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BACKGROUND: The incidence of early-stage lung adenocarcinoma (ES-LUAD) is steadily increasing among non-smokers. Previous research has identified dysbiosis in the gut microbiota of patients with lung cancer. However, the local microbial profile of non-smokers with ES-LUAD remains largely unknown. In this study, we systematically characterized the local microbial community and its associated features to enable early intervention. METHODS: A prospective collection of ES-LUAD samples (46 cases) and their corresponding normal tissues adjacent to the tumor (41 cases), along with normal lung tissue samples adjacent to pulmonary bullae in patients with spontaneous pneumothorax (42 cases), were subjected to ultra-deep metagenomic sequencing, host transcriptomic sequencing, and proteomic sequencing. The obtained omics data were subjected to both individual and integrated analysis using Spearman correlation coefficients. RESULTS: We concurrently detected the presence of bacteria, fungi, and viruses in the lung tissues. The microbial profile of ES-LUAD exhibited similarities to NAT but demonstrated significant differences from the healthy controls (HCs), characterized by an overall reduction in species diversity. Patients with ES-LUAD exhibited local microbial dysbiosis, suggesting the potential pathogenicity of certain microbial species. Through multi-omics correlations, intricate local crosstalk between the host and local microbial communities was observed. Additionally, we identified a significant positive correlation (rho > 0.6) between Methyloversatilis discipulorum and GOLM1 at both the transcriptional and protein levels using multi-omics data. This correlated axis may be associated with prognosis. Finally, a diagnostic model composed of six bacterial markers successfully achieved precise differentiation between patients with ES-LUAD and HCs. CONCLUSIONS: Our study depicts the microbial spectrum in patients with ES-LUAD and provides evidence of alterations in lung microbiota and their interplay with the host, enhancing comprehension of the pathogenic mechanisms that underlie ES-LUAD. The specific model incorporating lung microbiota can serve as a potential diagnostic tool for distinguishing between ES-LUAD and HCs.
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Adenocarcinoma de Pulmão , Neoplasias Pulmonares , Metagenômica , Microbiota , Proteômica , Transcriptoma , Humanos , Adenocarcinoma de Pulmão/microbiologia , Adenocarcinoma de Pulmão/genética , Neoplasias Pulmonares/microbiologia , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/genética , Metagenômica/métodos , Masculino , Feminino , Transcriptoma/genética , Microbiota/genética , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Disbiose/microbiologia , Perfilação da Expressão Gênica , Interações entre Hospedeiro e Microrganismos/genética , IdosoRESUMO
Arachidonic acid metabolism plays a crucial role in the development and progression of inflammatory and metabolic liver diseases. However, its role in hepatocellular carcinoma (HCC) remains unclear. In this study, we investigated the expression of key genes involved in the arachidonic acid metabolism pathway in HCC using a combination of bioinformatics, proteomics and immunohistochemistry analyses. Through a comprehensive analysis of publicly available datasets, clinical HCC tissues, and tissue microarrays, we compared the expression of hepatic arachidonic acid metabolic genes. We observed significant downregulation of cytochrome P450 (CYP450) pathway genes at both the messenger RNA and protein levels in HCC tissues compared to normal liver tissues. Furthermore, we observed a strong correlation between the deregulation of the arachidonic acid metabolism CYP450 pathway and the pathological features and prognosis of HCC. Specifically, the expression of CYP2C8/9/18/19 was significantly correlated with pathological grade (r = -.484, p < .0001), vascular invasion (r = -.402, p < .0001), aspartate transaminase (r = -.246, p = .025), gamma-glutamyl transpeptidase (r = -.252, p = .022), alkaline phosphatase (r = -.342, p = .002), alpha-fetoprotein (r = -.311, p = .004) and carbohydrate antigen 19-9 (r = -.227, p = .047). Moreover, we discovered a significant association between CYP450 pathway activity and vascular invasion in HCC. Collectively, these data indicate that arachidonic acid CYP450 metabolic pathway deregulation is implicated in HCC progression and may be a potential predictive factor for early recurrence in patients with HCC.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , Ácido Araquidônico , Sistema Enzimático do Citocromo P-450/genéticaRESUMO
We propose a low loss, wideband silicon transverse magnetic (TM) polarizer with high polarization extinction ratio and low reflection based on subwavelength grating. By arranging and optimizing a mutually perpendicular subwavelength grating with different duty cycles as the core and cladding, efficient waveguiding and radiation can be achieved for the TM and transverse electric (TE) injection, respectively. In simulation, the proposed TM polarizer has a footprint of 40µm×16.68µm, an insertion loss <0.7d B, a polarization extinction ratio ≥20d B, and an unwanted TE reflection <-17.4d B in the wavelength range of 1230-1700 nm. Moreover, the fabrication tolerance of the proposed device is also investigated.
