Cultured Mouse Cells Research Articles

Characterization of the TLR9-Activating Potential of LNA-Modified Antisense Oligonucleotides.

Early characterization of the immunostimulatory potential of therapeutic antisense oligonucleotides (ASOs) is crucial. At present, little is known about the toll-like receptor 9 (TLR9)-mediated immunostimulatory potential of third-generation locked nucleic acid (LNA)-modified ASOs. In this study, we have systematically investigated the TLR9-activating potential of LNA-modified oligonucleotides using different mouse and human cell culture systems. Although it has been reported that LNA modifications as well as cytosine methylation of 5'-cytosine-phosphate-guanine-3' (CpG) motifs can reduce TLR9 stimulation by phosphorothioate (PTO)-modified oligonucleotides, we identified CpG-containing LNA gapmers with substantial TLR9-stimulatory activity. We further identified immunostimulatory LNA gapmers without CpG motifs. Unexpectedly, methylation of cytosines only within the CpG motif did not necessarily reduce but could even increase TLR9 activation. In contrast, systematic methylation of all cytosines reduced or even abrogated TLR9 activation in most cases. Context dependently, the introduction of LNA-modifications into the flanks could either increase or decrease TLR9 stimulation. Overall, our results indicate that TLR9-dependent immunostimulatory potential is an individual feature of an oligonucleotide and needs to be investigated on a case-by-case basis.

Role of transforming growth factor-β1 pathway in angiogenesis induced by chronic stress in colorectal cancer

ABSTRACT Background Chronic stress can induce stress-related hormones; norepinephrine (NE) is considered to have the highest potential in cancer. NE can stimulate the expression of hypoxia-inducible factor-1α (HIF-1α), which is associated with vascular endothelial growth factor (VEGF) secretion and tumor angiogenesis. However, the underlying mechanisms are poorly understood. Methods Tumor-bearing mice were subjected to chronic restraint stress and treated with normal saline, human monoclonal VEGF-A neutralizing antibody bevacizumab, or β-adrenergic receptor (β-AR) antagonist (propranolol). Tumor growth and vessel density were also evaluated. Human colorectal adenocarcinoma cells were treated with NE, propranolol, or the inhibitor of transforming growth factor-β (TGF-β) receptor Type I kinase (Ly2157299) in vitro. TGF-β1 in mouse serum and cell culture supernatants was quantified using ELISA. The expression of HIF-1α was measured using Real time-PCR and western blotting. Cell migration and invasion were tested. Results Chronic restraint stress attenuated the efficacy of bevacizumab and promoted tumor growth and angiogenesis in a colorectal tumor model. Propranolol blocked this effect and inhibited TGF-β1 elevation caused by chronic restraint stress or NE. NE upregulated HIF-1α expression, which was reversed by propranolol or Ly2157299. Propranolol and Ly2157199 blocked NE-stimulated cancer cell migration and invasion. Conclusions Our results demonstrate the effect of NE on tumor angiogenesis and the critical role of TGF-β1 signaling during this process. In addition, β-AR/TGF-β1 signaling/HIF-1α/VEGF is a potential signaling pathway. This study also indicates that psychosocial stress might be a risk factor which weakens the efficacy of anti-angiogenic therapy.

The basement membrane regulates the cellular localization and the cytoplasmic interactome of Yes-Associated Protein (YAP) in mammary epithelial cells.

