Flow Cytometry Experiments Research Articles

Triptolide Promotes Ferroptosis in Cervical Cancer Cell via NRF2/xCT/GPX4.

Cervical cancer (CC) is a serious risk to women's health; it is necessary to explore less toxic and more effective therapies to cure CC. Triptolide (Tri) is the principal active constituent found in "Tripterygium Wilford," has been shown to have antitumor effects. This study set up to demonstrate whether Tri is capable of inducing ferroptosis in CC cells and its potential mechanism. Invitro, Tri was used to treat CC cells, and lipid peroxidation levels in CC cells were detected by flow cytometry, immunofluorescence, and other experiments; the molecular mechanism of Tri treatment of CC was explored by western blot; moreover, the regulatory effects of Tri on the NRF2/GPX4/xCT axis were verified by overexpressing NRF2 in reverse. Invivo, CC cells tumor-bearing mice were constructed to observe the effect of Tri treatment on tumor growth. Invitro, we have demonstrated that Tri prevents the growth and migration of CC cells. Further investigation revealed that Tri substantially enhances ferroptosis in CC cells by increasing lipid peroxidation accumulation. Mechanically, Tri significantly reduced the expression of NRF2, leading to a corresponding repression of the NRF2 downstream targets GPX4 and xCT. Moreover, overexpressing of NRF2 effectively reversed the impact of Tri on ferroptosis in CC cells. Additionally, animal experiments indicted that Tri markedly inhibited tumor size in nude mice by inhibiting the NRF2/GPX4/xCT axis. Tri exerts antitumor effects by triggering ferroptosis in CC cells through the NRF2/GPX4/xCT axis.

Self-Assembled Eutectogel with Cell Permeation and Multiple Anti-Inflammatory Abilities for Treating Chronic Periodontitis.

Eutectogels represent an attractive option for various industrial applications that use deep eutectic solvents (DESs) as effective liquid active ingredients and offer remarkable stability, cost-effectiveness, and environmental friendliness. However, the biological applications of these compounds are limited. DESs are highly structurally tunable and exhibit remarkable biofunctionality and biocompatibility, conferring substantial benefits in the treatment of diseases. In this study, choline-chloride and mannose are used to fabricate ChCl/M DES, followed by introduction of lysozyme fibers and gallic acid for self-assembly into injectable eutectogels through hydrogen bonding and hydrophilic/hydrophobic interaction interactions. The eutectogels demonstrate almost 100% bactericidal activities against three strains and significant immune-regulation. This is supported by a decrease in the proportion of CD86-expressing cells from 64.02% to 18.17% and an increase in CD206-expressing cells from 2.53% to 29.96% through flow cytometry experiments. The eutectogels effectively inhibit alveolar bone loss and alleviated local inflammation in a rat model of chronic periodontitis owing to the promotion of gallic acid in the cell membrane by the ChCl/M DES. Hence, self-assembled eutectogels exhibit the potential to enhance the efficacy if treatments/therapies against inflammatory diseases by facilitating bacterial control, reactive oxygen species scavenging, and the regulation of macrophages by promoting cell permeation of small-molecule drugs.

Rho and riboswitch-dependent regulations of mntP gene expression evade manganese and membrane toxicities

The trace metal ion manganese (Mn) in excess is toxic. Therefore, a small subset of factors tightly maintains its cellular level, among which an efflux protein MntP is the champion. Multiple transcriptional regulators and a manganese-dependent translational riboswitch regulate the MntP expression in Escherichia coli. As riboswitches are untranslated RNAs, they are often associated with the Rho-dependent transcription termination in bacteria. Here, performing in vitro transcription and in vivo reporter assays, we demonstrate that Rho efficiently terminates transcription at the mntP riboswitch region. Excess manganese activates the riboswitch, restoring the coupling between transcription and translation to evade Rho-dependent transcription termination partially. RT-PCR and western blot experiments revealed that the deletion of the riboswitch abolishes Rho-dependent termination and thereby overexpresses MntP. Interestingly, deletion of the riboswitch also renders bacteria sensitive to manganese. This manganese sensitivity is linked with the overexpression of MntP. Further spot assays, confocal microscopy, and flow cytometry experiments revealed that the high level of MntP expression was responsible for slow growth, cell filamentation, and reactive oxygen species (ROS) production. We posit that manganese-dependent transcriptional activation of mntP in the absence of Rho-dependent termination leads to excessive MntP expression, a membrane protein, causing membrane protein toxicity. Thus, we show a regulatory role of Rho-dependent termination, which prevents membrane protein toxicity by limiting MntP expression.

