-
Bio-protocol Jun 2024The intricate composition, heterogeneity, and hierarchical organization of the human bone marrow hematopoietic microenvironment (HME) present challenges for...
The intricate composition, heterogeneity, and hierarchical organization of the human bone marrow hematopoietic microenvironment (HME) present challenges for experimentation, which is primarily due to the scarcity of HME-forming cells, notably bone marrow stromal cells (BMSCs). The limited understanding of non-hematopoietic cell phenotypes complicates the unraveling of the HME's intricacies and necessitates a precise isolation protocol for systematic studies. The protocol presented herein puts special emphasis on the accuracy and high quality of BMSCs obtained for downstream sequencing analysis. Utilizing CD45 and CD235a as negative markers ensures sufficient enrichment of non-hematopoietic cells within the HME. By adding positive selection based on CD271 expression, this protocol allows for selectively isolating the rare and pivotal stromal cell population with high precision. The outlined step-by-step protocol provides a robust tool for isolating and characterizing non-hematopoietic cells, including stromal cells, from human bone marrow preparations. This approach thus contributes valuable information to promote research in a field that is marked by a scarcity of studies and helps to conduct important experimentation that will deepen our understanding of the intricate cellular interactions within the bone marrow niche. Key features • Isolation of high-quality human non-hematopoietic bone marrow cells for scRNAseq • Targeted strategy for enriching low-frequency stromal cells.
PubMed: 38948257
DOI: 10.21769/BioProtoc.5020 -
World Journal of Stem Cells Jun 2024Necrotizing enterocolitis (NEC) is a severe gastrointestinal disease that affects premature infants. Although mounting evidence supports the therapeutic effect of...
BACKGROUND
Necrotizing enterocolitis (NEC) is a severe gastrointestinal disease that affects premature infants. Although mounting evidence supports the therapeutic effect of exosomes on NEC, the underlying mechanisms remain unclear.
AIM
To investigate the mechanisms underlying the regulation of inflammatory response and intestinal barrier function by umbilical cord mesenchymal stem cell (UCMSCs) exosomes, as well as their potential in alleviating NEC in neonatal mice.
METHODS
NEC was induced in 5-d-old C57BL/6 pups through hypoxia and gavage feeding of formula containing lipopolysaccharide (LPS), after which the mice received human UCMSC exosomes (hUCMSC-exos). The control mice were allowed to breastfeed with their dams. Ileal tissues were collected from the mice and analyzed by histopathology and immunoblotting. Colon tissues were collected from NEC neonates and analyzed by immunofluorescence. Molecular biology and cell culture approaches were employed to study the related mechanisms in intestinal epithelial cells.
RESULTS
We found that autophagy is overactivated in intestinal epithelial cells during NEC, resulting in reduced expression of tight junction proteins and an increased inflammatory response. The ability of hUCMSC-exos to ameliorate NEC in a mouse model was dependent on decreased intestinal autophagy. We also showed that hUCMSC-exos alleviate the inflammatory response and increase migration ability in intestinal epithelial cells induced by LPS.
CONCLUSION
These results contribute to a better understanding of the protective mechanisms of hUCMSC-exos against NEC and provide a new theoretical and experimental foundation for NEC treatment. These findings also enhance our understanding of the role of the autophagy mechanism in NEC, offering potential avenues for identifying new therapeutic targets.
PubMed: 38948093
DOI: 10.4252/wjsc.v16.i6.728 -
Frontiers in Physiology 2024Proximal tubule (PT) cells maintain a high-capacity apical endocytic pathway to recover essentially all proteins that escape the glomerular filtration barrier. The multi...
