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AIM: Biotechnical processes in Escherichia coli often operate with artificial plasmids. However, these bioprocesses frequently encounter plasmid loss. To ensure stable expression of heterologous genes in E. coli BL21(DE3), a novel plasmid addiction system (PAS) was developed. METHODS AND RESULTS: This PAS employed an essential gene grpE encoding a cochaperone in the DnaK-DnaJ-GrpE chaperone system as the selection marker, which represented a chromosomal ΔgrpE mutant harboring episomal expression plasmids that carry supplementary grpE alleles to restore the deficiency. To demonstrate the feasibility of this system, it was implemented in phloroglucinol (PG) biosynthesis, manifesting improved host tolerance to PG and increased PG production. Specifically, PG titer significantly improved from 0.78 ± 0.02 to 1.34 ± 0.04 g l-1, representing a 71.8% increase in shake-flask fermentation. In fed-batch fermentation, the titer increased from 3.71 ± 0.11 to 4.54 ± 0.10 g l-1, showing a 22.4% increase. RNA sequencing and transcriptome analysis revealed that the improvements were attributed to grpE overexpression and upregulation of various protective chaperones and the biotin acetyl-CoA carboxylase ligase coding gene birA. CONCLUSION: This novel PAS could be regarded as a typical example of nonanabolite- and nonmetabolite-related PAS. It effectively promoted plasmid maintenance in the host, improved tolerance to PG, and increased the titer of this compound.
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Proteínas de Escherichia coli , Proteínas de Choque Térmico , Floroglucinol , Plasmídeos , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Fermentação , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Floroglucinol/metabolismo , Floroglucinol/análogos & derivados , Plasmídeos/genéticaRESUMO
Bone metastatic disease of prostate cancer (PCa) is incurable and progression in bone is largely dictated by tumor-stromal interactions in the bone microenvironment. We showed previously that bone neutrophils initially inhibit bone metastatic PCa growth yet metastatic PCa becomes resistant to neutrophil response. Further, neutrophils isolated from tumor-bone lost their ability to suppress tumor growth through unknown mechanisms. With this study, our goal was to define the impact of metastatic PCa on neutrophil function throughout tumor progression and to determine the potential of neutrophils as predictive biomarkers of metastatic disease. Using patient peripheral blood polymorphonuclear neutrophils (PMNs), we identified that PCa progression dictates PMN cell surface markers and gene expression, but not cytotoxicity against PCa. Importantly, we also identified a novel phenomenon in which second generation androgen deprivation therapy (ADT) suppresses PMN cytotoxicity via increased transforming growth factor beta receptor I (TßRI). High dose testosterone and genetic or pharmacologic TßRI inhibition rescued androgen receptor-mediated neutrophil suppression and restored neutrophil anti-tumor immune response. These studies highlight the ability to leverage standard-care ADT to generate neutrophil anti-tumor responses against bone metastatic PCa.
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Neoplasias Ósseas , Neoplasias da Próstata , Masculino , Humanos , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Receptores Androgênicos/metabolismo , Androgênios , Neutrófilos/metabolismo , Antagonistas de Androgênios/farmacologia , Antagonistas de Androgênios/uso terapêutico , Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/secundário , Linhagem Celular Tumoral , Microambiente TumoralRESUMO
BACKGROUND: Formyl peptide receptor 2 (Fpr2) plays a crucial role in colon homeostasis and microbiota balance. Commensal E. coli is known to promote the regeneration of damaged colon epithelial cells. The aim of the study was to investigate the connection between E. coli and Fpr2 in the recovery of colon epithelial cells. RESULTS: The deficiency of Fpr2 was associated with impaired integrity of the colon mucosa and an imbalance of microbiota, characterized by the enrichment of Proteobacteria in the colon. Two serotypes of E. coli, O22:H8 and O91:H21, were identified in the mouse colon through complete genome sequencing. E. coli O22:H8 was found to be prevalent in the gut of mice and exhibited lower virulence compared to O91:H21. Germ-free (GF) mice that were pre-orally inoculated with E. coli O22:H8 showed reduced susceptibility to chemically induced colitis, increased proliferation of epithelial cells, and improved mouse survival. Following infection with E. coli O22:H8, the expression of Fpr2 in colon epithelial cells was upregulated, and the products derived from E. coli O22:H8 induced migration and proliferation of colon epithelial cells through Fpr2. Fpr2 deficiency increased susceptibility to chemically induced colitis, delayed the repair of damaged colon epithelial cells, and heightened inflammatory responses. Additionally, the population of E. coli was observed to increase in the colons of Fpr2-/- mice with colitis. CONCLUSION: Commensal E. coli O22:H8 stimulated the upregulation of Fpr2 expression in colon epithelial cells, and the products from E. coli induced migration and proliferation of colon epithelial cells through Fpr2. Fpr2 deficiency led to an increased E. coli population in the colon and delayed recovery of damaged colon epithelial cells in mice with colitis. Therefore, Fpr2 is essential for the effects of commensal E. coli on colon epithelial cell recovery.