Peptide Dimerization as a Strategy for the Development of Antileishmanial Compounds.

Leishmaniasis is recognized as a serious public health problem in Brazil and around the world. The limited availability of drugs for treatment, added to the diversity of side effects and the emergence of resistant strains, shows the importance of research focused on the development of new molecules, thus contributing to treatments. Therefore, this work aimed to identify leishmanicidal compounds using a peptide dimerization strategy, as well as to understand their mechanisms of action. Herein, it was demonstrated that the dimerization of the peptide TSHa, (TSHa)2K, presented higher potency and selectivity than its monomeric form when evaluated against Leishmania mexicana and Leishmania amazonensis. Furthermore, these compounds are capable of inhibiting the parasite cysteine protease, an important target explored for the development of antileishmanial compounds, as well as to selectively interact with the parasite membranes, as demonstrated by flow cytometry, permeabilization, and fluorescence microscopy experiments. Based on this, the identified molecules are candidates for use in in vivo studies with animal models to combat leishmaniasis.

Nonalcoholic fatty liver disease worsens cardiac repair after myocardial infarction

Abstract Background There is a strong epidemiological link between nonalcoholic fatty liver disease (NAFLD) and ischemic heart disease such as myocardial infarction (MI). Yet, a causal relationship between liver injury and post-MI cardiac repair remains to be established. Purpose NAFLD impairs post-MI myocardial repair processes. In particular, we hypothesized that the pathological liver could transmit deleterious signals shaping the inflammatory reaction in the infarcted milieu. Methods To characterize post-MI cardiac function and remodeling in three mouse models of liver damage mimicking different stages of NAFLD without cardiovascular (CV) risk factors : i) choline-deficient, L-amino-acid-defined, high fat diet (CDA-HFD) causing severe non-alcoholic steatohepatitis; ii) NEMOLPC-KO mice deficient for NF-κB in liver parenchymal cells, exhibiting liver inflammation and fibrosis, but mild steatosis; and ATGLLPC-KO animals deficient for Adipose Triglyceride Lipase in liver parenchymal cells displaying strong steatosis. Results In our three experimental models, liver damages were characterized by inflammation, steatosis and fibrosis but were not associated with metabolic syndrome. More interestingly, left ventricular ejection fraction were reduced by 1.2-fold, 1.3-fold and 1.3-fold in CDA-HFD, NEMOLPC-KO and ATGLLPC-KO male and female mice, respectively when compared to their respective controls, at 35 days post-MI (n=12-25 per group). NAFLD-induced cardiac dysfunction was not associated with worsen cardiac remodeling as infarct size, fibrosis and vascular density were not significantly affected. On the same note, flow cytometry experiments, at 7- and 14-days post-MI, showed no changes in the number or type of inflammatory cells, particularly that of cardiac resident macrophages, in CDAHFD, ATGLLPC-KO and NEMOLPC-KO mice. We next hypothesized that reduced left ventricular function could be induced by altered cardiomyocyte contractility. To corroborate this hypothesis, we isolated cardiomyocytes from hearts of infarcted mice with or without NAFLD and assessed cell contractility using IonOptix technology. Our preliminary results, in the CDAHFD model, showed that there was a reduction in contraction amplitude, defining by the ratio of the difference in sarcomere length during contraction/relaxation to the sarcomere length at the time of relaxation. The speed of contraction and relaxation were also decreased in CDAHFD mice compared with control animals. Conclusions NAFLD favors cardiac dysfunction post-MI. We now aim to identify molecular and cellular entities participating to such pathogenic dialogue between the dysfunctional liver and the infarcted heart.

Effect of Hepatic Lipid Overload on Accelerated Hepatocyte Proliferation Promoted by HGF Expression via the SphK1/S1PR2 Pathway in MCD-diet Mouse Partial Hepatectomy.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is becoming a major health problem worldwide. Liver regeneration is crucial for restoring liver function, and is regulated by extraordinary complex process, involving numerous factors under both physiologic and pathologic conditions. Sphingosine-1-phosphate (S1P), a bioactive sphingolipid synthesized by sphingosine kinase 1 (SphK1), plays an important role in liver function through S1P receptors (S1PRs)-expressing cells. In this study, we investigated the effect of lipid overload on hepatocyte proliferation in a mouse hepatic steatosis model induced by feeding a methionine- and choline-deficient (MCD) diet. After 50% partial hepatectomy (PHx), liver tissues were sampled at various timepoints and then analyzed by immunohistochemistry, oil Red-O staining, quantitative-polymerase chain reaction (qPCR), and flow cytometry. In mice fed the MCD-diet, significantly exacerbated hepatic steatosis and accelerated liver regeneration were observed. After PHx, hepatocyte proliferation peaked at 48 and 36 hr in the liver of chow- and MCD-diet fed mice, respectively. By contrast, increased expression of S1PR2 was observed in hepatic neutrophils and macrophages of MCD-diet fed mice. Flow cytometry and qPCR experiments demonstrated that levels of HGF and FGF2 released by neutrophils and macrophages were significantly higher in MCD-diet fed mice. In conclusion, hepatic lipid overload recruits Kupffer cells and neutrophils that release HGF and FGF2 via SphK1/S1PR2 activation to accelerate hepatocyte proliferation.

