Cells Stained Research Articles

Enabling high-sensitivity live single-cell mass spectrometry using an integrated electrical lysis and nano electrospray ionization interface

BackgroundLive single-cell metabolomic studies encounter inherent difficulties attributed to the limited sample volume, minimal compound quantity, and insufficient sensitivity in the Mass Spectrometry (MS) method used to obtain single-cell data. However, understanding cellular heterogeneity, functional diversity, and metabolic processes within individual cells is essential. Exploring how individual cells respond to stimuli, including drugs, environmental changes, or signaling molecules, offers insights into biology, oncology, and drug discovery. Efficient release of cell contents (lysis) is vital for accurate metabolite detection at the single-cell level. Despite this, traditional approaches in live single cell metabolomics methods do not emphasize efficient lysis to prevent sample dilution. Instead, current live single cell metabolomics methods use direct infusion to introduce the cell into the mass spectrometry without prior chromatographic separation or a lysis step, which adversely affects sensitivity and metabolic coverage. ResultsTo address this, we developed an integrated single-cell electrical lysis and nano spray (SCEL-nS) platform coupled to an Orbitrap MS capable of efficiently lysing a single cell after being sampled with specially manufactured micropipettes. Lysis efficiency was validated by comparing live cell stain fluorescent intensities of intact and electrically lysed cells through microscopy imaging. The SCEL-nS platform successfully induced the breakdown of a single cell, significantly reducing the live cell stain's fluorescent intensity indicating cell membrane breakdown. Additionally, SCEL-nS was validated by measuring single cells spiked with the anti-cancer drug tamoxifen by MS. SCEL-nS use resulted in statistically significant increase in the peak measured by the method compared to the traditional non-lysis method. SignificanceOverall, our results demonstrate that the newly incorporated SCEL-nS platform achieved higher sensitivities compared to traditional live single cell analysis methods.

The effects of combustible cigarettes and electronic nicotine delivery systems on immune cell-driven inflammation and mucosal healing in ulcerative colitis.

The effects of combustible cigarettes (CCs) and electronic nicotine delivery systems (ENDS) on immune cell-driven colon inflammation and intestinal healing of patients with ulcerative colitis (UC) are still unknown and, therefore, were examined in this study. Intracellular staining and flow cytometry analysis of immune cells isolated from UC patients who used ENDS (UCENDS), CCs (UCCC) and who were non-smokers (UCAIR) were performed to elucidate cellular mechanisms which were responsible for CCs and ENDS-dependent modulation of immune response during UC progression. Additionally, dextran sulfate sodium (DSS)-colitis was induced in ENDS/CC/air-exposed mice (DSSENDS/ DSSCC/DSSAIR groups) to support clinical findings. Significantly increased number of immunosuppressive, IL-10, TGF-β and IL-35-producing, FoxP3-expressing CD3+CD4+T regulatory cells (Tregs) was observed in the blood of UCENDS patients while reduced presence of inflammatory, TNF-α and IFN-γ-producing, Tbx21-expressing CD3+CD4+ Th1, IL-4-producing Gata3-expresing Th2 and IL-17, IL-22-producing, RORγT, IL-23R-expressing Th17 cells were noticed in the blood of UCCC patients. Exposure to either CCs or ENDS was associated with enhanced mucosal healing, ameliorated spontaneous recovery and improved survival of DSS-treated mice. An expansion of immunosuppressive cells (IL-10-producing tolerogenic CD11c+ dendritic cells, alternatively activated CD206, Arginase 1-expressing, IL-10-producing F4/80+macrophages, IL-10-producing FoxP3-expressing Tregs) was noticed in the colons of DSSENDS-treated mice, while reduced number of inflammatory, IL-17- and IL-4-producing T lymphocytes was observed in the colons of DSSCC-compared to DSSAIR-treated mice. Despite different mechanisms of action, both ENDS and CCs attenuated on-going colon inflammation, enhanced healing and ameliorated recovery of injured intestines of DSS-treated mice and UC patients. This is the first study that compared the effects of CCs and ENDS on immune cells of patients suffering from ulcerative colitis, providing new information about molecular and cellular mechanisms which were responsible for ENDS and CCs-dependent modulation of immune cell-driven colon injury and inflammation. Obtained results showed that both ENDS and CCs had capacity to attenuate detrimental immune response, enhanced healing and ameliorated recovery of injured intestines.