Proximal tubule (PT) cells maintain a high-capacity apical endocytic pathway to recover essentially all proteins that escape the glomerular filtration barrier. The multi ligand receptors megalin and cubilin play pivotal roles in the endocytic uptake of normally filtered proteins in PT cells but also contribute to the uptake of nephrotoxic drugs, including aminoglycosides. We previously demonstrated that opossum kidney (OK) cells cultured under continuous fluid shear stress (FSS) are superior to cells cultured under static conditions in recapitulating essential functional properties of PT cells . To identify drivers of the high-capacity, efficient endocytic pathway in the PT, we compared FSS-cultured OK cells with less endocytically active static-cultured OK cells. Megalin and cubilin expression are increased, and endocytic uptake of albumin in FSS-cultured cells is > 5-fold higher compared with cells cultured under static conditions. To understand how differences in receptor expression, distribution, and trafficking rates contribute to increased uptake, we used biochemical, morphological, and mathematical modeling approaches to compare megalin traffic in FSS- versus static-cultured OK cells. Our model predicts that culturing cells under FSS increases the rates of all steps in megalin trafficking. Importantly, the model explains why, despite seemingly counterintuitive observations (a reduced fraction of megalin at the cell surface, higher colocalization with lysosomes, and a shorter half-life of surface-tagged megalin in FSS-cultured cells), uptake of albumin is dramatically increased compared with static-grown cells. We also show that FSS-cultured OK cells more accurately exhibit the mechanisms that mediate uptake of nephrotoxic drugs compared with static-grown cells. This culture model thus provides a useful platform to understand drug uptake mechanisms, with implications for developing interventions in nephrotoxic injury prevention.
PubMed: 38948083
DOI: 10.3389/fphys.2024.1404248 -
Frontiers in Molecular Biosciences 2024Acute ischemic stroke is the most common cause of neurologic dysfunction caused by focal brain ischemia and tissue injury. Diabetes is a major risk factor of stroke,...
Acute ischemic stroke is the most common cause of neurologic dysfunction caused by focal brain ischemia and tissue injury. Diabetes is a major risk factor of stroke, exacerbating disease management and prognosis. Therefore, discovering new diagnostic markers and therapeutic targets is critical for stroke prevention and treatment. Extracellular vesicles (EVs), with their distinctive properties, have emerged as promising candidates for biomarker discovery and therapeutic application. This case-control study utilized mass spectrometry-based proteomics to compare EVs from non-diabetic stroke (nDS = 14), diabetic stroke (DS = 13), and healthy control (HC = 12) subjects. Among 1288 identified proteins, 387 were statistically compared. Statistical comparisons using a general linear model (log2 foldchange ≥0.58 and FDR-p≤0.05) were performed for nDS vs HC, DS vs HC, and DS vs nDS. DS vs HC and DS vs nDS comparisons produced 123 and 149 differentially expressed proteins, respectively. Fibrinogen gamma chain (FIBG), Fibrinogen beta chain (FIBB), Tetratricopeptide repeat protein 16 (TTC16), Proline rich 14-like (PR14L), Inhibitor of nuclear factor kappa-B kinase subunit epsilon (IKKE), Biorientation of chromosomes in cell division protein 1-like 1 (BD1L1), and protein PR14L exhibited significant differences in the DS group. The pathway analysis revealed that the complement system pathways were activated, and blood coagulation and neuroprotection were inhibited in the DS group (z-score ≥2; ≤ 0.05). These findings underscore the potential of EVs proteomics in identifying biomarkers for stroke management and prevention, warranting further clinical investigation.
PubMed: 38948080
DOI: 10.3389/fmolb.2024.1387859 -
Oncology Research 2024Osteosarcoma is the most common malignant primary bone tumor. The prognosis for patients with disseminated disease remains very poor despite recent advancements in...
BACKGROUND
Osteosarcoma is the most common malignant primary bone tumor. The prognosis for patients with disseminated disease remains very poor despite recent advancements in chemotherapy. Moreover, current treatment regimens bear a significant risk of serious side effects. Thus, there is an unmet clinical need for effective therapies with improved safety profiles. Taurolidine is an antibacterial agent that has been shown to induce cell death in different types of cancer cell lines.
METHODS
In this study, we examined both the antineoplastic and antiangiogenic effects of taurolidine in animal models of osteosarcoma. K7M2 murine osteosarcoma cells were injected, both intramuscular and intraperitoneal, into 60 BALB/c mice on day zero. Animals were then randomized to receive treatment with taurolidine 2% (800 mg/kg), taurolidine 1% (400 mg/kg), or NaCl 0.9% control for seven days by intravenous or intraperitoneal administration.
