) affinity makes it difficult for an opioid of lower affinity (e.g., fentanyl) to displace it from the receptor. In the last decade, synthetic opioids e.g., fentanyl, its potent analogs such as carfentanil and the benzimidazole derivative nitazene 'superagonists' have contributed to the exponential growth in opioid deaths due to RD. The MOR, encoded by gene Oprm1, is widely expressed in the central and peripheral nervous systems including centers that modulate breathing. Opioids bind to the receptors, but consensus is lacking on which site(s) makes the most significant contribution to induction of OIRD. Both the preBötzinger complex (preBötC), the inspiratory rhythm generator, and Kölliker-Fuse nucleus (KFN), the respiratory modulator, contribute to RD but receptor binding is not restricted to a single site. Breathing is composed of three phases, inspiration, post-inspiration, and active expiration, each generated by distinct rhythm-generating networks: the preBötC, the post-inspiratory complex (PiCo), and the lateral parafacial nucleus (pF L), respectively. Somatostatin expressing mouse cells involved in breathing regulation are not involved in opioid-induced RD.

Deficiency of myeloid discoidin domain receptor 2 aggravates melanoma lung and bone metastasis.

Melanoma, one of the most prevalent cancers worldwide, frequently metastasizes to the lung and bones. Tumor-associated macrophages play essential roles in melanoma metastasis but the underlying mechanism remains obscure. We previously demonstrated that specific knockout of Ddr2, a receptor tyrosine kinase, exacerbates systemic inflammation via modulating macrophage repolarization. To investigate whether myeloid Ddr2 regulates melanoma growth and metastasis, we injected B16BL6 melanoma cells into Ddr2LysM (cKO) mice via subcutaneous neck, tail vein, and left ventricle, respectively. We found that the growth of melanoma cells in cKO mice was significantly retarded, as demonstrated by the subcutaneous transplantation tumor model. Unexpectedly, the melanoma metastasis to the lung or bone was significantly stimulated in cKO mice, indicating the complicated role of Ddr2 in macrophages in melanoma development. Furthermore, Ddr2 in macrophages regulated the migration of B16BL6 cells in the co-culture system. Bioinformatics analysis showed that Ddr2 expression correlates with improved prognostic outcomes in melanoma, and high expression of Ddr2 is protective in melanoma metastasis. Our results enrich the current knowledge of Ddr2 in tumor biology and indicate that more consideration should be taken when applying Ddr2 inhibition as a melanoma treatment strategy.

Human collagen type I‑based scaffold retains human‑derived fibroblasts in a patient‑derived tumor xenograft mouse model.

The present study aimed to investigate the role of a recombinant protein based on human collagen type I (RCPhC1) as a scaffold in maintaining the human tumor microenvironment within a patient-derived tumor xenograft (PDTX) model. RCPhC1, synthesized under animal component-free conditions, was explored for its potential to support the human-specific stroma associated with tumor growth. PDTX models were established using resected colorectal cancer liver metastasis specimens, and stromal cell populations from humans and mice were compared using three scaffolds: No scaffold (control), Matrigel and recombinant human collagen type I, across two passages. Specific antibodies for human Lamin B and mouse Lamin B were used for immunostaining to distinguish between human and mouse cells. Additionally, the impact of each scaffold on the invasive ability of mouse fibroblasts was assessed using an invasion assay. Patient-derived tumor tissues embedded with RCPhC1 hydrogels had significantly more human Lamin B-positive cells and fewer mouse Lamin B cells than those embedded with no scaffolds or Matrigel. The human Lamin B-positive cells in PDTX tumors with RCPhC1 hydrogels were recognized as fibroblasts. Additionally, these hydrogels significantly reduced the invasion of mouse fibroblast cell lines in vitro compared with Matrigel. The present study investigated RCPhC1 hydrogels as a new scaffold material for tumor engraftment in PDTX mouse models, and identified a promising experimental tool for maintaining the tumor microenvironment.

Excess Ub-K48 Induces Neuronal Apoptosis in Alzheimer's Disease.