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Abnormal cholesterol synthesis plays a crucial role in the development of hepatocellular carcinoma (HCC). Sterol regulatory element-binding protein 2 (SREBP2) is involved in cholesterol synthesis by translocating to the nucleus where it stimulates the transcription of genes encoding enzymes involved in the cholesterol synthesis pathway. However, the function and regulatory mechanism of SREBP2 in HCC remain unclear. In this study, we aimed to gain a better understanding of the effects of SREBP2 and its functional mechanism in HCC. In 20 HCC patients, we demonstrated that SREBP2 was highly expressed in HCC specimens, relative to their peritumoral tissue, and that higher expression correlated positively with a poor prognosis in these patients. Moreover, higher SREBP2 levels in the nucleus enhanced the occurrence of microvascular invasion, whereas inhibition of SREBP2 nuclear translocation by fatostatin markedly suppressed the migration and invasion of HCC cells via the epithelial-mesenchymal transition (EMT) process. The effects of SREBP2 were subject to functional activity of large tumor suppressor kinase (LATS), whereas inhibition of LATS promoted nuclear translocation of SREBP2, as observed in hepatoma cells and a subset of subcutaneous tumor samples from nude mice. In conclusion, SREBP2 enhances the invasion and metastasis of HCC cells by promoting EMT, which can be strengthened by the repression of LATS. Therefore, SREBP2 may serve as a novel therapeutic target for HCC.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Animais , Camundongos , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células , Transição Epitelial-Mesenquimal/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Hepáticas/patologia , Camundongos Nus , HumanosRESUMO
In this paper, the physicochemical properties, surface charge, and crystal defects of MIL-88A (Al) were controlled by adjusting the ratio of metal ligands and temperature in the synthetic system without the addition of surfactants. The adsorption properties of different crystals for Congo red (CR) were studied. Among them, MIL-88A (Al)-130 and MIL-88A (Al)-d have the best adsorption properties. The maximum adsorption capacities are 600.8 and 1167 mg · g-1, respectively. Compared with MIL-88A (Al)-130, the adsorption performance of MIL-88A (Al)-d was increased by 94.2%, and the adsorption rate was increased by about 4 times. It can be seen that increasing the proportion of metal ligands within a certain range will improve the adsorption capacity. The structure and morphology of the adsorbent were characterized by XRD, FTIR, SEM, EDS, TGA, BET, and zeta potential. The effects of time, temperature, pH, initial solution concentration, and dosage on CR adsorption properties were systematically discussed. The pseudo-second-order kinetic model and Langmuir isothermal model can well describe the adsorption process, which indicates that the adsorption process is a single-layer chemisorption occurring on a uniform surface. According to thermodynamics, this adsorption is an endothermic process. The mechanism of CR removal is proposed as the electrostatic attraction, hydrogen bond, metal coordination effect, π-π conjugation, crystal defect, and pore-filling effect. In addition, MIL-88A (Al)-d has good repeatability, indicating that it is a good material for treating anionic dye wastewater.