The Hippo pathway, a signaling cascade involved in the regulation of organ size and several other processes, acts as a conduit between extracellular matrix (ECM) cues and cellular responses. We asked whether the basement membrane (BM),a specialized ECM component known to induce quiescence and differentiation in mammary epithelial cells, would regulate the localization, activity, and interactome of YAP, a Hippo pathway effector. To address this question, we used a broad range of experimental approaches, including 2D and 3D cultures of both mouse and human mammary epithelial cells, as well as the developing mouse mammary gland. In contrast to malignant cells, nontumoral cells cultured with a reconstituted BM (rBM)displayed higher concentrations of YAP in the cytoplasm. Incidentally, when in the nucleus of rBM-treated cells, YAP resided preferentially at the nuclear periphery. In agreement with our cell culture experiments, YAP exhibited cytoplasmic predominance in ductal cells of developing mammary epithelia, where a denser BM is found. Conversely, terminal end bud (TEB)cells with a thinner BM displayed higher nucleus-to-cytoplasm ratios of YAP. Bioinformatic analysis revealed that genes regulated by YAP were overrepresented in the transcriptomes of microdissected TEBs. Consistently, mouse epithelial cells exposed to the rBM expressed lower levels of YAP-regulated genes, although the protein level of YAP and Hippo components were slightly altered by the treatment. Mass spectrometry analysis identified a differential set of proteins interacting with YAP in cytoplasmic fractions of mouse epithelial cells in the absence or presence of rBM. In untreated cells, YAP interactants were enriched in processes related to ubiquitin-mediated proteolysis, whereas in cells exposed to rBM YAP interactants were mainly key proteins related to amino acid, amino sugar, and carbohydrate metabolism. Collectively, we unraveled that the BM induces YAP translocation or retention in the cytoplasm of nontumoral epithelial cells and that in the cytoplasm YAP seems to undertake novel functions in metabolic pathways.

Validation of GCN5L1/BLOC1S1/BLOS1 antibodies using knockout cells and tissue.

GCN5L1, also known as BLOC1S1 and BLOS1, is a small intracellular protein involved in many key biological processes. Over the last decade, GCN5L1 has been implicated in the regulation of protein lysine acetylation, energy metabolism, endo-lysosomal function, and cellular immune pathways. An increasing number of published papers have used commercially-available reagents to interrogate GCN5L1 function. However, in many cases these reagents have not been rigorously validated, leading to potentially misleading results. In this report we tested several commercially-available antibodies for GCN5L1, and found that two-thirds of those available did not unambiguously detect the protein by western blot in cultured mouse cells or ex vivo liver tissue. These data suggest that previously published studies which used these unverified antibodies to measure GCN5L1 protein abundance, in the absence of other independent methods of corroboration, should be interpreted with appropriate caution.

Endothelium-specific SIRT7 targeting ameliorates pulmonary hypertension through Krüpple-like factor 4 deacetylation.

Pulmonary hypertension (PH) is a pulmonary vascular disease characterized by a high mortality rate. Pulmonary arterial endothelium cells (PAECs) serve as a primary sensor of various environmental cues, such as shear stress and hypoxia, but PAEC dysfunction may trigger vascular remodelling during the onset of PH. This study aimed to illustrate the role of Sirtuin 7 (SIRT7) in endothelial dysfunction during PH and explore the potential therapeutic strategy for PH. SIRT7 levels were measured in human and murine experimental PH samples. Bioinformatic analysis, immunoprecipitation, and deacetylation assay were used to identify the association between SIRT7 and Krüpple-like factor 4 (KLF4), a key transcription factor essential for endothelial cell (EC) homeostasis. Sugen5416 + hypoxia (SuHx)-induced PH mouse models and cell cultures were used for the study of the therapeutic effect of SIRT7 for PH. SIRT7 level was significantly reduced in lung tissues and PAECs from PH patients and the SuHx-induced PH mouse model as compared with healthy controls. Pulmonary endothelium-specific depletion of Sirt7 increased right ventricular systolic pressure and exacerbated right ventricular hypertrophy in the SuHx-induced PH model. At the molecular level, we identified KLF4 as a downstream target of SIRT7, which deacetylated KLF4 at K228 and inhibited the ubiquitination-proteasome degradation. Thus, the SIRT7/KLF4 axis maintained PAEC homeostasis by regulating proliferation, migration, and tube formation. PAEC dysfunction was reversed by adeno-associated virus type 1 vector-mediated endothelial overexpression of Sirt7 or supplementation with nicotinamide adenine dinucleotide (NAD)+ intermediate nicotinamide riboside which activated Sirt7; both approaches successfully reversed PH phenotypes. The SIRT7/KLF4 axis ensures PAEC homeostasis, and pulmonary endothelium-specific SIRT7 targeting might constitute a PH therapeutic strategy.