Abstract We008: Hallmark Maturation of the Human Pluripotent Stem Cells-derived Cardiovascular Progenitors in Myocardial Infarcted Large Animal Model

For the goal of regenerative cardiology, cellular therapy may be used to restore injured heart muscle. The molecular mechanisms behind the engraftment and maturation of these transplanted cells, however, remain poorly understood. Thus, to obtain a comprehensive understanding of the complex transcriptional landscapes within their transplanted spatial context, we utilized spatial transcriptomics in this study. We have previously demonstrated the safety and effectiveness of transplanting our cardiovascular progenitors (CVPs) produced from laminin-221 into pig hearts that had suffered myocardial infarction (MI). Here, we undertake 10x spatial transcriptomics in chronic MI pig models at 1-, 4-, and 12 weeks post-transplantation to further our understanding of these cells in vivo. With confidence, we can distinguish between the transcripts from the pigs and the humans in the infarcted region. By using human-specific anti-Ku80 antibodies for immunohistochemical labeling of the pig tissue samples, this in silico prediction was verified. The CVPs' engraftment and longevity in the infracted region were shown by the long-term identification and staining of human cells at various time points. It is interesting to note that after 12 weeks, ribosomal and mitochondrial activity were higher than at 1 and 2 weeks. These results are consistent with the hallmarks of cardiac maturation, in which cells transit from immature fetal-like cells to mature cardiomyocytes (CMs) by switching their metabolic energy source from glucose to fatty acids. Also, by comparing the heart-specific genes, including MYH6 and MYH7, we were able to quantify the myosin isoform switches in the xenograft over time. Furthermore, to delineate the fate of the transplanted cells, we performed a ligand-receptor interaction analysis. Intriguing, we identified the midkine protein as a possible secreted human paracrine factor that enhances angiogenesis in the graft. In conclusion, we have shown the successful engraftment and maturation of human CVPs toward CMs using spatial transcriptomic technology. These data will be used to understand other aspects of regenerative cardiology such as the mechanism of VTs and in vivo cellular repair mechanisms. A shiny app atlas has been generated to share our transcriptomics data for all to explore gene expression of human transplanted cells in myocardial infarcted pig hearts.

Antioxidant properties and bioactivity of three marine microalgae on human cancer cell lines

ABSTRACT Marine microalgae offer significant potential as sustainably cultivable microorganisms for the biorefining of a diverse array of functional metabolites. In this study, three species of marine microalgae, Tetraselmis chuii Butcher CCAP 66/21B (Chlorophyte), Phaeodactylum tricornutum Bohlin CCAP 1052/1 (Diatom) and Amphidinium carterae Hulburt CCAP 1102/8 (Dinoflagellate), were batch cultivated and their biomass retrieved for analysis in terms of pigment and fatty acid composition as well as bioactivity potential. Sequential solvent maceration of the biomass returned extracts was used to determine antioxidant capacity and assess their growth inhibition potential against A549 (a human lung carcinoma) and MCF-7 (a breast carcinoma) cell lines. The extracts of A. carterae showed higher antioxidant capacity (≤ 33.5 μmol Trolox Equivalent mg Recovered Dried Material‒1) compared to the other extracts. No major cytotoxic activity nor production of reactive oxygen species was observed using the extracts of T. chuii and P. tricornutum. However, the methanolic extraction of A. carterae inhibited the growth of both A549 and MCF-7 cells (< 4%) at 25, 50 and 100 µg ml−1 concentrations, as determined using the MTS assay and AO/PI cell staining. Microalgal metabolites have the potential for practical applications in a variety of industrial sectors. The absence of toxicity and substantial antioxidant potential of extracts of T. chuii and P. tricornutum confirm their suitability for incorporation into animal feeds or human nutrition supplements. A. carterae also appears to be a promising source of bioactives that can be further valorized via multi-compound biorefinery pipelines.

The Expression of Cyclooxygenase-2 in Cervical Intraepithelial Neoplasia and Cervical Cancer.

Aim To examine the relationship between tumor differentiation, parametrial, and lymphovascular invasion, as well as the differential expression pattern of cyclooxygenase-2 (COX-2) in cervical intraepithelial neoplasia and various forms of cervical cancer. Methods Histologically diagnosed cases of in-situ and malignant lesions of the cervix were included in the study. Two sections were cut from paraffin blocks. One section was stained with Haematoxylin and Eosin (H&E) for morphologic diagnosis, and the other sections were subjected to COX-2 immunohistochemical staining. Cases of colon carcinoma were taken as positive controls. Cytoplasmic and membrane staining of tumor cells were considered as positive staining, and grading was done. Results Out of the 62 patients, 40 cases (64.5%) showed positive expression of COX-2 in squamous cell carcinoma when compared to in-situ cervical intraepithelial neoplasia and adenocarcinoma. The results were statistically significant, with a p-value of 0.003. Conclusion COX-2 expression is directly proportional to the level of grading of the tumor. The higher the grading, the higher the expression of COX-2. Selective COX-2 inhibitors increase the efficacy of chemotherapy or radiotherapy.