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Over the past thirty years, the importance of chemokines and their seven-transmembrane G protein-coupled receptors (GPCRs) has been increasingly recognized. Chemokine interactions with receptors trigger signaling pathway activity to form a network fundamental to diverse immune processes, including host homeostasis and responses to disease. Genetic and nongenetic regulation of both the expression and structure of chemokines and receptors conveys chemokine functional heterogeneity. Imbalances and defects in the system contribute to the pathogenesis of a variety of diseases, including cancer, immune and inflammatory diseases, and metabolic and neurological disorders, which render the system a focus of studies aiming to discover therapies and important biomarkers. The integrated view of chemokine biology underpinning divergence and plasticity has provided insights into immune dysfunction in disease states, including, among others, coronavirus disease 2019 (COVID-19). In this review, by reporting the latest advances in chemokine biology and results from analyses of a plethora of sequencing-based datasets, we outline recent advances in the understanding of the genetic variations and nongenetic heterogeneity of chemokines and receptors and provide an updated view of their contribution to the pathophysiological network, focusing on chemokine-mediated inflammation and cancer. Clarification of the molecular basis of dynamic chemokine-receptor interactions will help advance the understanding of chemokine biology to achieve precision medicine application in the clinic.
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COVID-19 , Medicina de Precisão , Humanos , Receptores de Quimiocinas/genética , Receptores de Quimiocinas/metabolismo , COVID-19/genética , Quimiocinas/genética , Quimiocinas/metabolismo , Epigênese GenéticaRESUMO
Background: Hepatocellular carcinoma (HCC) is one of the most lethal human tumors with extensive intratumor heterogeneity (ITH). Serine protease 3 (PRSS3) is an indispensable member of the trypsin family and has been implicated in the pathogenesis of several malignancies, including HCC. However, the paradoxical effects of PRSS3 on carcinogenesis due to an unclear molecular basis impede the utilization of its biomarker potential. We hereby explored the contribution of PRSS3 transcripts to tumor functional heterogeneity by systematically dissecting the expression of four known splice variants of PRSS3 (PRSS3-SVs, V1~V4) and their functional relevance to HCC. Methods: The expression and DNA methylation of PRSS3 transcripts and their associated clinical relevance in HCC were analyzed using several publicly available datasets and validated using qPCR-based assays. Functional experiments were performed in gain- and loss-of-function cell models, in which PRSS3 transcript constructs were separately transfected after deleting PRSS3 expression by CRISPR/Cas9 editing. Results: PRSS3 was aberrantly differentially expressed toward bipolarity from very low (PRSS3Low ) to very high (PRSS3High ) expression across HCC cell lines and tissues. This was attributable to the disruption of PRSS3-SVs, in which PRSS3-V2 and/or PRSS3-V1 were dominant transcripts leading to PRSS3 expression, whereas PRSS3-V3 and -V4 were rarely or minimally expressed. The expression of PRSS3-V2 or -V1 was inversely associated with site-specific CpG methylation at the PRSS3 promoter region that distinguished HCC cells and tissues phenotypically between hypermethylated low-expression (mPRSS3-SVLow ) and hypomethylated high-expression (umPRSS3-SVHigh ) groups. PRSS3-SVs displayed distinct functions from oncogenic PRSS3-V2 to tumor-suppressive PRSS3-V1, -V3 or PRSS3-V4 in HCC cells. Clinically, aberrant expression of PRSS3-SVs was translated into divergent relevance in patients with HCC, in which significant epigenetic downregulation of PRSS3-V2 was seen in early HCC and was associated with favorable patient outcome. Conclusions: These results provide the first evidence for the transcriptional and functional characterization of PRSS3 transcripts in HCC. Aberrant expression of divergent PRSS3-SVs disrupted by site-specific CpG methylation may integrate the effects of oncogenic PRSS3-V2 and tumor-suppressive PRSS3-V1, resulting in the molecular diversity and functional plasticity of PRSS3 in HCC. Dysregulated expression of PRSS3-V2 by site-specific CpG methylation may have potential diagnostic value for patients with early HCC.