CYP2J2-derived epoxyeicosatrienoic acids protect against doxorubicin-induced cardiotoxicity by reducing oxidative stress and apoptosis via activation of the AMPK pathway

ObjectiveDespite the widespread use of doxorubicin (DOX) in chemotherapy, it can cause cardiotoxicity, which severely limits its potential clinical use. CYP2J2-derived epoxyeicosatrienoic acids (EETs) exert cardioprotective effects by maintaining cardiac homeostasis. The roles and latent mechanisms of EETs in DOX cardiotoxicity remain uncertain. We investigated these aspects using mouse tissue and cell culture models. MethodsC57BL/6J mice were injected with rAAV9-CYP2J2 or a control vector via the caudal vein. A five-week intraperitoneal course of DOX (5 mg/kg per week) was administered. After pretreatment with 14,15-EET, H9C2 cells were treated for 24-h with DOX, to use as a cell model to verify the role of EETs in cardiotoxicity in vitro. ResultsCYP2J2 overexpression mitigated DOX-induced cardiotoxicity, as shown by the diminished cardiac injury marker levels, improved heart function, reduced oxidative stress, and inhibition of myocardial apoptosis in vivo. These protective roles are associated with the enhancement of antioxidant and anti-apoptotic abilities and the activation of the AMPK pathway. 14,15-EET suppresses DOX-induced oxidative stress, mitochondrial dysfunction, and apoptosis in H9C2 cells. AMPK knockdown partially abolished the cardioprotective effects of 14,15-EET against oxidative damage and apoptosis in DOX-treated cells, suggesting that AMPK is responsible for EET-mediated protection against cardiotoxicity. ConclusionCYP2J2-derived EETs confer myocardial protection against DOX-induced toxicity by activating the AMPK pathway, which reduces oxidative stress, mitochondrial dysfunction, and apoptosis.

CHEX-seq detects single-cell genomic single-stranded DNA with catalytical potential

Genomic DNA (gDNA) undergoes structural interconversion between single- and double-stranded states during transcription, DNA repair and replication, which is critical for cellular homeostasis. We describe “CHEX-seq” which identifies the single-stranded DNA (ssDNA) in situ in individual cells. CHEX-seq uses 3’-terminal blocked, light-activatable probes to prime the copying of ssDNA into complementary DNA that is sequenced, thereby reporting the genome-wide single-stranded chromatin landscape. CHEX-seq is benchmarked in human K562 cells, and its utilities are demonstrated in cultures of mouse and human brain cells as well as immunostained spatially localized neurons in brain sections. The amount of ssDNA is dynamically regulated in response to perturbation. CHEX-seq also identifies single-stranded regions of mitochondrial DNA in single cells. Surprisingly, CHEX-seq identifies single-stranded loci in mouse and human gDNA that catalyze porphyrin metalation in vitro, suggesting a catalytic activity for genomic ssDNA. We posit that endogenous DNA enzymatic activity is a function of genomic ssDNA.

A Method for Eliminating Fibroblast Contamination in Mouse and Human Primary Corneal Epithelial Cell Cultures

Purpose This study was designed to determine if previous approaches to eliminate fibroblast contamination in different cells types would be successful in eliminating fibroblast contamination from human and mouse primary corneal epithelial cell cultures, with the primary goal being to describe a simple, easy, and effective method to culture fibroblast-free primary mouse and human corneal epithelial cell cultures. Methods Primary human and mouse corneal stromal cells and epithelial cells were isolated and cultured from human corneal rims and mouse corneas, respectively. Several approaches previously used in other tissue types were evaluated using corneal epithelial cells and mixtures of fibroblasts and epithelial cells to determine the most effective purification method. Methods evaluated included 0.25% trypsin-EDTA, low temperature, mitomycin-C, and dispase. Degree of fibroblast contamination was examined using light microscopy evaluation of cell phenotype, immunofluorescence and western blotting using cell type-specific markers. Anti-pancytokeratin (PanCK) was used as the epithelial immunofluorescence label, and anti-α smooth muscle actin (αSMA) as the fibroblast immunofluorescence label. Epithelial western blot antibodies included PanCK, keratin 12, and E-cadherin, while αSMA, collagen 1A1 and collagen 3A1 were used to identify fibroblasts. Results Fibroblast contamination of human and mouse primary cornea epithelial cell cultures was best controlled using the 0.25% trypsin-EDTA method. The other methods examined were not effective at eliminating cornea fibroblast contamination. Conclusions Trypsin-EDTA digestion is a simple and effective method for controlling fibroblast contamination of cultured primary human and mouse corneal epithelial cells.