Sphingosine-1-phosphate reduces arterial thrombosis via enhanced endothelial thrombomodulin expression by S1PR1 signaling

Abstract Background Sphingosine 1-phosphate (S1P) is an important lipid mediator in the cardiovascular system. Numerous links between S1P and blood coagulation have been described. However, results regarding the role of S1P in coagulation are inconclusive. A physiological antithrombotic regulator expressed by endothelial cells (EC) is thrombomodulin (TM). To date, the impact of S1P on TM-expression is unknown. Methods We evaluated S1P effects on TM-expression and subsequent platelet adhesion and thrombus formation. For this, we conducted cell culture with human umbilical cord venous endothelial cells (HUVECs), flow cytometry and flow chamber experiments. Additionally, in vivo arterial thrombus formation in mice was analyzed. Results Sphingosine kinase-1 deficient mice (SphK1-/-) have about 70% less circulating plasma S1P and show a significant reduction in TM expression on the aorta as compared to their littermates (WT: 207.2 ± 45.8 pg/g Aorta vs. SphK1-/- 124.5 ± 40.2 pg/g Aorta, p<0.0001). In contrast, S1P incubation of 24 hours significantly increased TM-expression on HUVECs by about 30 percent. This effect was abolished by S1PR1-inhibition with W146. Treatment with S1P reduced platelet adhesion from whole blood to endothelial cells in the flow chamber experiments (Con: 100.00 ± 28.09% vs. S1P: 80.91 ± 22.85%, p<0.0001). This effect could be reversed both by S1PR1 inhibition and with a TM-neutralizing antibody. P-selectin-expression on platelets was decreased after incubation with TM suggesting (Ctrl: 62.79 ± 13.30% vs. TM: 43.47 ± 11.36%, p=0.0222). Finally, to assess the in vivo effects of our findings, arterial thrombus formation in mice was performed. TM inhibited in vivo thrombus formation. Occlusion of the carotid artery could not be detected during the 30 minutes recording. In contrast, SphK1-/- mice with low S1P levels showed decreased times to first occlusion. Treatment with TM in these animals was able to reverse this effect. Conclusion We can conclude that the main findings of our study were (i) S1P increased endothelial TM-expression, (ii) which subsequently leads to reduced platelet adhesion and (iii) inhibition of arterial thrombus formation in vivo.

Performance and potential mechanisms for reactive electrochemical ceramic membrane system to inhibit resistance transmission in antibiotic-resistant contaminated wastewater: From a microbial perspective

Due to the widespread presence of antibiotic resistance genes (ARGs) in traditional biological wastewater treatment systems, electrochemical disinfection techniques have received extensive attention. The rTNA/Sb-SnO2/PbO2 Reactive Electrochemical Ceramic Membrane (RECM) system has demonstrated outstanding performance in effectively removing refractory antibiotics. However, its capacity to remove antibiotic resistant bacteria (ARB) remains unclear. In this study, an antibiotic resistant strain of Escherichia coli (AR E. coli) was employed to investigate the disinfection efficiency of the RECM system at a macroscopic level, along with its disinfection mechanism at a microscopic level. Furthermore, the secondary effluent from an antibiotic production wastewater treatment plant was collected to validate the disinfection performance of this system against ARB in actual wastewater. In the RECM system, the inactivation of AR E. coli and the removal of ARGs were primarily governed by the electrocatalytic process, with notable influences from critical factors such as current density, electrolyte type, and hydraulic retention time. Morphological observation and flow cytometry experiments confirmed that the AR E. coli in the RECM system was inactivated by alterations in cell morphology, leading to fragmentation, and by improvement in cell membrane permeability. More significantly, this system exhibited noteworthy efficiency in treating wastewater contaminated with antibiotic resistant pollutants, effectively eliminating both antibiotics and ARGs from the secondary effluent. Overall, our study comprehensively investigated the performance and underlying mechanisms of the RECM system in mitigating the dissemination of ARGs, as well as its potential feasibility of practical application.