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Medulloblastoma (MB) is one of the most common childhood malignant brain tumors (WHO grade IV), traditionally divided into WNT, SHH, Group 3, and Group 4 subgroups based on the transcription profiles, somatic DNA alterations, and clinical outcomes. Unlike WNT and SHH subgroup MBs, Group 3 and Group 4 MBs have similar transcriptomes and lack clearly specific drivers and targeted therapeutic options. The recently revised WHO Classification of CNS Tumors has assigned Group 3 and 4 to a provisional non-WNT/SHH entity. In the present study, we demonstrate that Kir2.1, an inwardly-rectifying potassium channel, is highly expressed in non-WNT/SHH MBs, which promotes tumor cell invasion and metastasis by recruiting Adam10 to enhance S2 cleavage of Notch2 thereby activating the Notch2 signaling pathway. Disruption of the Notch2 pathway markedly inhibited the growth and metastasis of Kir2.1-overexpressing MB cell-derived xenograft tumors in mice. Moreover, Kir2.1high/nuclear N2ICDhigh MBs are associated with the significantly shorter lifespan of the patients. Thus, Kir2.1high/nuclear N2ICDhigh can be used as a biomarker to define a novel subtype of non-WNT/SHH MBs. Our findings are important for the modification of treatment regimens and the development of novel-targeted therapies for non-WNT/SHH MBs.
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Neoplasias Cerebelares , Meduloblastoma , Animais , Neoplasias Cerebelares/genética , Neoplasias Cerebelares/metabolismo , Neoplasias Cerebelares/patologia , Criança , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Humanos , Meduloblastoma/genética , Meduloblastoma/metabolismo , Meduloblastoma/patologia , Camundongos , Mutação , Canais de Potássio Corretores do Fluxo de Internalização , Transdução de SinaisRESUMO
Swift recruitment of phagocytic leucocytes is critical in preventing infection when bacteria breach through the protective layers of the skin. According to canonical models, this occurs via an indirect process that is initiated by contact of bacteria with resident skin cells and which is independent of the pathogenic potential of the invader. Here we describe a more rapid mechanism of leucocyte recruitment to the site of intrusion of the important skin pathogen Staphylococcus aureus that is based on direct recognition of specific bacterial toxins, the phenol-soluble modulins (PSMs), by circulating leucocytes. We used a combination of intravital imaging, ear infection and skin abscess models, and in vitro gene expression studies to demonstrate that this early recruitment was dependent on the transcription factor EGR1 and contributed to the prevention of infection. Our findings refine the classical notion of the non-specific and resident cell-dependent character of the innate immune response to bacterial infection by demonstrating a pathogen-specific high-alert mechanism involving direct recruitment of immune effector cells by secreted bacterial products.