Borrelia burgdorferi Engages Mammalian Type I IFN Responses via the cGAS-STING Pathway.

Borrelia burgdorferi, the etiologic agent of Lyme disease, is a spirochete that modulates numerous host pathways to cause a chronic, multisystem inflammatory disease in humans. B. burgdorferi infection can lead to Lyme carditis, neurologic complications, and arthritis because of the ability of specific borrelial strains to disseminate, invade, and drive inflammation. B. burgdorferi elicits type I IFN (IFN-I) responses in mammalian cells and tissues that are associated with the development of severe arthritis or other Lyme-related complications. However, the innate immune sensors and signaling pathways controlling IFN-I induction remain unclear. In this study, we examined whether intracellular nucleic acid sensing is required for the induction of IFN-I to B. burgdorferi. Using fluorescence microscopy, we show that B. burgdorferi associates with mouse and human cells in culture, and we document that internalized spirochetes colocalize with the pattern recognition receptor cyclic GMP-AMP synthase (cGAS). Moreover, we report that IFN-I responses in mouse macrophages and murine embryonic fibroblasts are significantly attenuated in the absence of cGAS or its adaptor stimulator of IFN genes (STING), which function to sense and respond to intracellular DNA. Longitudinal invivo tracking of bioluminescent B. burgdorferi revealed similar dissemination kinetics and borrelial load in C57BL/6J wild-type, cGAS-deficient, or STING-deficient mice. However, infection-associated tibiotarsal joint pathology and inflammation were modestly reduced in cGAS-deficient compared with wild-type mice. Collectively, these results indicate that the cGAS-STING pathway is a critical mediator of mammalian IFN-I signaling and innate immune responses to B. burgdorferi.

Sorting states of environmental DNA: Effects of isolation method and water matrix on the recovery of membrane‐bound, dissolved, and adsorbed states of eDNA

AbstractEnvironmental DNA (eDNA) once shed can exist in numerous states with varying behaviors including degradation rates and transport potential. In this study, we consider three states of eDNA: (1) a membrane‐bound state referring to DNA enveloped in a cellular or organellar membrane, (2) a dissolved state defined as the extracellular DNA molecule in the environment without any interaction with other particles, and (3) an adsorbed state defined as extracellular DNA adsorbed to a particle surface in the environment. Capturing, isolating, and analyzing a target state of eDNA provides utility for better interpretation of eDNA degradation rates and transport potential. While methods for separating different states of DNA have been developed, they remain poorly evaluated due to the lack of state‐controlled experimentation. We evaluated the methods for separating states of eDNA from a single sample by spiking DNA from three different species to represent the three states of eDNA as state‐specific controls. We used chicken DNA to represent the dissolved state, cultured mouse cells for the membrane‐bound state, and salmon DNA adsorbed to clay particles as the adsorbed state. We performed the separation in three water matrices, two environmental and one synthetic, spiked with the three eDNA states. The membrane‐bound state was the only state that was isolated with minimal contamination from nontarget states. The membrane‐bound state also had the highest recovery (54.11 ± 19.24%), followed by the adsorbed state (5.08 ± 2.28%), and the dissolved state had the lowest total recovery (2.21 ± 2.36%). This study highlights the potential to sort the states of eDNA from a single sample and independently analyze them for more informed biodiversity assessments. However, further method development is needed to improve recovery and reduce cross‐contamination.