AB043. BRD4 promotes immune escape of glioma cells by upregulating PD-L1 expression.

Glioblastoma multiforme (GBM) poses significant challenges in treatment due to its aggressive nature and immune escape mechanisms. Despite recent advances in immune checkpoint blockade therapies, GBM prognosis remains poor. The role of bromodomain and extraterminal domain protein 4 (BRD4) in GBM, especially its interaction with immune checkpoints, is not well understood. Bioinformatic gene expression and survival analysis for BRD4 was utilized in The Cancer Genome Atlas (TCGA) and Chinese Glioma Genome Atlas (CGGA) databases. Clone formation assay, Transwell, Cell Counting Kit-8 (CCK8), and wound healing assay were utilized to validate BRD4's promotion of glioma cell proliferation, invasion, and migration. Chromatin immunoprecipitation (ChIP) assay was conducted to confirm BRD4 binding to the programmed death ligand 1 (PD-L1) promoter. A co-culture model was utilized with activated cluster of differentiation 8 (CD8)+ T cells and glioma cells. GL261 cells with BRD4 short hairpin RNA (shRNA) and/or PD-L1 cDNA were intracranially injected into mice to investigate tumor growth and survival time. Tumor tissue characteristics were analyzed using hematoxylin-eosin (H&E) and immunohistochemistry (IHC) staining and immune cell infiltration were assessed by flow cytometry. Bioinformatics analyses reveal elevated BRD4 expression in high-grade gliomas, correlating with poor patient survival. In vitro studies confirm that BRD4 promotes proliferation, invasion, and migration in GBM cells. BRD4 is a regulator of PD-L1 at the transcriptional level, implying its involvement in GBM's immune escape mechanisms. Co-culture experiments with CD8+ T cells demonstrate that BRD4 inhibition enhances tumor cell apoptosis. In vivo studies indicate that BRD4 knockout reduces immunosuppression, improves prognosis. Simultaneous manipulation of BRD4 and PD-L1 levels provides insights into their intertwined roles in shaping the immune landscape of GBM. BRD4 has the capability to regulate the growth of glioblastoma and enhance immune suppression by promoting PD-L1 expression. Targeting BRD4 represents a promising direction for future research and treatment.

Semiquantitative assessment of phosphatase and tensin homolog value with immunohistochemistry in colorectal cancer.

Colorectal cancer has emerged as a concerning health problem, ranking the third most common form of cancer in both men and women. The phosphatase and tensin homologue (PTEN) protein is widely known for its role as an inhibitor of the phosphatidylinositol 3-kinase/protein kinase-B/mammalian target of rapamycin (PI3K/Akt/mTOR) pathway, playing a major role inhibiting tumor development. Previous studies investigated the role of this protein in the PI3K pathway and how it affected colorectal cancer. However, a standardized cut-off value for PTEN expression has not been established. Immunohistochemistry was used in examining PTEN. The staining grade ranging from 0 to 3 was then multiplied by the number of 100 cancer cells counted, with total score between 0 and 300. In this study, receiver operating characteristic (ROC) curve was employed to determine the expression cut-off value for PTEN in colorectal cancer. This study showed statistically significant results (P < 0.001) in either tumor or non-tumor tissues by using the ROC curve with a cut-off value of 199.0. This study also revealed significant correlation between nodal status with PTEN (P = 0.008) and stage with PTEN (P = 0.019) with sensitivity 0.753 and specificity 0.728. Semiquantitative assessment with cell counting multiplied by color intensity is a good method in determining PTEN expression. The use of immunohistochemical staining intensity and cell scoring with ROC cut-off is effective to elaborate the effects of PTEN in colorectal cancer (PTEN value > 199.0 was classified as strong and ≤ 199.0 as weak).

Generation of Murine Chimeric Antigen Receptor-Modified T Cells for In Vivo Studies in Syngeneic Tumor Models.