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Toxinas Bacterianas/imunologia , Linfócitos/imunologia , Infiltração de Neutrófilos/imunologia , Pele/imunologia , Pele/microbiologia , Infecções Cutâneas Estafilocócicas/imunologia , Staphylococcus aureus/imunologia , Animais , Feminino , Humanos , Microscopia Intravital/métodos , Camundongos Endogâmicos C57BL , Staphylococcus aureus/patogenicidade , Fatores de VirulênciaRESUMO
Human cathelicidin antimicrobial peptide LL-37 (LL-37) is an antimicrobial peptide derived from its precursor protein hCAP18, which is an only cathelicidin in human. LL-37 not only serves as a mediator of innate immune defense against invading microorganisms, but it also plays an essential role in tissue homeostasis, regenerative processes, regulation of proinflammatory responses, and inhibition of cancer progression. Therefore, LL-37 has been considered as a drug lead for diseases. However, high levels of LL-37 may reduce cell viability and promote apoptosis of osteoblasts, vascular smooth muscle cells, periodontal ligament cells, neutrophils, airway epithelial cells and T cells. Recent evidence reveals that LL-37-derived short peptides possess similar biological activities as the whole LL-37 with reduced cytotoxicity. Thus, such small molecules constitute a pool of potential therapeutic agents for diseases.
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Peptídeos Antimicrobianos , Catelicidinas , Células Epiteliais , Humanos , NeutrófilosRESUMO
INTRODUCTION: Glutathione reductase (GSR) provides reduced glutathione (GSH) to maintain redox homeostasis. Inhibition of GSR disrupts this balance, resulting in cell damage, which benefits cancer therapy. However, the effect of GSR inhibition on the tumorigenicity of human cervical cancer is not fully understood. MATERIALS AND METHODS: Tissue microarray analysis was employed to determine GSR expression in cervical cancer tissues by immunohistochemical staining. Cell death was measured with PI/FITC-annexin V staining. mRNA levels were measured via quantitative RT-PCR. Protein expression was measured by Western blotting and flow cytometry. STAT3 deletion was performed with CRISPR/Cas9 technology. GSR knockdown was achieved by RNA interference. Reactive oxygen species (ROS) levels were measured by DCF staining. GSR enzymatic activity was measured with a GSR assay kit. The effect of GSR inhibition on the growth of tumors formed by cervical cancer cells was investigated using a xenograft model. RESULTS: The expression of GSR was increased in human cervical cancer tissues, as shown by immunohistochemical staining. GSR knockdown by RNA interference in human cervical cancer cell lines resulted in cell death, suggesting the ability of GSR to maintain cancer cell survival. The STAT3 inhibitor 6-nitrobenzo[b]thiophene 1,1-dioxide (Stattic) also inhibited the enzymatic activity of GSR and induced the death of cervical cancer cells. More importantly, Stattic decreased the growth of xenograft tumors formed by cervical cancer cells in nude mice. Mechanistically, tumor cell death induced by Stattic-mediated GSR inhibition was ROS-dependent, since the ROS scavengers GSH and N-acetyl cysteine (NAC) reversed the effect of Stattic. In contrast, pharmacological and molecular inhibition of STAT3 did not induce the death of cervical cancer cells, suggesting a STAT3-independent activity of Stattic. CONCLUSION: Stattic inhibits the enzymatic activity of GSR and induces STAT3-independent but ROS-dependent death of cervical cancer cells, suggesting its potential application as a therapeutic agent for human cervical cancers.
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BACKGROUND: Robot-assisted and laparoscopic surgery are the most minimally invasive surgical approaches for the removal of liver lesions. Minor hepatectomy is a common surgical procedure. In this study, we evaluated the advantages and disadvantages of robot-assisted vs laparoscopic minor hepatectomy (LMH). METHODS: A systematic literature search was performed in PubMed, Embase, and the Cochrane Library to identify comparative studies on robot-assisted vs. laparoscopicminor hepatectomy up to February, 2020. The odds ratios (OR) and mean differences with 95% confidence intervals were calculated using the fixed-effects model or random-effects model. RESULTS: A total of 12 studies involving 751 patients were included in the meta-analysis. Among them, 297 patients were in the robot-assisted minor hepatectomy (RMH) group and 454 patients were in the LMH group. There were no significant differences in intraoperative blood loss (Pâ=â.43), transfusion rates (Pâ=â.14), length of hospital stay (Pâ>â.64), conversion rate (Pâ=â.62), R0 resection rate (Pâ=â.56), complications (Pâ=â.92), or mortaliy (Pâ=â.37) between the 2 groups. However, the RMH group was associated with a longer operative time (Pâ=â.0003), and higher cost (Pâ<â.00001) compared to the LMH group. No significant differences in overall survival or disease free survival between the 2 groups were observed. In the subgroup analysis of left lateral sectionectomies, RMH was still associated with a longer operative time, but no other differences in clinical outcomes were observed. CONCLUSIONS: Although RMH is associated with longer operation times and higher costs, it exhibits the same safety and effectiveness as LMH. Prospective randomized controlled clinical trials should now be considered to obtain better evidence for clinical consensus.