Zaluzanin-D enriched Vernonia arborea extract mediated copper oxide nanoparticles synthesis and their anti-oxidant, anti-inflammatory and DNA methylation altering properties.

Metal oxide nanoparticles synthesized with the aid of medicinal plant extracts are showing potential as treatment options for inflammatory diseases. Two key benefits of this synthesis method is: the synthesis process is environmentally benign and the utilization of medicinal plant-derived extracts adds to the medicinal value of the synthesized nanoparticle. Earlier, sesquiterpene lactone zaluzanin-D (ZD) has been isolated from leaves of Vernonia arborea. ZD showed ability to reduce inflammation in activated monocytes. Copper oxide nanoparticles (bCuO-NPs) were synthesized using ZD-enriched leaf extract of V. arborea and characterized by UV-vis spectroscopy, FT-IR, XRD, particle size analyzer, and TEM. Synthesized bCuO-NPs did not show significant toxicity to human monocytic cell lines (THP-1) at the tested concentrations. The bCuO-NPs showed radical scavenging ability indicating anti-oxidant properties. Flow cytometry experiments proved the capability of bCuO-NPs to reduce intracellular ROS in peroxide-activated THP-1 cells. The NPs also showed a significant ability to reduce inflammatory adhesion in PMA-activated THP-1 cells. In the DNA methylation studies, bCuO-NPs behaved similarly to ZD and prevented DNA hypomethylation at the MMP-9 promoter region. These properties strongly indicate the ability of bCuO-NPs to reduce inflammation in the activated monocytes. Furthermore, in zebrafish (Danio rerio) embryos, the developmental toxicity of bCuO-NPs was assessed. The studies indicated the reduced toxicity and compatibility of the NPs with biological organisms. Based on the results, it can be concluded that the bCuO-NPs produced from ZD-enriched leaf extract have significant anti-oxidant capabilities and the ability to reduce inflammation in monocytic cell lines. Overall, reduced in vitro and in vivo toxicity, along with its antioxidant and anti-inflammatory properties, makes bCuO-NPs a potential candidate for anti-inflammatory drugs.

Role of hypothetical protein PA1-LRP in antibacterial activity of endolysin from a new Pantoea phage PA1.

Pantoea ananatis has emerged as a significant plant pathogen affecting various crops worldwide, causing substantial economic losses. Bacteriophages and their endolysins offer promising alternatives for controlling bacterial infections, addressing the growing concerns of antibiotic resistance. This study isolated and characterized the Pantoea phage PA1 and investigated the role of PA1-LRP in directly damaging bacteria and assisting endolysin PA1-Lys in cell lysis, comparing its effect to exogenous transmembrane domains following the identification and analysis of the PA1-Lys and the PA1-LRP based on whole genome analysis of phage PA1. Additionally, this study also explored how hydrophobic region of PA1-LRP (HPP) contributes to bacterial killing when combined with PA1-Lys and examined the stability and lytic spectrum of PA1-Lys under various conditions. Phage PA1 belonging to the Chaseviridae family exhibited a broad host range against P. ananatis strains, with a latent period of 40 minutes and a burst size of 17.17 phages per infected cell. PA1-Lys remained stable at pH 6-10 and temperatures of 20-50°C and showed lytic activity against various Gram-negative bacteria, while PA1-Lys alone could not directly lyse bacteria, its lytic activity was enhanced in the presence of EDTA. Surprisingly, PA1-LRP inhibited bacterial growth when expressed alone. After 24 h of incubation, the OD600 value of pET28a-LRP decreased by 0.164 compared to pET28a. Furthermore, the lytic effect of co-expressed PA1-LRP and PA1-Lys was significantly stronger than each separately. After 24 h of incubation, compared to pET28a-LRP, the OD600 value of pET28a-Lys-LRP decreased by 0.444, while the OD420 value increased by 3.121. Live/dead cell staining, and flow cytometry experiments showed that the fusion expression of PA1-LRP and PA1-Lys resulted in 41.29% cell death, with bacterial morphology changing from rod-shaped to filamentous. Notably, PA1-LRP provided stronger support for endolysin-mediated cell lysis than exogenous transmembrane domains. Additionally, our results demonstrated that the HPP fused with PA1-Lys, led to 40.60% cell death, with bacteria changing from rod-shaped to spherical and exhibiting vacuolation. Taken together, this study provides insights into the lysis mechanisms of Pantoea phages and identifies a novel lysis-related protein, PA1-LRP, which could have potential applications in phage therapy and bacterial disease control.