K48-linked ubiquitin chain (Ub-K48) is a crucial ubiquitin chain implicated in protein degradation within the ubiquitin-proteasome system. However, the precise function and molecular mechanism underlying the role of Ub-K48 in the pathogenesis of Alzheimer's disease (AD) and neuronal cell abnormalities remain unclear. The objective of this study was to examine the function of K48 ubiquitination in the etiology of AD, and its associated mechanism of neuronal apoptosis. A mouse model of AD was constructed, and behavioral phenotypic changes were detected using an open field test (OFT). The expression of glial fibrillary acidic protein (GFAP), an early marker of AD, was detected by western blotting (WB). Neuronal apoptosis in the hippocampal region was assessed by hematoxylin and eosin (HE) and Nissl staining. Immunohistochemistry and immunofluorescence were performed to observe the changes in Phosphorylated tubulin associated unit (p-Tau) and Ub-K48 colocalization in neurons of the hippocampal region of AD mice. WB was further applied to detect the degree of ubiquitylation of K48 and the expression of Tau, p-Tau, B-cell lymphoma-2 (Bcl-2) and Bcl-2-associated X (Bax) proteins in neuronal cells of the hippocampus and cortical regions of mice. Mice with AD exhibited significantly longer resting times (p < 0.05) and shorter average speeds (p < 0.01), total distances travelled (p < 0.01), and distances travelled (p < 0.01) in the central region than those in the control group. This indicated cognitive impairment, which occurred concurrent with an increased expression of the AD marker GFAP protein (p < 0.001). The hippocampal region of AD mice showed abnormalities with sparsely and irregularly arranged cells, large gaps between cells, lighter staining, unclear boundaries of the cell membranes and nuclei, and agglutinated and condensed nuclei (p < 0.01). The neuronal cells of AD mice exhibited significantly elevated levels of p-Tau (p < 0.01) and Ub-K48 (p < 0.01), as well as a notable degree of co-localization within the cells. The intracellular pro-inflammatory protein Bax was significantly upregulated (p < 0.05), while the Bcl-2/Bax ratio was significantly lower than that in the control group (p < 0.05), thus inducing apoptosis in AD neuronal cells. Ub-K48 is strongly linked to the development of AD. p-Tau aggregate in neuronal cells in the hippocampal region of the AD brain and colocalize with Ub-K48, which in turn leads to cellular inflammation and the induction of apoptosis in neuronal cells.

Pulmonary lysyl oxidase expression and its role in seeding Lewis lung carcinoma cells.

Copper promotes tumor growth and metastasis through a variety of mechanisms, most notably as a cofactor within the lysyl oxidase (LOX) family of secreted cuproenzymes. Members of this family, which include LOX and LOX-like enzymes LOXL1-4, catalyze the copper-dependent crosslinking of collagens and elastin within the extracellular matrix (ECM). Elevated LOX expression is associated with higher incidence and worse prognosis in multiple cancers, including colorectal, breast, pancreatic, and head and neck. In this study, we demonstrated that elevated LOX expression correlates with decreased overall survival and shorter median time to first progression in patients with lung cancer. Previous studies have demonstrated that LOX secreted from tumors is critical for pre-metastatic niche formation by promoting ECM remodeling and the recruitment of immune cells and endothelial precursors. Here, we demonstrated that ablation of the LOX gene in Lewis lung carcinoma (LLC) cells diminishes tumor growth and metastasis compared to wild-type LLC cells in a syngeneic mouse model. Although the role of tumor-derived LOX in tumor formation and metastasis is well established, little is known regarding the possible contribution of LOX produced by the parenchymal tissue of metastatic organs. Thus, this report describes our findings that host-derived LOX produced by the lung contributes to the pulmonary metastasis of LLC cells in mice. The suppression of pulmonary lysyl oxidase expression reduces the metastatic potential of Lewis Lung Carcinoma cells in mice, revealing a previously unknown influence of LOX expression in the parenchymal tissue of metastatic target organs on the seeding of tumor cells.