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Hepatectomia/métodos , Laparoscopia/estatística & dados numéricos , Procedimentos Cirúrgicos Robóticos/estatística & dados numéricos , Adulto , Idoso , Idoso de 80 Anos ou mais , Perda Sanguínea Cirúrgica/estatística & dados numéricos , Transfusão de Sangue/estatística & dados numéricos , Feminino , Humanos , Laparoscopia/métodos , Masculino , Pessoa de Meia-Idade , Estudos Observacionais como Assunto , Duração da Cirurgia , Complicações Pós-Operatórias/etiologia , Ensaios Clínicos Controlados Aleatórios como Assunto , Procedimentos Cirúrgicos Robóticos/métodos , Resultado do TratamentoRESUMO
Disruption in mucins (MUCs) is involved in cancer development and metastasis and is thus used as a biomarker. Nonsmall cell lung carcinoma (NSCLC) is characterized by heterogeneous genetic and epigenetic alterations. Lung adenocarcinoma (LUAD) and squamous cell carcinoma (LUSC) are the two primary subtypes of NSCLC that require different therapeutic interventions. Here, we report distinct expression and epigenetic alterations in mucin 22 (MUC22), a new MUC family member, in LUSC vs. LUAD. In lung cancer cell lines and tissues, MUC22 was downregulated in LUSC (MUC22Low) but upregulated in LUAD (MUC22High) with coexpression of MUC21. The aberrant expression of MUC22 was inversely correlated with its promoter hypermethylation in LUSC and hypomethylation in LUAD cells and tissues, respectively. Decreased MUC22 expression in NSCLC cell lines was restored upon treatment with epigenetic modifiers 5aza2'deoxycytidine (5Aza) or trichostatin A (TSA), accompanied by reduction in global protein level of histone deacetylase 1 (HDAC1) but increased enrichment of histone H3 lysine 9 acetylation (H3K9ac) specifically in the MUC22 promoter in the SKMES1 cell line. MUC22 knockdown increased the growth and motility of lung cancer cells and an immortalized human bronchial epithelial BEAS2B cell line via NFκB activation. Clinically, MUC22Low in LUSC and MUC22High in LUAD were shown to be indicators of unfavorable overall survival for patients with early cancer stages. Our study reveals that changes in MUC22 expression due to epigenetic alterations in NSCLC may have important biological significance and prognostic potential in LUSC when compared to LUAD. Thus, MUC22 expression and epigenetic alterations may be used for molecular subtyping of NSCLC in precision medicine.