MRI Tracking of Marine Proliferating Cells In Vivo Using Anti-Oct4 Antibody-Conjugated Iron Nanoparticles for Precision in Regenerative Medicine

Marine invertebrates are multicellular organisms consisting of a wide range of marine environmental species. Unlike vertebrates, including humans, one of the challenges in identifying and tracking invertebrate stem cells is the lack of a specific marker. Labeling stem cells with magnetic particles provides a non-invasive, in vivo tracking method using MRI. This study suggests antibody-conjugated iron nanoparticles (NPs), which are detectable with MRI for in vivo tracking, to detect stem cell proliferation using the Oct4 receptor as a marker of stem cells. In the first phase, iron NPs were fabricated, and their successful synthesis was confirmed using FTIR spectroscopy. Next, the Alexa Fluor anti-Oct4 antibody was conjugated with as-synthesized NPs. Their affinity to the cell surface marker in fresh and saltwater conditions was confirmed using two types of cells, murine mesenchymal stromal/stem cell culture and sea anemone stem cells. For this purpose, 106 cells of each type were exposed to NP-conjugated antibodies and their affinity to antibodies was confirmed by an epi-fluorescent microscope. The presence of iron-NPs imaged with the light microscope was confirmed by iron staining using Prussian blue stain. Next, anti-Oct4 antibodies conjugated with iron NPs were injected into a brittle star, and proliferating cells were tracked by MRI. To sum up, anti-Oct4 antibodies conjugated with iron NPs not only have the potential for identifying proliferating stem cells in different cell culture conditions of sea anemone and mouse cell cultures but also has the potential to be used for in vivo MRI tracking of marine proliferating cells.

Role of FGF10/FGFR2b Signaling in Homeostasis and Regeneration of Adult Lacrimal Gland and Corneal Epithelium Proliferation.

Fibroblast growth factor 10 (FGF10) is involved in eye, meibomian, and lacrimal gland (LG) development, but its function in adult eye structures remains unknown. This study aimed to characterize the role of FGF10 in homeostasis and regeneration of adult LG and corneal epithelium proliferation. Quantitative reverse transcription PCR was used for analysis of FGF10 expression in both early postnatal and adult mouse LG, and RNA sequencing was used to analyze gene expression during LG inflammation. FGF10 was injected into the LG of two mouse models of Sjögren's syndrome and healthy controls. Flow cytometry, BrdU cell proliferation assay, immunostaining, and hematoxylin and eosin staining were used to evaluate the effects of FGF10 injection on inflammation and cell proliferation in vivo. Mouse and human epithelial cell cultures were treated with FGF10 in vitro, and cell viability was assessed using WST-8 and adenosine triphosphate (ATP) quantification assays. The level of Fgf10 mRNA expression was lower in adult LG compared to early postnatal LG and was downregulated in chronic inflammation. FGF10 injection into diseased LGs significantly increased cell proliferation and decreased the number of B cells. Mouse and human corneal epithelial cell cultures treated with FGF10 showed significantly higher cell viability and greater cell proliferation. FGF10 appears to promote regeneration in damaged adult LGs. These findings have therapeutic potential for developing new treatments for dry eye disease targeting the ability of the cornea and LG to regenerate.

Protrusion growth driven by myosin-generated force.

Actin-based protrusions extend from the surface of all eukaryotic cells, where they support diverse activities essential for life. Models of protrusion growth hypothesize that actin filament assembly exerts force for pushing the plasma membrane outward. However, membrane-associated myosin motors are also abundant in protrusions, although their potential for contributing, growth-promoting force remains unexplored. Using an inducible system that docks myosin motor domains to membrane-binding modules with temporal control, we found that application of myosin-generated force to the membrane is sufficient for driving robust protrusion elongation in human, mouse, and pig cell culture models. Protrusion growth scaled with motor accumulation, required barbed-end-directed force, and was independent of cargo delivery or recruitment of canonical elongation factors. Application of growth-promoting force was also supported by structurally distinct myosin motors and membrane-binding modules. Thus, myosin-generated force can drive protrusion growth, and this mechanism is likely active in diverse biological contexts.