RESULTS
After 35 days, mice were euthanized, and the tumors were harvested for analysis. Eighteen mice were excluded from the analysis due to complications. Body weight was significantly lower in the 2% taurolidine intraperitoneal treatment group from day 9 to 21, consistent with elevated mortality in this group. Intraperitoneal tumor weight was significantly lower in the 1% ( = 0.003) and 2% ( = 0.006) intraperitoneal taurolidine treatment groups compared to the control. No antineoplastic effects were observed on intramuscular tumors or for intravenous administration of taurolidine. There were no significant differences in microvessel density or mitotic rate between treatment groups. Reduced body weight and elevated mortality in the 2% taurolidine intraperitoneal group suggest that the lower 1% dose is preferable.
CONCLUSIONS
In conclusion, there is no evidence of antiangiogenic activity, and the antitumor effects of taurolidine on osteosarcoma observed in this study are limited. Moreover, its toxic profile grants further evaluation. Given these observations, further research is necessary to refine the use of taurolidine in osteosarcoma treatment.
PubMed: 38948019
DOI: 10.32604/or.2024.050907 -
World Journal of Transplantation Jun 2024In this editorial, we talk about a compelling case focusing on posterior reversible encephalopathy syndrome (PRES) as a complication in patients undergoing liver...
In this editorial, we talk about a compelling case focusing on posterior reversible encephalopathy syndrome (PRES) as a complication in patients undergoing liver transplantation and treated with Tacrolimus. Tacrolimus (FK 506), derived from , is a potent immunosuppressive macrolide. It inhibits T-cell transcription by binding to FK-binding protein, and is able to amplify glucocorticoid and progesterone effects. Tacrolimus effectively prevents allograft rejection in transplant patients but has adverse effects such as Tacrolimus-related PRES. PRES presents with various neurological symptoms alongside elevated blood pressure, and is primarily characterized by vasogenic edema on neuroimaging. While computed tomography detects initial lesions, magnetic resonance imaging, especially the Fluid-Attenuated Inversion Recovery sequence, is superior for diagnosing cortical and subcortical edema. Our discussion centers on the incidence of PRES in solid organ transplant recipients, which ranges between 0.5 to 5 +ACU-, with varying presentations, from seizures to visual disturbances. The case of a 66-year-old male status post liver transplantation highlights the diagnostic and management challenges associated with Tacrolimus-related PRES. Radiographically evident in the parietal and occipital lobes, PRES underlines the need for heightened vigilance among healthcare providers. This editorial emphasizes the importance of early recognition, accurate diagnosis, and effective management of PRES to optimize outcomes in liver transplant patients. The case further explores the balance between the efficacy of immunosuppression with Tacrolimus and its potential neurological risks, underlining the necessity for careful monitoring and intervention strategies in this patient population.
PubMed: 38947962
DOI: 10.5500/wjt.v14.i2.91146 -
Frontiers in Oncology 2024Hepatoblastoma (HB) is the most common pediatric hepatic malignancy. Despite the progress in HB treatment, investigating HB pathomechanisms to optimize stratification...
BACKGROUND
Hepatoblastoma (HB) is the most common pediatric hepatic malignancy. Despite the progress in HB treatment, investigating HB pathomechanisms to optimize stratification and therapies remains a focal point to improve the outcome for high-risk patients.
METHODS
Here, we pointed to explore the impact of these mechanisms in HB. An observational study was performed on liver samples from a cohort of 17 patients with a diagnosis of HB and two normal liver samples. The experiments were executed on the Huh6 human HB cell line treated with the FAK inhibitor TAE226.
RESULTS
Our results highlight a significant up-regulation of mRNA and protein expression of FAK in livers from HB with respect to normal livers. The increased protein expression of total and Tyr397 phosphorylated FAK (pTyr397FAK) was significantly correlated with the expression of some epigenetic regulators of histone H3 methylation and acetylation. Of note, the expression of pTyr397FAK, N-methyltransferase enzyme (EZH2) and tri-methylation of the H3K27 residue correlated with tumor size and alpha-fetoprotein (AFP) levels. Finally, TAE226 caused a significant reduction of pTyr397FAK, epigenetic regulators, , , , and , in association with anti-proliferative and pro-apoptotic effects on HB cells.
CONCLUSION
Our results suggest a role of FAK in HB that requires further investigations mainly focused on the exploration of its effective diagnostic and therapeutic translatability.