CAR-T cell therapy has emerged as a potent and effective tool in the immunotherapy of refractory cancers. However, challenges exist in their clinical application, necessitating extensive preclinical research to optimize their function. Various preclinical in vitro and in vivo models have been proposed for such purpose; among which immunocompetent mouse models serve as an invaluable tool in studying host immune interactions within a more realistic simulation of the tumor milieu. We hereby describe a standardized protocol for the generation of high-titer γ-retroviral vectors through transfection of the HEK293T packaging cell line. The virus-containing supernatant is further concentrated using an inhouse concentrator solution, titrated, and applied to mouse T cells purified via a convenient and rapid method by nylon-wool columns. Using the method presented here, we were able to achieve high titer γ-retrovirus and highly pure mouse T cells with desirable CAR transduction efficiency. The mouse CAR T cells produced through this protocol demonstrate favorable CAR expression and viability, thus making them suitable for further in vitro/in vivo assays. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Production of γ-retroviral vectors from retrovirus-backbone plasmids Basic Protocol 2: Concentration of γ-retrovirus-containing supernatants Basic Protocol 3: Titration of concentrated γ-retrovirus Basic Protocol 4: Isolation and activation of mouse T cells Basic Protocol 5: Transduction of activated mouse T cells, assessment of CAR expression, and expansion of CAR T cells for further in vitro/in vivo studies Support Protocol: Surface staining of cells for flow cytometric assessment of CAR expression.

Possible mechanisms underlying the regulation of postmenopausal osteoporosis by follicle-stimulating hormone

ObjectiveTo explore the possible mechanisms by which follicle-stimulating hormone (FSH) regulates postmenopausal osteoporosis through the FSH/FSH receptor (FSHr)/G protein/C/EBPβ/heat shock protein 90 alpha (HSP90α) signalling pathways. MethodsWe measured serum FSH, luteinising hormone (LH), and HSP90α levels in the serum and adipose tissue of women of childbearing age and menopausal status. In the in vivo studies, 12 B57CL female mice were divided equally into Sham, OVX, and OVX + FSHr Blocker groups. Serum levels of alkaline phosphatase, FSH, and HSP90α, along with StRACP vitality, were determined, and femur micro-computed tomography was performed. Additionally, FSH, FSHr, G protein, C/EBPβ, and HSP90α levels were assessed using quantitative polymerase chain reaction. Finally, we divided the human multiple myeloma cell line U266 into three groups. The activity of tartrate-resistant acid phosphatase (TRAP) in the supernatant at different stages was detected, and myeloma cells were stained with TRAP. ResultsHSP90α levels in adipose tissue supernatant and serum were lower in women of childbearing age than in menopausal women (P < 0.05). Serum FSH and HSP90α levels demonstrated a strong correlation. Treatment with FSHr blockers resulted in decreased FSH, FSHr, G protein, C/EBPβ, and HSP90α levels in mice. TRAP staining of osteoclast-like cells exhibited a significantly higher intensity in the M-CSF + RANKL + recombinant HSP90α group than in the M-CSF + RANKL and blank control groups (P < 0.05). ConclusionsOur results indicate that FSH promotes HSP90α secretion by adipocytes via the FSHr/G protein/C/EBPβ pathway. This mechanism affects osteoclast activity and exacerbates osteoporosis.

Rhus coriaria induces autophagic and apoptotic cell death in pancreatic cancer cells.

Background:Pancreatic cancer is a leading cause of cancer-related mortality worldwide with increasing global incidence. We previously reported the anticancer effect of Rhus coriaria ethanolic extract (RCE) in triple negative breast and colon cancer cells. Herein, we investigated the anticancer effect of RCE on human pancreatic cancer cells. Methods: Cell viability was measured using Cell Titer-Glo and staining of viable and dead cells based on differential permeability to two DNA binding dyes. Cell cycle distribution and annexin V staining was carried out in Muse cell analyzer. Protein level was determined by Western blot. Tumor growth was assessed by in ovo chick embryo chorioallantoic membrane assay. Results: We found that RCE significantly inhibited the viability and colony growth of pancreatic cancer cells (Panc-1, Mia-PaCa-2, S2-013, AsPC-1). The antiproliferative effects of RCE in pancreatic cancer cells (Panc-1 and Mia-PaCa-2) were mediated through induction of G1 cell cycle arrest, Beclin-1-independent autophagy, and apoptosis. RCE activated both the extrinsic and intrinsic pathways of apoptosis and regulated the Bax/Bcl-2 apoptotic switch. Mechanistically, we found that RCE inhibited the AKT/mTOR pathway, downstream of which, inactivation of the cell cycle regulator p70S6K and downregulation of the antiapoptotic protein survivin was observed. Additionally, we found that RCE-induced autophagy preceded apoptosis. Further, we confirmed the anticancer effect of RCE in a chick embryo xenograft model and found that RCE inhibited the growth of pancreatic cancer xenografts without affecting embryo survival. Conclusion: Collectively, our findings demonstrate that Rhus coriaria exerts potent anti-pancreatic cancer activity though cell cycle impairment, autophagy, and apoptosis, and is hence a promising source of anticancer phytochemicals.