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Many bacterial pathogens hijack macrophages to egress from the port of entry to the lymphatic drainage and/or bloodstream, causing dissemination of life-threatening infections. However, the underlying mechanisms are not well understood. Here, we report that Salmonella infection generates directional electric fields (EFs) in the follicle-associated epithelium of mouse cecum. In vitro application of an EF, mimicking the infection-generated electric field (IGEF), induces directional migration of primary mouse macrophages to the anode, which is reversed to the cathode upon Salmonella infection. This infection-dependent directional switch is independent of the Salmonella pathogenicity island 1 (SPI-1) type III secretion system. The switch is accompanied by a reduction of sialic acids on glycosylated surface components during phagocytosis of bacteria, which is absent in macrophages challenged by microspheres. Moreover, enzymatic cleavage of terminally exposed sialic acids reduces macrophage surface negativity and severely impairs directional migration of macrophages in response to an EF. Based on these findings, we propose that macrophages are attracted to the site of infection by a combination of chemotaxis and galvanotaxis; after phagocytosis of bacteria, surface electrical properties of the macrophage change, and galvanotaxis directs the cells away from the site of infection.
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Trato Gastrointestinal/imunologia , Macrófagos/fisiologia , Resposta Táctica/fisiologia , Animais , Proteínas de Bactérias , Movimento Celular/fisiologia , Condutividade Elétrica , Eletricidade , Epitélio/imunologia , Epitélio/metabolismo , Feminino , Macrófagos/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fagocitose , Salmonella/patogenicidade , Infecções por Salmonella/metabolismo , Infecções por Salmonella/fisiopatologiaRESUMO
BACKGROUND: The risk of chronic hepatitis B (CHB) infection is influenced by aberrant DNA methylation and altered nucleotide synthesis and repair, possibly caused by polymorphic variants in one-carbon metabolism genes. In the present study, we investigated the relationship between polymorphisms belonging to the one-carbon metabolic pathway and CHB infection. METHODS: A case-control study using 230 CHB patients and 234 unrelated healthy controls was carried out to assess the genetic association of 24 single nucleotide polymorphisins (SNPs) determined by mass spectrometry. RESULTS: Three SNPs, comprising rs10717122 and rs2229717 in serine hydroxymethyltransferase1/2 (SHMT2) and rs585800 in betaine-homocysteine S-methyltransferase (BHMT), were associated with the risk of CHB. Patients with DEL allele, DEL.DEL and DEL.T genotypes of rs10717122 had a 1.40-, 2.00- and 1.83-fold increased risk for CHB, respectively. Cases inheriting TA genotype of rs585800 had a 2.19-fold risk for CHB infection. The T allele of rs2229717 was less represented in the CHB cases (odds ratio = 0.66, 95% confidence interval = 0.48-0.92). The T allele of rs2229717 was less in patients with a low hepatitis B virus-DNA level compared to the control group (odds ratio = 0.49, 95% confidence interval = 0.25-0.97) and TT genotype of rs2229717 had a significant correlation with hepatitis B surface antigen level (p = 0.0195). Further gene-gene interaction analysis showed that subjects carrying the rs10717122 DEL.DEL/DEL.T and rs585800 TT/TA genotypes had a 2.74-fold increased risk of CHB. CONCLUSIONS: The results of the present study suggest that rs10717122, rs585800 and rs2229717 and gene-gene interactions of rs10717122 and rs585800 affect the outcome of CHB infection, at the same time as indicating their usefulness as a predictive and diagnostic biomarker of CHB infection.