Modification with IL-21 and CCL19 enhances killing efficiency and tumor infiltration of NKP30 CAR-T cells in lung cancer

To investigate whether modification with IL-21 and CCL19 enhances killing and tumor-infiltrating efficiency of NKP30 CAR-T cells in lung cancer. The modified IL-21-CCL19 NKP30 CAR-T cells expressing IL-21 and CCL19 fusion gene was constructed based on NKP30 CAR-T cells and stimulated with CD3CD28 antibodies and IL-2. The immunophenotype and migration of the cells in the presence of IL-21 were investigated using flow cytometry and migration experiments. Lactate dehydrogenase (LDH) release and sphere formation assays were used to assess the killing and infiltration capabilities of CAR-T cells, and the secretion levels of IFN-γ, IL-21 and CCL19 were determined with enzyme-linked immunospot assay (ELISPOT) and ELISA. A zebrafish model bearing HCG-27 cell xenograft was established by microinjection of the tumor cells into the yolk sac followed 24 h later by injection of the immune cells at the same site, and the fluorescence signals were captured using a fluorescent microscopy. The NKP30 ligand B7H6, which was almost undetectable in normal tissues and blood cells, was highly expressed (over 90%) in lung cancer cells. Compared with NKP30 CAR-T cells and conventional T cells, IL-21-CCL19 NKP30 CAR-T cells exhibited stronger proliferative and migration capabilities with the formation of central memory T cells. The reduced expressions of CTLA4 and PD1 in the constructed cells resulted in enhanced killing efficiency against lung cancer cells accompanied by significantly increased production of IFN-γ, IL-21 and CCL19. In the zebrafish models, CAR-T cells exhibited stronger cytotoxicity and proliferative abilities than typical T cells, but these differences were not statistically significant between the two CAR-T cells. Modification of NKP30 CAR-T cells with IL-21 and CCL19 facilitates their access into solid tumors for more effective tumor cell killing while producing a large number of memory T cells.

Harnessing the targeting potential of hyaluronic acid for augmented anticancer activity and safety of duvelisib-loaded nanoparticles in hematological malignancies

Duvelisib (DUV) is effective against numerous hematological malignancies; however, it suffers from numerous setbacks like poor aqueous solubility, low cellular uptake and adverse effects. Hyaluronic acid is an excellent ligand for CD44 receptors that are overexpressed on cancer cell surfaces. Thus, for the targeted delivery of DUV in hematological malignancies, we have fabricated hyaluronic acid-coated polylactide-co-glycolide nanoparticles (DUV-P/CH/HA-NPs) through electrostatic interactions. DUV-P/CH/HA-NPs exhibited optimum characteristics such as mean particle size of 183.63 ± 0.23 nm, polydispersity index of 0.261 ± 0.02 and drug loading capacity of 5.75 ± 0.05 %. An in-vitro release study demonstrated sustained release behavior of DUV-P/CH/HA-NPs (77.65 ± 2.89 % release in 48 h). The flow cytometry experiments revealed 1.62-fold and 1.50-fold enhanced uptake of DUV-P/CH/HA-NPs compared to non-coated nanoparticles in MOLT-4 and HH cells, respectively. The DUV-P/CH/HA-NPs showed higher cytotoxicity, arrested the cell cycle in G0/G1 phase and showed increased apoptosis compared to non-coated nanoparticles and free DUV. An in-vivo pharmacokinetic study revealed 2.9-fold and 3.6-fold enhancement in AUC0-t and MRT with the DUV-P/CH/HA-NPs compared to free DUV. Further, toxicity evaluation and hemolysis assessment of DUV-P/CH/HA-NPs indicated good safety for intravenous administration. Conclusively, DUV-P/CH/HA-NPs are an excellent option for selectively targeting hematological malignant cells.

Cepharanthine-mediated endoplasmic reticulum stress inhibits Notch1 via binding GRP78 for suppressing hepatocellular carcinoma metastasis