How Can "No Specific Molecular Profile" Heterogeneity be Reduced in Molecularly Classified Endometrial Cancer?: Prognostic Significance of L1 Cell Adhesion Molecule.

This study aims to investigate the role of L1 cell adhesion molecule (L1CAM) in the prognostic assessment of endometrial cancers that have been depicted as having no specific molecular profile (NSMP) in molecular classification. This is a retrospective review of 150 patients who received the diagnosis of endometrial cancer and underwent surgery at the study center between January 2008 and January 2022. When evaluating L1CAM immunohistochemical staining, scoring was done according to the percentage of positivity in tumor cells. Accordingly, score 0 = 0%, score 1=1% to 10%, score 2 = >10% to 50% and score 3 = >50%. If the staining in tumor cells was ≥10% (scores 2 and 3), it was considered positive. The patients with L1CAM positivity had significantly more frequent lymphovascular space invasion and lymph node metastasis than patients with L1CAM negativity (P = 0.013 and P = 0.007). L1CAM expression was strongly associated with mutant p53 (P = 0.003). Recurrence was significantly higher (P = 0.001) and overall survival and progression-free survival were significantly lower in patients with L1CAM positivity (P = 0.001 for both). Seventy-nine patients (52.7%) were put into NSMP group. About 84.8% of them (n = 67) were L1CAM negative and 15.2% of them (n = 12) were L1CAM-positive. Recurrence was significantly higher (P = 0.001) and overall survival and progression-free survival were significantly lower in patients with NSMP who were positive for L1CAM (P = 0.002 and P = 0.001, respectively). This study demonstrates that L1CAM expression status may add prognostic information to endometrial cancer, particularly in the NSMP subgroup. Considering the prognostic importance of L1CAM, its use as a marker may make significant contributions to reducing prognostic heterogeneity, especially in the NSMP subgroup.

Porphyromonas gingivalis promote microglia M1 polarization through the NF-кB signaling pathway

BackgroundPorphyromonasgingivalis (P.gingivalis) is associated with the onset of Alzheimer's disease (AD), but the underlying molecular mechanism is unclear. Neuroinflammation in the brain from the microglial immune response induces the pathological progression of AD. In this study, the roles and molecular mechanism of P.gingivalis in microglial inflammation in vitro were investigated. MethodsIn this study, a P.gingivalis oral administration mouse model was generated, and microglia were stimulated with P.gingivalis in vitro. The viability of the microglia after P.gingivalis treatment was evaluated through CCK-8 and live/dead cell staining. Inflammation in brain tissue after P.gingivalis treatment and the immune response of microglia in vitro were detected by RT‒PCR, Western blotting and IF. Moreover, the RNA sequence was used, and the role of the NF-κB signalling pathway in microglial activation was analysed after P.gingivalis stimulation. ResultsThe mRNA and protein levels of IL-6 and IL-17 were increased, and the expression of IL-10 was decreased in brain tissue after P.gingivalis oral administration. The viability of the HMC3 cells significantly decreased with 5% P.gingivalis after stimulation. The results of live/dead cell staining also showed the inhibitory effect of 5% P.gingivalis supplementation on cell viability. Moreover, 5% P.gingivalis supplementation increased the mRNA and protein levels of IL-6 and IL-17 and decreased IL-10 expression in HMC3 cells. P.gingivalis supplementation increased the mRNA and protein levels of iNOS and CD86 and decreased CD206 expression in HMC3 cells. RNA sequencing revealed that the NF-κB signalling pathway was involved in this process. Furthermore, p-P65 was upregulated and p-IKBα was downregulated in brain tissue and HMC3 cells after P.gingivalis stimulation, and an NF-κB signalling pathway inhibitor (QNZ) reversed the viability, M1 polarization and inflammatory factors of microglia in HMC3 cells in vitro. ConclusionsIn conclusion, P.gingivalis induced neuroinflammation in the brain, possibly through promotion of M1 polarization of microglia via activation of the NF-κB signalling pathway during the progression of AD.

Fu-zi decoction attenuate rheumatoid arthritis in vivo and in vitro by modulating RANK/RANKL signaling pathway.