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Betaína-Homocisteína S-Metiltransferase/genética , Carbono/metabolismo , Glicina Hidroximetiltransferase/genética , Hepatite B Crônica/genética , 5-Metiltetra-Hidrofolato-Homocisteína S-Metiltransferase/genética , Adenosil-Homocisteinase/genética , Adulto , Povo Asiático/genética , Estudos de Casos e Controles , Metilases de Modificação do DNA/genética , Enzimas Reparadoras do DNA/genética , Feminino , Predisposição Genética para Doença , Glicina N-Metiltransferase/genética , Hepatite B Crônica/metabolismo , Humanos , Masculino , Metionina Adenosiltransferase/genética , Metilenotetra-Hidrofolato Redutase (NADPH2)/genética , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Proteínas Supressoras de Tumor/genéticaRESUMO
Genetically encoded fluorescent voltage indicators, such as ArcLight, have been used to report action potentials (APs) in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). However, the ArcLight expression, in all cases, relied on a high number of lentiviral vector-mediated random genome integrations (8-12 copy/cell), raising concerns such as gene disruption and alteration of global and local gene expression, as well as loss or silencing of reporter genes after differentiation. Here, we report the use of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 nuclease technique to develop a hiPSC line stably expressing ArcLight from the AAVS1 safe harbor locus. The hiPSC line retained proliferative ability with a growth rate similar to its parental strain. Optical recording with conventional epifluorescence microscopy allowed the detection of APs as early as 21 days postdifferentiation, and could be repeatedly monitored for at least 5 months. Moreover, quantification and analysis of the APs of ArcLight-CMs identified two distinctive subtypes: a group with high frequency of spontaneous APs of small amplitudes that were pacemaker-like CMs and a group with low frequency of automaticity and large amplitudes that resembled the working CMs. Compared with FluoVolt voltage-sensitive dye, although dimmer, the ArcLight reporter exhibited better optical performance in terms of phototoxicity and photostability with comparable sensitivities and signal-to-noise ratios. The hiPSC line with targeted ArcLight engineering design represents a useful tool for studying cardiac development or hiPSC-derived cardiac disease models and drug testing.
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Potenciais de Ação/fisiologia , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Miócitos Cardíacos/metabolismo , Células Cultivadas , Terapia Genética , HumanosRESUMO
Inflammasomes are cytosolic multiprotein complexes that sense microbial infection and trigger cytokine production and cell death. However, the molecular components of inflammasomes and what they sense remain poorly defined. Here we demonstrate that 35 amino acids of the carboxyl terminus of flagellin triggered inflammasome activation in the absence of bacterial contaminants or secretion systems. To further elucidate the host flagellin-sensing pathway, we generated mice deficient in the intracellular sensor Naip5. These mice failed to activate the inflammasome in response to the 35 amino acids of flagellin or in response to Legionella pneumophila infection. Our data clarify the molecular basis for the cytosolic response to flagellin.
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Flagelina/imunologia , Macrófagos/imunologia , Complexos Multiproteicos/imunologia , Proteína Inibidora de Apoptose Neuronal/imunologia , Motivos de Aminoácidos/imunologia , Animais , Proteínas Reguladoras de Apoptose/imunologia , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas de Ligação ao Cálcio/imunologia , Proteínas de Ligação ao Cálcio/metabolismo , Citosol , Ensaio de Imunoadsorção Enzimática , Flagelina/química , Immunoblotting , Legionella pneumophila/imunologia , Doença dos Legionários/imunologia , Macrófagos/microbiologia , Camundongos , Proteína Inibidora de Apoptose Neuronal/genética , Receptor 5 Toll-Like/imunologia , Receptor 5 Toll-Like/metabolismo , Transdução GenéticaRESUMO
Proper control of cell migration is critically important in many biologic processes, such as wound healing, immune surveillance, and development. Much progress has been made in the initiation of cell migration; however, little is known about termination and sometimes directional reversal. During active cell migration, as in wound healing, development, and immune surveillance, the integrin expression profile undergoes drastic changes. Here, we uncovered the extensive regulatory and even opposing roles of integrins in directional cell migration in electric fields (EFs), a potentially important endogenous guidance mechanism. We established cell lines that stably express specific integrins and determined their responses to applied EFs with a high throughput screen. Expression of specific integrins drove cells to migrate to the cathode or to the anode or to lose migration direction. Cells expressing αMß2, ß1, α2, αIIbß3, and α5 migrated to the cathode, whereas cells expressing ß3, α6, and α9 migrated to the anode. Cells expressing α4, αV, and α6ß4 lost directional electrotaxis. Manipulation of α9 molecules, one of the molecular directional switches, suggested that the intracellular domain is critical for the directional reversal. These data revealed an unreported role for integrins in controlling stop, go, and reversal activity of directional migration of mammalian cells in EFs, which might ensure that cells reach their final destination with well-controlled speed and direction.-Zhu, K., Takada, Y., Nakajima, K., Sun, Y., Jiang, J., Zhang, Y., Zeng, Q., Takada, Y., Zhao, M. Expression of integrins to control migration direction of electrotaxis.