BackgroundThe metastasis of hepatocellular carcinoma (HCC) leads to a poor prognosis, wherein the activation of Notch1 is an essential contributor. Cepharanthine (Cep) has been identified for its effective antiviral function and versatile intracellular targets. Our previous study has only reported the anti-cancer efficacy of Cep in lung cancer, without an in-depth exploration. Herein, the present study aims to investigate the anti-metastasis effect in HCC, the target involved, and the molecular mechanism of Cep. MethodsStable over-expression of Notch1-N1ICD yielded C5WN1 cells compared with C5WBF344 cells. The C5WN1 cells and C5WN1 cell-bearing mice were applied as the HCC model. The bioinformatics analysis, RNA sequencing, molecular docking, cellular thermal shift assay (CETSA), drug affinity responsive target stability (DARTS), microscale thermophoresis (MST), and transient knockdown techniques were carried out to identify the underlying target. The apoptosis assay, immunofluorescent staining, qRT-PCR, Western blots, Elisa, flow cytometry, migration and scratching experiments, Transmission electron microscopy (TEM), laser scanning confocal microscopy (LSCM), micro-computed tomography (micro-CT), and histopathological experiments were conducted to assay the anti-HCC efficacy, functions, and mechanism. ResultsNotch1 had an increased expression in HCC and contributed to metastasis thereupon. Surprisingly, Cep (2 μg/ml in vitro, 5 mg kg-1in vivo) presented potent Notch1 signaling pathway inhibitory effect and anti-metastasis efficacy in C5WN1 cells and in situ mice models as evidenced by reduced Notch1/MMP-2/MMP-9 expression, TGF-β release, decreased cell migration, diminished pulmonary metastases, and prolonged survival. RNA sequencing showed that the differential gene of Cep-treated HCC cells was positioned in the endoplasmic reticulum (ER). Molecular docking, CETSA, DARTS, and MST further identified that the possible target of Cep was GRP78, which was distributed in the ER. As expected, Cep (2 μg/ml) up-regulated the critical molecules of ER stress such as GRP78, induced β-amyloid accumulation, and promoted calcium burst in HCC. In contrast, suppression of GRP78 attenuated Cep-induced ER stress. Furthermore, inhibition of ER stress abated Cep-induced Notch1 inactivation and HCC cells’ migration. ConclusionsTaken together, the present study finds that Cep possesses excellent anti-metastasis of HCC, wherein the GRP78 could be directly bound and activated by Cep, leading to ER stress and Notch1 blockage. This study reveals for the first time the effect, critical target, and mechanism of the Cep-mediated anti-cancer effect, providing novel insights into the molecular target therapy by phytomedicine.

Synthetic Peptides Induce Human Colorectal Cancer Cell Death via Proapoptotic Pathways.

Cancer resistance to drugs and chemotherapy is a problem faced by public health systems worldwide. Repositioning antimicrobial peptides could be an efficient strategy to overcome that problem. This study aimed at repurposing antimicrobial peptides PepGAT and PepKAA for cancer treatment. After screening against several cancers, PepGAT and PepKAA presented IC50 values of 125.42 and 40.51 μM at 72 h toward colorectal cancer (CRC) cells. The mechanisms of action revealed that both peptides induced cell cycle arrest in G2/M and drove HCT-116 cells to death by triggering apoptosis. qPCR analysis revealed that peptides modulated gene expression in apoptosis, corroborating the data from caspase 3/7 and flow cytometry experiments. Yet, peptides induced ROS overaccumulation and increased membrane permeabilization, pore formation, and loss of internal content, leading to death. Additionally, peptides were able to inhibit cell invasion. Previous studies from the same group attested to no toxicity to normal human cells. Thus, PepGAT and PepKAA have great potential as anticancer molecules.

Mechanism of Guishao Yigong decoction in treating colorectal cancer based on network pharmacology and experimental validation.

To explore the effective components of Guishao Yigong decoction (GYD) in the treatment of colorectal cancer and reveal its potential mechanism of action. Through network pharmacology, the main target and signaling pathway of GYD therapy for colorectal cancer (CRC) were found. Subsequently, the effect of GYD was verified by in vitro cell viability measurements, colony formation, and scratch healing tests. The effects of GYD on metabolic pathways in vivo were found through plasma metabolomics. Finally, flow cytometry and qPCR experiments were used to verify the cycle-blocking effect of GYD on CRC cells. Based on the network pharmacological analysis and molecular docking technology, it was found that GYD could restrain the growth of CRC cells by affecting lipid metabolic pathways and mitogen-activated protein kinase (MAPK) signaling pathways. A series of cell experiments showed that GYD could inhibit the proliferation, migration and clonogenic ability of CRC cells. Furthermore, the plasma metabolomics results showed that GYD could affect the production of unsaturated fatty acids in mice. Flow cytometry and qPCR experiments further proved that GYD blocked the CRC cells in the G1 phase and modulated the expression of cell cycle-related targets, such as AKT, TP53, CDKN1A, and CDK2. All the results indicated that GYD could regulate the related metabolism of unsaturated fatty acids. Thus, the cell cycle was blocked and the expressions of the key proteins such as AKT and TP53 were regulated, which achieved the purpose of intervention in colorectal cancer.