Fu-zi decoction (FZD) has a long history of application for treating Rheumatoid arthritis (RA) as a classic formulation. However, its underlying mechanisms have not been fully elucidated. This study aimed to decipher the potential mechanism of FZD in treating RA, with a specific focus on receptor activator of nuclear factor κB/receptor activator of nuclear factor κB ligand (RANK/RANKL) signaling pathway. The impact of FZD on RA was investigated in collagen-induced arthritis rats (CIA), and the underlying mechanism was investigated in an osteoclast differentiation cell model. In vivo, the antiarthritic effect of FZD at various doses (2.3, 4.6, 9.2g/kg/day) was evaluated by arthritis index score, paw volume, toe thickness and histopathological examination of inflamed joints. Additionally, the ankle joint tissues were determined with micro-CT and safranin O fast green staining to evaluate synovial hyperplasia and articular cartilage damage. In vitro, osteoclast differentiation and maturation were evaluated by TRAP staining in RANKL-induced bone marrow mononuclear cells. The levels of pro- and anti-inflammatory cytokines as well as RANKL and OPG were evaluated by ELISA kits. In addition, Western blotting was used to investigate the effect of FZD on RANK/RANKL pathway activation both in vivo and in vitro. FZD significantly diminished the arthritis index score, paw volume, toe thickness and weigh loss in CIA rats, alleviated the pathological joint alterations. Consistent with in vivo results, FZD markedly inhibited RANKL-induced osteoclast differentiation by decreasing osteoclast numbers in a dose-dependent manner. Moreover, FZD decreased the levels of pro-inflammatory cytokines IL-6, IL-1β and TNF-α, while increasing anti-inflammatory cytokine IL-10 level both in serum and culture supernatants. Treatment with FZD significantly reduced serum RANKL levels, increased OPG levels, and decreased the RANKL/OPG ratio. In both in vivo and in vitro settings, FZD downregulated the protein expressions of RANK, RANKL, and c-Fos, while elevating OPG levels, further decreasing the RANKL/OPG ratio. In conclusion, FZD exerts a therapeutic effect in CIA rats by inhibiting RANK/RANKL-mediated osteoclast differentiation, which suggested that FZD is a promising treatment for RA.

β-Hydroxybutyrate Improves the Redox Status, Cytokine Production and Phagocytic Potency of Glucose-Deprived HMC3 Human Microglia-like Cells.

Brain glucose deprivation is a component of the pathophysiology of ischemia, glucose transporter1 (GLUT1) deficiency, neurological disorders and occurs transiently in diabetes. Microglia, the neuroimmune cells must function effectively to offer immune defence and debris removal in low-energy settings. Brain glucose deprivation may compromise microglial functions further escalating the disease pathology and deteriorating the overall mental health. In the current study, HMC3 human microglia-like cells were culturedin vitro and exposed to glucose deprivation to investigate the effects of glucose deprivation on phenotypic state, redox status, secretion of cytokines and phagocytic capabilities of HMC3 cells. However, HMC3 cells were able to proliferate in the absence of glucose but showed signs of redox imbalance and mitochondrial dysfunction, as demonstrated by decreased MTT reduction and Mito Tracker™ staining of cells, along with a concomitant reduction in NOX2 protein, superoxide, and nitrite levels. Reduced levels of secreted TNF and IL-1β were the signs of compromised cytokine secretion by glucose-deprived HMC3 microglia-like cells. Moreover, glucose-deprived HMC3 cells also showed reduced phagocytic activity as assessed by fluorescently labelled latex beads-based functional phagocytosis assay. β-hydroxybutyrate (BHB) supplementation restored the redox status, mitochondrial health, cytokine secretion, and phagocytic activity of glucose-deprived HMC3 microglia-like cells. Overall, impaired brain glucose metabolism may hinder microglia's capacity to release diffusible immune factors and perform phagocytosis. This could escalate the mental health issues in neurological diseases where brain glucose metabolism is compromised. Moreover, nutritional ketosis or exogenous ketone supplementation such as BHB may be utilized as a potential metabolic therapies for these conditions.

Identification of Quiescent Cells in a Zebrafish T-Cell Acute Lymphoblastic Leukemia Model Using Cell Proliferation Staining.