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Movimento Celular/fisiologia , Integrinas/fisiologia , Animais , Células CHO , Movimento Celular/genética , Cricetulus , Eletricidade , Corantes Fluorescentes , Humanos , Cadeias alfa de Integrinas/genética , Cadeias alfa de Integrinas/fisiologia , Integrinas/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Resposta Táctica/fisiologia , Imagem com Lapso de Tempo , TranscriptomaRESUMO
For directional movement, eukaryotic cells depend on the proper organization of their actin cytoskeleton. This engine of motility is made up of highly dynamic nonequilibrium actin structures such as flashes, oscillations, and traveling waves. In Dictyostelium, oscillatory actin foci interact with signals such as Ras and phosphatidylinositol 3,4,5-trisphosphate (PIP3) to form protrusions. However, how signaling cues tame actin dynamics to produce a pseudopod and guide cellular motility is a critical open question in eukaryotic chemotaxis. Here, we demonstrate that the strength of coupling between individual actin oscillators controls cell polarization and directional movement. We implement an inducible sequestration system to inactivate the heterotrimeric G protein subunit Gß and find that this acute perturbation triggers persistent, high-amplitude cortical oscillations of F-actin. Actin oscillators that are normally weakly coupled to one another in wild-type cells become strongly synchronized following acute inactivation of Gß. This global coupling impairs sensing of internal cues during spontaneous polarization and sensing of external cues during directional motility. A simple mathematical model of coupled actin oscillators reveals the importance of appropriate coupling strength for chemotaxis: moderate coupling can increase sensitivity to noisy inputs. Taken together, our data suggest that Gß regulates the strength of coupling between actin oscillators for efficient polarity and directional migration. As these observations are only possible following acute inhibition of Gß and are masked by slow compensation in genetic knockouts, our work also shows that acute loss-of-function approaches can complement and extend the reach of classical genetics in Dictyostelium and likely other systems as well.
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Actinas/metabolismo , Movimento Celular , Polaridade Celular , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Modelos Biológicos , Relógios Biológicos , Citoesqueleto/metabolismo , Dictyostelium , Transdução de Sinais , SirolimoRESUMO
Stationary symmetrical fish keratocyte cells break symmetry and become motile spontaneously but slowly. We found that applying electric field (EF) accelerates the polarization by an order of magnitude. While spontaneously polarized cells move persistently for hours, the EF-induced polarity is lost in a majority of cells when the EF is switched off. However, if the EF is applied for a long time and then switched off, the majority of cell move stably. Myosin inhibition abolishes spontaneous polarization, but does not slow down EF-induced polarization, and after the EF is turned off, motility does not stop; however, the cell movements are erratic. Our results suggest that the EF rapidly polarizes the cells, but that resulting polarization becomes stable slowly, and that the EF bypasses the requirement for myosin action in motility initiation.