Putative role of 6-chogaol against tramadol-induced hepatotoxicity in albino rats via anti-inflammatory, antifibrotic, and antiapoptotic effects

Tramadol is a commonly used drug to relieve pain and avoid premature ejaculation in males with hepatotoxic effects, and 6-chogaol has potent anti-inflammatory and hepatoprotective properties.The work impetus is probing the hepatoprotective mechanisms of 6-chogaol against tramadol hepatoxicity. Twenty adult male rats were enrolled to obtain four equal groups [control group (G1), 6-chogaol group (G2), tramadol group (G3), and 6-chogaol+tramadol group (G4)]. Liver specimens were excised and processed to evaluate hepatocyte injury through histopathological (HP), immunohistochemical (IHC), flow cytometry, and biochemical investigations. The HP study exhibited hepatic injury in G3 hepatocytes (inflammatory cell infiltration, hepatic fibrosis, and disturbed liver structure). The IHC study showed a significant rise in caspase-3 and reduced PCNA immuno-expression (IE). Likewise, the flow cytometry and biochemical experiments exhibited a substantial elevation of apoptotic hepatocytes and the serum levels of IL-1β, IL-6, TNF-α, ALP, ALT, and AST in G3. In contrast, G4 rats significantly improved in all HP, IHC, flow cytometry, and biochemical parameters. Collectively, tramadol intake exerted harmful toxic effects on hepatocytes, whereas 6-Shogaol hampered these changes and served as a natural hepatoprotective agent. Therefore, we advise concurrent intake of 6-Shogaol supplement with tramadol to preserve the integrity of hepatic tissues.

The study on 4D culture system of squamous cell carcinoma of tongue

Traditional cell culture methods often fail to accurately replicate the intricate microenvironments crucial for studying specific cell growth patterns. In our study, we developed a 4D cell culture model-a precision instrument comprising an electromagnet, a force transducer, and a cantilever bracket. The experimental setup involves placing a Petri dish above the electromagnet, where gel beads encapsulating magnetic nanoparticles and tongue cancer cells are positioned. In this model, a magnetic force is generated on the magnetic nanoparticles in the culture medium to drive the gel to move and deform when the magnet is energized, thereby exerting an external force on the cells. This setup can mimic the microenvironment of tongue squamous cell carcinoma CAL-27 cells under mechanical stress induced by tongue movements. Electron microscopy and rheological analysis were performed on the hydrogels to confirm the porosity of alginate and its favorable viscoelastic properties. Additionally, Calcein-AM/PI staining was conducted to verify the biosafety of the hydrogel culture system. It mimics the microenvironment where tongue squamous cell carcinoma CAL-27 cells are stimulated by mechanical stress during tongue movement. Electron microscopy and rheological analysis experiments were conducted on hydrogels to assess the porosity of alginate and its viscoelastic properties. Calcein-AM/PI staining was performed to evaluate the biosafety of the hydrogel culture system. We confirmed that the proliferation of CAL-27 tongue squamous cells significantly increased with increased matrix stiffness after 5 d as assessed by MTT. After 15 d of incubation, the tumor spheroid diameter of the 1%-4D group was larger than that of the hydrogel-only culture. The Transwell assay demonstrated that mechanical stress stimulation and increased matrix stiffness could enhance cell aggressiveness. Flow cytometry experiments revealed a decrease in the number of cells in the resting or growth phase (G0/G1 phase), coupled with an increase in the proportion of cells in the preparation-for-division phase (G2/M phase). RT-PCR confirmed decreased expression levels of P53 and integrinβ3 RNA in the 1%-4D group after 21 d of 4D culture, alongside significant increases in the expression levels of Kindlin-2 and integrinαv. Immunofluorescence assays confirmed that 4D culture enhances tissue oxygenation and diminishes nuclear aggregation of HIF-1α. This device mimics the microenvironment of tongue cancer cells under mechanical force and increased matrix hardness during tongue movement, faithfully reproducing cell growthin vivo, and offering a solid foundation for further research on the pathogenic matrix of tongue cancer and drug treatments.

A unique human cord blood CD8+CD45RA+CD27+CD161+ T-cell subset identified by flow cytometric data analysis using Seurat.