Cellular quiescence is a state of growth arrest or slowed proliferation that is described in normal and cancer stem cells (CSCs). Quiescence may protect CSCs from antiproliferative chemotherapy drugs. In T-cell acute lymphoblastic leukemia (T-ALL) patient-derived xenograft (PDX) mouse models, quiescent cells are associated with treatment resistance and stemness. Cell proliferation dyes are popular tools for the tracking of cell division. The fluorescent dye is covalently anchored into amine groups on the membrane and macromolecules inside the cell. This allows for the tracking of labeled cells for up to 10 divisions, which can be resolved by flow cytometry. Ultimately, cells with the highest proliferation rates will have low dye retention, as it will be diluted with each cell division, while dormant, slower-dividing cells will have the highest retention. The use of cell proliferation dyes to isolate dormant cells has been optimized and described in T-ALL mouse models. Complementary to the existing mouse models, the rag2:Myc-derived zebrafish T-ALL model provides an excellent venue to interrogate self-renewal in T-ALL due to the high frequency of leukemic stem cells (LSCs) and the convenience of zebrafish for large-scale transplant experiments. Here, we describe the workflow for the staining of zebrafish T-ALL cells with a cell proliferation dye, optimizing the concentration of the dye for zebrafish cells, passaging successfully stained cells in vivo, and the collection of cells with varying levels of dye retention by live cell sorting from transplanted animals. Given the absence of well-established cell surface makers for LSCs in T-ALL, this approach provides a functional means to interrogate quiescent cells in vivo. For representative results, we describe the engraftment efficiency and the LSC frequency of high and low dye-retaining cells. This method can help investigate additional properties of quiescent cells, including drug response, transcriptional profiles, and morphology.

Ribosomal Incorporation of Lithocholic Acid into Peptides for the De Novo Discovery Of Peptide-Lithocholic Acid Hybrid Macrocyclic Peptides.

Peptide-bile acid hybrids offer promising drug candidates due to enhanced pharmacological properties, such as improved protease resistance and oral bioavailability. However, it remains unknown whether bile acids can be incorporated into peptide chains by the ribosome to produce a peptide-bile acid hybrid macrocyclic peptide library for target-based de novo screening. In this study, we achieved the ribosomal incorporation of lithocholic acid (LCA)-d-tyrosine into peptide chains. This led to the construction of a peptide-LCA hybrid macrocyclic peptide library, which enabled the identification of peptides TP-2C-4L3 (targeting Trop2) and EP-2C-4L5 (targeting EphA2) with strong binding affinities. Notably, LCA was found to directly participate in binding to EphA2 and confer on the peptides improved stability and resistance to proteases. Cell staining experiments confirmed the high specificity of the peptides for targeting Trop2 and EphA2. This study highlights the benefits of LCA in peptides and paves the way for de novo discovery of stable peptide-LCA hybrid drugs.

Oxytocin Attenuates Sympathetic Innervation with Inhibition of Cardiac Mast Cell Degranulation in Rats after Myocardial Infarction.

Sympathetic hyperinnervation is the leading cause of fatal ventricular arrhythmia (VA) after myocardial infarction (MI). Cardiac mast cells cause arrhythmias directly through degranulation. However, the role and mechanism of mast cell degranulation in sympathetic remodeling remain unknown. We investigated the role of oxytocin (OT) in stabilizing cardiac mast cells and improving sympathetic innervation in rats. MI was induced by coronary artery ligation. Western blotting, immunofluorescence, and toluidine staining of mast cells were performed to determine the expression and location of target protein. Mast cells accumulated significantly in peri-infarcted tissues and were present in a degranulated state. They expressed OT receptor (OTR), and OT infusion reduced the number of degranulated cardiac mast cells post-MI. Sympathetic hyperinnervation was attenuated as assessed by immunofluorescence for tyrosine hydroxylase (TH). Seven days post-MI, the arrhythmia score of programmed electrical stimulation was higher in vehicle-treated rats with MI than in rats treated with OT. An in vitro study showed that OT stabilized mast cells via the phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) signaling pathway. Further in vivo studies on OTR-deficient mice showed worsening mast cell degranulation and worsening sympathetic innervation. OT pretreatment inhibited cardiac mast cell degranulation post-MI and prevented sympathetic hyperinnervation, along with mast cell stabilization via the PI3K/Akt pathway. SIGNIFICANCE STATEMENT: This is the first study to elucidate the role and mechanism of oxytocin (OT) in inflammatory-sympathetic communication mediated sympathetic hyperinnervation after myocardial infarction (MI), providing new approaches to prevent fatal arrhythmias.