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Escamas de Animais/metabolismo , Movimento Celular , Polaridade Celular , Estimulação Elétrica , Células Epiteliais/metabolismo , Miosinas/metabolismo , Escamas de Animais/citologia , Animais , Células Cultivadas , Ciclídeos , Fenótipo , Fatores de TempoRESUMO
When a constraint is removed, confluent cells migrate directionally into the available space. How the migration directionality and speed increase are initiated at the leading edge and propagate into neighboring cells are not well understood. Using a quantitative visualization technique-Particle Image Velocimetry (PIV)-we revealed that migration directionality and speed had strikingly different dynamics. Migration directionality increases as a wave propagating from the leading edge into the cell sheet, while the increase in cell migration speed is maintained only at the leading edge. The overall directionality steadily increases with time as cells migrate into the cell-free space, but migration speed remains largely the same. A particle-based compass (PBC) model suggests cellular interplay (which depends on cell-cell distance) and migration speed are sufficient to capture the dynamics of migration directionality revealed experimentally. Extracellular Ca2+ regulated both migration speed and directionality, but in a significantly different way, suggested by the correlation between directionality and speed only in some dynamic ranges. Our experimental and modeling results reveal distinct directionality and speed dynamics in collective migration, and these factors can be regulated by extracellular Ca2+ through cellular interplay. Quantitative visualization using PIV and our PBC model thus provide a powerful approach to dissect the mechanisms of collective cell migration.
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Cálcio/metabolismo , Comunicação Celular , Movimento Celular , Epitélio Corneano/citologia , Materiais Biocompatíveis/química , Contagem de Células , Linhagem Celular , Dimetilpolisiloxanos/química , Epitélio Corneano/metabolismo , Humanos , Modelos Biológicos , CicatrizaçãoRESUMO
Cell fragments devoid of the nucleus and major organelles are found in physiology and pathology, for example platelets derived from megakaryocytes, and cell fragments from white blood cells and glioma cells. Platelets exhibit active chemotaxis. Fragments from white blood cells display chemotaxis, phagocytosis, and bactericidal functions. Signaling mechanisms underlying migration of cell fragments are poorly understood. Here we used fish keratocyte fragments and demonstrated striking differences in signal transduction in migration of cell fragments and parental cells in a weak electric field. cAMP or cGMP agonists completely abolished directional migration of fragments, but had no effect on parental cells. The inhibition effects were prevented by pre-incubating with cAMP and cGMP antagonists. Blocking cAMP and cGMP downstream signaling by inhibition of PKA and PKG also recovered fragment galvanotaxis. Both perturbations confirmed that the inhibitory effect was mediated by cAMP or cGMP signaling. Inhibition of cathode signaling with PI3K inhibitor LY294002 also prevented the effects of cAMP or cGMP agonists. Our results suggest that cAMP and cGMP are essential for galvanotaxis of cell fragments, in contrast to the signaling mechanisms in parental cells.
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Movimento Celular , Micropartículas Derivadas de Células/metabolismo , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Fibroblastos/metabolismo , Sistemas do Segundo Mensageiro , Animais , Movimento Celular/efeitos dos fármacos , Micropartículas Derivadas de Células/efeitos dos fármacos , Células Cultivadas , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de GMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Estimulação Elétrica , Fibroblastos/efeitos dos fármacos , Peixes , Fosfatidilinositol 3-Quinase/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/farmacologia , Sistemas do Segundo Mensageiro/efeitos dos fármacos , Fatores de TempoRESUMO
Brucella are facultative intracellular bacteria that cause chronic infections by limiting innate immune recognition. It is currently unknown whether Brucellaâ FliC flagellin, the monomeric subunit of flagellar filament, is sensed by the host during infection. Here, we used two mutants of Brucella melitensis, either lacking or overexpressing flagellin, to show that FliC hinders bacterial replication in vivo. The use of cells and mice genetically deficient for different components of inflammasomes suggested that FliC was a target of the cytosolic innate immune receptor NLRC4â in vivo but not in macrophages in vitro where the response to FliC was nevertheless dependent on the cytosolic adaptor ASC, therefore suggesting a new pathway of cytosolic flagellin sensing. However, our work also suggested that the lack of TLR5 activity of Brucella flagellin and the regulation of its synthesis and/or delivery into host cells are both part of the stealthy strategy of Brucella towards the innate immune system. Nevertheless, as a flagellin-deficient mutant of B. melitensis wasfound to cause histologically demonstrable injuries in the spleen of infected mice, we suggested that recognition of FliC plays a role in the immunological stand-off between Brucella and its host, which is characterized by a persistent infection with limited inflammatory pathology.