Advances in single-cell level analytical techniques, especially cytometric approaches, have led to profound innovation in biomedical research, particularly in the field of clinical immunology. This has resulted in an expansion of high-dimensional data, posing great challenges for comprehensive and unbiased analysis. Conventional manual analysis is thus becoming untenable to handle these challenges. Furthermore, most newly developed computational methods lack flexibility and interoperability, hampering their accessibility and usability. Here, we adapted Seurat, an R package originally developed for single-cell RNA sequencing (scRNA-seq) analysis, for high-dimensional flow cytometric data analysis. Based on a 20-marker antibody panel and analyses of T-cell profiles in both adult blood and cord blood (CB), we showcased the robust capacity of Seurat in flow cytometric data analysis, which was further validated by Spectre, another high-dimensional cytometric data analysis package, and conventional manual analysis. Importantly, we identified a unique CD8+ T-cell population defined as CD8+CD45RA+CD27+CD161+ T cell that was predominantly present in CB. We characterised its IFN-γ-producing and potential cytotoxic properties using flow cytometry experiments and scRNA-seq analysis from a published dataset. Collectively, we identified a unique human CB CD8+CD45RA+CD27+CD161+ T-cell subset and demonstrated that Seurat, a widely used package for scRNA-seq analysis, possesses great potential to be repurposed for cytometric data analysis. This facilitates an unbiased and thorough interpretation of complicated high-dimensional data using a single analytical pipeline and opens a novel avenue for data-driven investigation in clinical immunology.

Cell type-specific and subcellular expression of phospholipid phosphatase-related proteins to modulate lyso-phosphatidic acid synaptic signaling in the developing and adult CNS.

Lysophosphatidic acid (LPA) is a bioactive phospholipid that participates in critical processes in neural development and adult brain function and is implicated in various pathophysiological conditions. Along with its six well-characterized receptors, atypical regulators of LPA signaling have also been suggested, including phospholipid phosphatase-related proteins (PLPPRs). PLPPRs have been mostly studied in the developing brain where they control LPA-dependent axon guidance, cortical network hyperexcitability, and glutamatergic neurotransmission. PLPPR4 and PLPPR3 represent two closely related proteins reported to localize predominantly in dendrites and axons, respectively, and differ in their developmental expression patterns. Herein, we have revised the expression patterns of PLPPRs in the cerebellum, dorsal and ventral hippocampus, prefrontal cortex (PFC), nucleus accumbens, and striatum during development and in the adult using quantitative PCR. Expression patterns of Plppr2,4 and 5 were consistent with previous studies, whereas Plppr3 and Plppr1 exhibited a unique expression profile in nucleus accumbens (NAc) and striatum in later developmental and adult stages, which we verified at the protein level for PLPPR3. To investigate neuron type-specific expression at the single cell level, we developed a bioinformatic tool to analyze recent single-cell RNA-sequencing data in the cerebral cortex and hippocampus of adult mice. Our analysis revealed a widespread but also selective adult neuron-type expression with higher expression levels of Plppr3, Plppr1, and Plppr5 in GABAergic and Plppr4 and Plppr2 in glutamatergic neurons. PLPPR4 has been identified as a post-synaptic modulator of LPA levels in glutamatergic synapses operating via an uptake mechanism, to control LPA-dependent cortical network hyperexcitability. Using subcellular fractionation experiments, we found that both PLPPR4 and PLPPR3 are co-expressed in adult synaptosomal membranes. Furthermore, flow cytometry experiments in HEK293 cells showed comparable LPA uptake by PLPPR4 and PLPPR3, whereas PLPRR3, but not PLPPR4, induced also uptake of monoacylglycerol, the dephosphorylation product of LPA. We propose that synaptic LPA may be subject to both pre-synaptic and post-synaptic mechanisms of regulation by PLPPRs in addition to LPARs in developing and adult synapses.

Knockdown of LncRNA-HAGLR restrains the viability and motility of pancreatic cancer via miR-625-5p/TAF15 axis in vitro and invivo

Dysregulation of long non-coding RNAs (lncRNAs) has been strongly involved to the development of pancreatic cancer (PC). However, the potential mechanisms by which lncRNA regulate PC development still need to be further explored. We attempted to elucidate the functional role and regulatory mechanism of lncRNA HAGLR on PC progression in vitro and vivo. RT-qPCR, Western blot, RNA pull-down, luciferase reporter assay, RNA immunoprecipitation assay, CCK-8 assay, EdU assay, flow cytometry, transwell assay and xenograft tumor experiment were performed in this study. We found that the expressions of HAGLR and TAF15 were increased in PC tissues and cells. HAGLR silencing restrained the PC cell growth and invasion, but induced cell apoptosis. Moreover, HAGLR targeted miR-625-5p to modulate the expression of TAF15. HAGLR overexpression partially eliminated the suppressive effect of TAF15 depletion on PC cell growth and the stimulative effect on apoptosis. In vivo assays showed that HAGLR knockdown inhibited PC cell growth by regulating the TAF15 expression. These findings suggest HAGLR could facilitate PC cell malignant behaviors through regulating the TAF15 expression, demonstrating that HAGLR might be a valuable target for the PC treatment.