In vitro cultivation of adherent cells on microscope slides for downstream applications

In vitro studies with cultured cells are often conducted as an important part of basic research. Adherent cells are typically cultivated in flasks or trays, for which cell staining and subsequent visualization become impractical. We here present a simple step-by-step method for growing adherent cells directly on glass microscope slides, using low-cost equipment readily available in most laboratories. Most parameters such as type of microscope slide (e.g. surface coating), cell seeding concentrations and incubation times can be adjusted according to cell line characteristics and experimental aims, reflecting the methods’ flexibility. Through our experiments, microscope slides proved to provide an acceptable surface for cell adhesion and growth of the tested cell lines, as well as being robust and functional with respect to downstream procedures. The method can potentially be combined with different techniques for visualization of experimental effects, such as histological staining methods, fluorescent staining, and immunochemistry. In our method development we have successfully cultivated three different cell lines directly on microscope slides – Atlantic salmon kidney cells (ASK), rainbow trout gill cells (RTgill-W1), and human cancerous lung cells (A549) – and subjected them to various experimental treatments. Finally, as proof-of-concept we provide examples of successful histological staining of the fixed cells.Experimental design in short:•Cultivate cells and calculate cell concentration•Seed a small volume of growth medium with an appropriate number of cells on microscope slide in an area confined by hydrophobic marker•Let cells adhere over night before adding more growth medium or directly conducting experiments and fixing cells for downstream applications

Hypoaldosteronism due to a novel SEC61A1 variant successfully treated with fludrocortisone.

Genetic variants in SEC61A1 are associated with autosomal dominant tubulointerstitial kidney disease. SEC61A1 is a translocon in the endoplasmic reticulum membrane and variants affect biosynthesis of renin and uromodulin. A patient is described that presented at 1 year of age with failure-to-thrive, kidney failure (glomerular filtration rate, GFR, 18 ml/min/1.73m2), hyperkalemia and acidosis. Genetic evaluation was performed by whole genome sequencing. The patient has a novel de novo heterozygous SEC61A1 variant, Phe458Val. Plasma renin was low or normal, aldosterone was low or undetectable and uromodulin was low. Kidney biopsy at 2 years exhibited subtle changes suggestive of tubular dysgenesis without tubulocystic or glomerulocystic lesions and with renin staining of the juxtaglomerular cells. The patient experienced extreme fatigue due to severe hypotension attributed to hypoaldosteronism and at 8 years of age fludrocortisone treatment was initiated with marked improvement in her well-being. Blood pressure and potassium normalized. Biopsy at 9 years showed extensive glomerulosclerosis and mild tubulointerstitial fibrosis, as well as tubular mitochondrial abnormalities, without specific diagnostic changes. Her GFR improved to 54 ml/min/1.73m2. As the renin-angiotensin system promotes aldosterone release, and the patient had repeatedly undetectable aldosterone levels, the SEC61A1 variant presumably contributed to severe hypotension. Treatment with a mineralocorticoid had a beneficial effect and corrected the electrolyte and acid-base disorder. We suggest that the increased blood pressure hemodynamically improved the patient's kidney function.

Kinin B1 receptor and TLR4 interaction in inflammatory response.

We aimed to broaden our understanding of a potential interaction between B1R and TLR4, considering earlier studies suggesting that lipopolysaccharide (LPS) may trigger B1R stimulation. We assessed the impact of DBK and LPS on the membrane potential of thoracic aortas from C57BL/6, B1R, or TLR4 knockout mice. Additionally, we examined the staining patterns of these receptors in the thoracic aortas of C57BL/6 and in endothelial cells (HBMEC). DBK does not affect the resting membrane potential of aortic rings in C57BL/6 mice, but it hyperpolarizes preparations in B1KO and TLR4KO mice. The hyperpolarization mechanism in B1KO mice involves B2R, and the TLR4KO response is independent of cytoplasmic calcium influx but relies on potassium channels. Conversely, LPS hyperpolarizes thoracic aorta rings in both C57BL/6 and B1KO mice, with the response unaffected by a B1R antagonist. Interestingly, the absence of B1R alters the LPS response to potassium channels. These activities are independent of nitric oxide synthase (NOS). While exposure to DBK and LPS does not alter B1R and TLR4 mRNA expression, treatment with these agonists increases B1R staining in endothelial cells of thoracic aortic rings and modifies the staining pattern of B1R and TLR4 in endothelial cells. Proximity ligation assay suggests a interaction between the receptors. Our findings provide additional support for a putative connection between B1R and TLR4 signaling. Given the involvement of these receptors and their agonists in inflammation, it suggests that drugs and therapies targeting their effects could be promising therapeutic avenues worth exploring.