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Brucella melitensis/patogenicidade , Brucelose/fisiopatologia , Flagelina/imunologia , Flagelina/metabolismo , Imunidade Inata/fisiologia , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Brucella melitensis/imunologia , Brucella melitensis/metabolismo , Brucelose/metabolismo , Brucelose/patologia , Proteínas de Ligação ao Cálcio/metabolismo , Linhagem Celular , Colo/microbiologia , Colo/patologia , Modelos Animais de Doenças , Feminino , Flagelina/genética , Humanos , Técnicas In Vitro , Macrófagos/microbiologia , Macrófagos/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Mutação/genética , Baço/microbiologia , Baço/patologia , Receptor 5 Toll-Like/metabolismoRESUMO
Salmonella translocate to the gut epithelium via microfold cells lining the follicle-associated epithelium (FAE). How Salmonella localize to the FAE is not well characterized. Here we use live imaging and competitive assays between wild-type and chemotaxis-deficient mutants to show that Salmonella enterica serotype Typhimurium (S. Typhimurium) localize to the FAE independently of chemotaxis in an ex vivo mouse caecum infection model. Electrical recordings revealed polarized FAE with sustained outward current and small transepithelial potential, while the surrounding villus is depolarized with inward current and large transepithelial potential. The distinct electrical potentials attracted S. Typhimurium to the FAE while Escherichia coli (E. coli) localized to the villi, through a process called galvanotaxis. Chloride flux involving the cystic fibrosis transmembrane conductance regulator (CFTR) generated the ionic currents around the FAE. Pharmacological inhibition of CFTR decreased S. Typhimurium FAE localization but increased E. coli recruitment. Altogether, our findings demonstrate that bioelectric cues contribute to S. Typhimurium targeting of specific gut epithelial locations, with potential implications for other enteric bacterial infections.
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Motile cells migrate directionally in the electric field in a process known as galvanotaxis, important and under-investigated phenomenon in wound healing and development. We previously reported that individual fish keratocyte cells migrate to the cathode in electric fields, that inhibition of PI3 kinase reverses single cells to the anode, and that large cohesive groups of either unperturbed or PI3K-inhibited cells migrate to the cathode. Here we find that small uninhibited cell groups move to the cathode, while small groups of PI3K-inhibited cells move to the anode. Small groups move faster than large groups, and groups of unperturbed cells move faster than PI3K-inhibited cell groups of comparable sizes. Shapes and sizes of large groups change little when they start migrating, while size and shapes of small groups change significantly, lamellipodia disappear from the rear edges of these groups, and their shapes start to resemble giant single cells. Our results are consistent with the computational model, according to which cells inside and at the edge of the groups pool their propulsive forces to move but interpret directional signals differently. Namely, cells in the group interior are directed to the cathode independently of their chemical state. Meanwhile, the edge cells behave like individual cells: they are directed to the cathode/anode in uninhibited/PI3K-inhibited groups, respectively. As a result, all cells drive uninhibited groups to the cathode, while larger PI3K-inhibited groups are directed by cell majority in the group interior to the cathode, while majority of the edge cells in small groups win the tug-of-war driving these groups to the anode.
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Macrophages can exhibit pro-inflammatory or pro-reparatory functions, contingent upon their specific activation state. This dynamic behavior empowers macrophages to engage in immune reactions and contribute to tissue homeostasis. Understanding the intricate interplay between macrophage motility and activation status provides valuable insights into the complex mechanisms that govern their diverse functions. In a recent study, we developed a classification method based on morphology, which demonstrated that movement characteristics, including speed and displacement, can serve as distinguishing factors for macrophage subtypes. In this study, we develop a deep learning model to explore the potential of classifying macrophage subtypes based solely on raw trajectory patterns. The classification model relies on the time series of x-y coordinates, as well as the distance traveled and net displacement. We begin by investigating the migratory patterns of macrophages to gain a deeper understanding of their behavior. Although this analysis does not directly inform the deep learning model, it serves to highlight the intricate and distinct dynamics exhibited by different macrophage subtypes, which cannot be easily captured by a finite set of motility metrics. Our study uses cell trajectories to classify three macrophage subtypes: M0, M1, and M2. This advancement holds promising implications for the future, as it suggests the possibility of identifying macrophage subtypes without relying on shape analysis. Consequently, it could potentially eliminate the necessity for high-quality imaging techniques and provide more robust methods for analyzing inherently blurry images.