774 TSG-6 and TSP-1 exhibit altered expression in the skin of diabetic patients

Diabetes Mellitus and chronic hyperglycemia are associated with many complications including delayed wound healing and diabetic foot ulcers. Mechanisms behind this wound healing failure remain poorly understood but may include differential expression of two molecules, tumor necrosis factor stimulated gene 6 (TSG-6) and thrombospondin 1 (TSP-1). These proteins, which interact with each other, play important roles in wound healing and display altered expression in diabetic mouse models and cell cultures.

Silencing the ADAM9 Gene through CRISPR/Cas9 Protects Mice from Alcohol-Induced Acute Liver Injury.

Alcoholic liver injury is a major global public health concern at present. The ADAM9 gene plays a crucial role in the occurrence and development of various liver diseases, but its role in acute alcoholic liver injury remains ambiguous. In this study, a chimeric single-guide RNA targeting the genomic regions of mouse ADAM9 was designed using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology. Next, the role of ADAM9 in acute alcoholic liver injury in vitro in cultured mouse cells and in vivo in a hydrodynamic injection-based alcoholic liver injury mouse model was documented. The findings of this study suggest that ADAM9 induces by regulating cell proliferation, apoptosis, and stress metabolism in mice. Thus, inhibiting the expression of ADAM9 gene using CRISPR/Cas9 can attenuate alcohol-induced acute liver injury in mice.

Systemic Delivery of an Adjuvant CXCR4-CXCL12 Signaling Inhibitor Encapsulated in Synthetic Protein Nanoparticles for Glioma Immunotherapy.

Glioblastoma (GBM) is an aggressive primary brain cancer, with a 5 year survival of ∼5%. Challenges that hamper GBM therapeutic efficacy include (i) tumor heterogeneity, (ii) treatment resistance, (iii) immunosuppressive tumor microenvironment (TME), and (iv) the blood-brain barrier (BBB). The C-X-C motif chemokine ligand-12/C-X-C motif chemokine receptor-4 (CXCL12/CXCR4) signaling pathway is activated in GBM and is associated with tumor progression. Although the CXCR4 antagonist (AMD3100) has been proposed as an attractive anti-GBM therapeutic target, it has poor pharmacokinetic properties, and unfavorable bioavailability has hampered its clinical implementation. Thus, we developed synthetic protein nanoparticles (SPNPs) coated with the transcytotic peptide iRGD (AMD3100-SPNPs) to target the CXCL2/CXCR4 pathway in GBM via systemic delivery. We showed that AMD3100-SPNPs block CXCL12/CXCR4 signaling in three mouse and human GBM cell cultures in vitro and in a GBM mouse model in vivo. This results in (i) inhibition of GBM proliferation, (ii) reduced infiltration of CXCR4+ monocytic myeloid-derived suppressor cells (M-MDSCs) into the TME, (iii) restoration of BBB integrity, and (iv) induction of immunogenic cell death (ICD), sensitizing the tumor to radiotherapy and leading to anti-GBM immunity. Additionally, we showed that combining AMD3100-SPNPs with radiation led to long-term survival, with ∼60% of GBM tumor-bearing mice remaining tumor free after rechallenging with a second GBM in the contralateral hemisphere. This was due to a sustained anti-GBM immunological memory response that prevented tumor recurrence without additional treatment. In view of the potent ICD induction and reprogrammed tumor microenvironment, this SPNP-mediated strategy has a significant clinical translation applicability.

An ALS-associated KIF5A mutant forms oligomers and aggregates and induces neuronal toxicity.