PubMed: 38947885
DOI: 10.3389/fonc.2024.1397647 -
ACS Omega Jun 2024The durability of Pt nanoparticle catalysts is currently the most important factor limiting the widespread use of polymer electrolyte fuel cells (PEFCs). Specifically,...
The durability of Pt nanoparticle catalysts is currently the most important factor limiting the widespread use of polymer electrolyte fuel cells (PEFCs). Specifically, the Pt nanoparticles in standard carbon black-supported Pt nanoparticle (Pt/CB) catalysts repeatedly aggregate on the CB surfaces during PEFC operation, thus, reducing the performance of the cell. Therefore, PEFCs must contain large quantities of Pt to maintain sufficient service lifetimes. This is the main factor hindering the reduction of the cost of PEFCs. The present research demonstrates that ultrafine Pt particles (Pt) having diameters of approximately 0.5 nm can be formed in situ from a platinum chloride complex (PtCl ) on a carbon-based material doped with Fe and N via the dissolution and reprecipitation of Pt in the PtCl during potential cycling in a 0.1 M HClO solution. The Pt are immobilized by both Fe and N in the support material. The mass-based catalytic activity of this material during the oxygen reduction reaction is eight times higher than that of a standard Pt/CB catalyst and is maintained even after 100,000 potential step cycles (0.6 ↔ 1.0 V). The present results provide guidelines for the development of highly durable yet active membrane electrode assemblies that minimize the use of Pt.
PubMed: 38947817
DOI: 10.1021/acsomega.4c02723 -
Journal of Plant Diseases and... 2024Septin GTPases are morphogenetic proteins that are widely conserved in eukaryotic organisms fulfilling diverse roles in cell division, differentiation and development....
Septin GTPases are morphogenetic proteins that are widely conserved in eukaryotic organisms fulfilling diverse roles in cell division, differentiation and development. In the filamentous fungal pathogen , the causal agent of the devastating blast diseases of rice and wheat, septins have been shown to be essential for plant infection. The blast fungus elaborates a specialised infection structure called an appressorium with which it mechanically ruptures the plant cuticle. Septin aggregation and generation of a hetero-oligomeric ring structure at the base of the infection cell is indispensable for plant infection. Furthermore, once the fungus enters host tissue it develops another infection structure, the transpressorium, enabling it to move between living host plant cells, which also requires septins for its function. Specific inhibition of septin aggregation-either genetically or with chemical inhibitors-prevents plant infection. Significantly, by screening for inhibitors of septin aggregation, broad spectrum anti-fungal compounds have been identified that prevent rice blast and a number of other cereal diseases in field trials. We review the recent advances in our understanding of septin biology and their potential as targets for crop disease control.
PubMed: 38947556
DOI: 10.1007/s41348-024-00883-4 -
The alarmin IL-33 exacerbates pulmonary inflammation and immune dysfunction in SARS-CoV-2 infection.IScience Jun 2024Dysregulated host immune responses contribute to disease severity and worsened prognosis in COVID-19 infection and the underlying mechanisms are not fully understood. In...
Dysregulated host immune responses contribute to disease severity and worsened prognosis in COVID-19 infection and the underlying mechanisms are not fully understood. In this study, we observed that IL-33, a damage-associated molecular pattern molecule, is significantly increased in COVID-19 patients and in SARS-CoV-2-infected mice. Using IL-33 mice, we demonstrated that IL-33 deficiency resulted in significant decreases in bodyweight loss, tissue viral burdens, and lung pathology. These improved outcomes in IL-33 mice also correlated with a reduction in innate immune cell infiltrates, i.e., neutrophils, macrophages, natural killer cells, and activated T cells in inflamed lungs. Lung RNA-seq results revealed that IL-33 signaling enhances activation of inflammatory pathways, including interferon signaling, pathogen phagocytosis, macrophage activation, and cytokine/chemokine signals. Overall, these findings demonstrate that the alarmin IL-33 plays a pathogenic role in SARS-CoV-2 infection and provides new insights that will inform the development of effective therapeutic strategies for COVID-19.
PubMed: 38947521
DOI: 10.1016/j.isci.2024.110117