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Betaine-homocysteine methyltransferase (BHMT) regulates protein methylation and is correlated with tumorigenesis; however, the effects and regulation of BHMT in hepatocarcinogenesis remain largely unexplored. Here, we determined the clinical significance of BHMT in the occurrence and progression of hepatocellular carcinoma (HCC) using tissue samples from 198 patients. BHMT was to be frequently found (86.6%) expressed at relatively low levels in HCC tissues and was positively correlated with the overall survival of patients with HCC. Bhmt overexpression effectively suppressed several malignant phenotypes in hepatoma cells in vitro and in vivo, whereas complete knockout of Bhmt (Bhmt-/-) produced the opposite effect. We combined proteomics, metabolomics, and molecular biological strategies and detected that Bhmt-/- promoted hepatocarcinogenesis and tumor progression by enhancing the activity of glucose-6-phosphate dehydrogenase (G6PD) and PPP metabolism in DEN-induced HCC mouse and subcutaneous tumor-bearing models. In contrast, restoration of Bhmt with an AAV8-Bhmt injection or pharmacological inhibition of G6PD attenuated hepatocarcinogenesis. Additionally, coimmunoprecipitation identified monomethylated modifications of the G6PD, and BHMT regulated the methylation of G6PD. Protein sequence analysis, generation and application of specific antibodies, and site-directed mutagenesis indicated G6PD methylation at the arginine residue 246. Furthermore, we established bidirectionally regulated BHMT cellular models combined with methylation-deficient G6PD mutants to demonstrate that BHMT potentiated arginine methylation of G6PD, thereby inhibiting G6PD activity, which in turn suppressed hepatocarcinogenesis. Taken together, this study reveals a new methylation-regulatory mechanism in hepatocarcinogenesis owing to BHMT deficiency, suggesting a potential therapeutic strategy for HCC treatment.
Assuntos
Betaína-Homocisteína S-Metiltransferase , Carcinogênese , Carcinoma Hepatocelular , Glucosefosfato Desidrogenase , Neoplasias Hepáticas , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/genética , Animais , Humanos , Metilação , Betaína-Homocisteína S-Metiltransferase/metabolismo , Betaína-Homocisteína S-Metiltransferase/genética , Glucosefosfato Desidrogenase/metabolismo , Glucosefosfato Desidrogenase/genética , Camundongos , Carcinogênese/genética , Carcinogênese/metabolismo , Arginina/metabolismo , Masculino , Linhagem Celular Tumoral , Camundongos Knockout , Regulação Neoplásica da Expressão GênicaRESUMO
Electrical signaling plays a crucial role in the cellular response to tissue injury in wound healing and an external electric field (EF) may expedite the healing process. Here, we have developed a standalone, wearable, and programmable electronic device to administer a well-controlled exogenous EF, aiming to accelerate wound healing in an in vivo mouse model to provide pre-clinical evidence. We monitored the healing process by assessing the re-epithelization rate and the ratio of M1/M2 macrophage phenotypes through histology staining. Following three days of treatment, the M1/M2 macrophage ratio decreased by 30.6% and the re-epithelization in the EF-treated wounds trended towards a non-statically significant 24.2% increase compared to the control. These findings provide point towards the effectiveness of the device in shortening the inflammatory phase by promoting reparative macrophages over inflammatory macrophages, and in speeding up re-epithelialization. Our wearable device supports the rationale for the application of programmed EFs for wound management in vivo and provides an exciting basis for further development of our technology based on the modulation of macrophages and inflammation to better wound healing.