KIF5A is a kinesin superfamily motor protein that transports various cargos in neurons. Mutations in Kif5a cause familial amyotrophic lateral sclerosis (ALS). These ALS mutations are in the intron of Kif5a and induce mis‐splicing of KIF5A mRNA, leading to splicing out of exon 27, which in human KIF5A encodes the cargo‐binding tail domain of KIF5A. Therefore, it has been suggested that ALS is caused by loss of function of KIF5A. However, the precise mechanisms regarding how mutations in KIF5A cause ALS remain unclear. Here, we show that an ALS‐associated mutant of KIF5A, KIF5A(Δexon27), is predisposed to form oligomers and aggregates in cultured mouse cell lines. Interestingly, purified KIF5A(Δexon27) oligomers showed more active movement on microtubules than wild‐type KIF5A in vitro. Purified KIF5A(∆exon27) was prone to form aggregates in vitro. Moreover, KIF5A(Δexon27)‐expressing Caenorhabditis elegans neurons showed morphological defects. These data collectively suggest that ALS‐associated mutations of KIF5A are toxic gain‐of‐function mutations rather than simple loss‐of‐function mutations.

Senolysis induced by 25-hydroxycholesterol targets CRYAB in multiple cell types

SummaryCellular senescence is a driver of many age-related pathologies. There is an active search for pharmaceuticals termed senolytics that can mitigate or remove senescent cells in vivo by targeting genes that promote the survival of senescent cells. We utilized single-cell RNA sequencing to identify CRYAB as a robust senescence-induced gene and potential target for senolysis. Using chemical inhibitor screening for CRYAB disruption, we identified 25-hydroxycholesterol (25HC), an endogenous metabolite of cholesterol biosynthesis, as a potent senolytic. We then validated 25HC as a senolytic in mouse and human cells in culture and in vivo in mouse skeletal muscle. Thus, 25HC represents a potential class of senolytics, which may be useful in combating diseases or physiologies in which cellular senescence is a key driver.

Evaluation of cancer stem cells markers expression in HCC trough real-time polymerase chain reaction.

Quantitative real-time polymerase chain reaction (qRT-PCR) flexibility, robustness and reproducibility have rapidly extended the scope of the method. Cancer stem cells are gaining increasing importance since their role in cancer initiation, treatment resistance and recurrence give rise to a wide range of potential diagnostic and therapeutic applications. The expression of several characteristic markers is proven a reliable method to assess stem-like-phenotype of cancer cells. Here, we provided a thorough protocol for the study of cancer stem cells in hepatocellular carcinoma mouse models and cell cultures using qRT-PCR.

Gut Flora Mediates the Rapid Tolerance of Electroacupuncture on Ischemic Stroke by Activating Melatonin Receptor through Regulating Indole-3-Propionic Acid.

Electroacupuncture (EA) is commonly used to treat cerebrovascular diseases. This study aimed to clarify the mechanisms of action of treatments of cerebral ischemic stroke from the perspective of gut microecology. We used a mouse model and cell cultures to investigate the effects of EA on the intestinal microflora in mice models of middle cerebral artery occlusion (MCAO) and the mechanisms underlying the antioxidant activities of metabolites. Fecal microbiota transplantation (FMT) was used to validate the roles of gut microbiota. Metabolomic analysis was performed to characterize the metabolic profile differences between the mice in the EA + MCAO and MCAO groups. Gavaging with feces relieved brain damage in mice that received EA (EA mice) more than in mice that did not (non-EA [NEA] mice). The gut microbial composition and metabolic profiles of the EA and NEA mice were different. In particular, the microbiota from the mice in the EA or EA-FMT groups generated more indole-3-propionic acid (IPA) than the microbiota from the mice in the MCAO or NEA-FMT groups. We confirmed that IPA binds to specific melatonin receptors (MTRs) in target cells and exerts antioxidant effects by adding MTR inhibitors or knocking out the MTR1 gene in vivo and in the oxygen and glucose deprivation/reperfusion models of N2a cell experiments. EA can prevent ischemic stroke by improving the composition of intestinal microbiota in MCAO mice. Moreover, this study reveals a new mechanism of intestinal flora regulation of stroke that differs from inflammation/immunity, namely gut microbiota regulates stroke by affecting IPA levels.