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Adenocarcinoma de Pulmão/genética , Biomarcadores Tumorais/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma de Células Escamosas/genética , Neoplasias Pulmonares/genética , Mucinas/genética , Adenocarcinoma de Pulmão/diagnóstico , Adenocarcinoma de Pulmão/mortalidade , Adenocarcinoma de Pulmão/terapia , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma Pulmonar de Células não Pequenas/diagnóstico , Carcinoma Pulmonar de Células não Pequenas/mortalidade , Carcinoma Pulmonar de Células não Pequenas/terapia , Carcinoma de Células Escamosas/diagnóstico , Carcinoma de Células Escamosas/mortalidade , Carcinoma de Células Escamosas/terapia , Linhagem Celular Tumoral , Metilação de DNA , Conjuntos de Dados como Assunto , Regulação para Baixo , Epigênese Genética , Feminino , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Heterogeneidade Genética , Humanos , Estimativa de Kaplan-Meier , Pulmão/patologia , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/terapia , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Medicina de Precisão/métodos , Prognóstico , Regiões Promotoras Genéticas/genética , RNA-Seq , Regulação para Cima , Adulto JovemRESUMO
Host-derived antimicrobial peptides play an important role in the defense against extracellular bacterial infections. However, the capacity of antimicrobial peptides derived from macrophages as potential antibacterial effectors against intracellular pathogens remains unknown. In this study, we report that normal (wild-type, WT) mouse macrophages increased their expression of cathelin-related antimicrobial peptide (CRAMP, encoded by Camp) after infection by viable E. coli or stimulation with inactivated E. coli and its product lipopolysaccharide (LPS), a process involving activation of NF-κB followed by protease-dependent conversion of CRAMP from an inactive precursor to an active form. The active CRAMP was required by WT macrophages for elimination of phagocytosed E. coli, with participation of autophagy-related proteins ATG5, LC3-II and LAMP-1, as well as for aggregation of the bacteria with p62 (also known as SQSTM1). This process was impaired in CRAMP-/- macrophages, resulting in retention of intracellular bacteria and fragmentation of macrophages. These results indicate that CRAMP is a critical component in autophagy-mediated clearance of intracellular E. coli by mouse macrophages.
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Peptídeos Catiônicos Antimicrobianos , Escherichia coli , Animais , Autofagia , Macrófagos , Camundongos , FagocitoseRESUMO
The cathelin-related antimicrobial peptide CRAMP protects the mouse colon from inflammation, inflammation-associated carcinogenesis, and disrupted microbiome balance, as shown in systemic Cnlp-/- mice (also known as Camp-/- mice). However, the mechanistic basis for the role and the cellular source of CRAMP in colon pathophysiology are ill defined. This study, using either epithelial or myeloid conditional Cnlp-/- mice, demonstrated that epithelial cell-derived CRAMP played a major role in supporting normal development of colon crypts, mucus production, and repair of injured mucosa. On the other hand, myeloid cell-derived CRAMP potently supported colon epithelial resistance to bacterial invasion during acute inflammation with exacerbated mucosal damage and higher rate of mouse mortality. Therefore, a well concerted cooperation of epithelial- and myeloid-derived CRAMP is essential for colon mucosal homeostasis. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.
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Peptídeos Catiônicos Antimicrobianos/metabolismo , Células Epiteliais/metabolismo , Homeostase/fisiologia , Mucosa Intestinal/metabolismo , Macrófagos/metabolismo , Animais , Colo/fisiologia , Camundongos , Camundongos Knockout , CatelicidinasRESUMO
The physiological homeostasis of gut mucosal barrier is maintained by both genetic and environmental factors and its impairment leads to pathogenesis such as inflammatory bowel disease. A cytokine like molecule, FAM3D (mouse Fam3D), is highly expressed in mouse gastrointestinal tract. Here, we demonstrate that deficiency in Fam3D is associated with impaired integrity of colonic mucosa, increased epithelial hyper-proliferation, reduced anti-microbial peptide production and increased sensitivity to chemically induced colitis associated with high incidence of cancer. Pretreatment of Fam3D-/- mice with antibiotics significantly reduces the severity of chemically induced colitis and wild type (WT) mice co-housed with Fam3D-/- mice phenocopy Fam3D-deficiency showing increased sensitivity to colitis and skewed composition of fecal microbiota. An initial equilibrium of microbiota in cohoused WT and Fam3D-/- mice is followed by an increasing divergence of the bacterial composition after separation. These results demonstrate the essential role of Fam3D in colon homeostasis, protection against inflammation associated cancer and normal microbiota composition.
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Carcinogênese , Colo , Citocinas/metabolismo , Animais , Colite , Colo/metabolismo , Colo/microbiologia , Colo/patologia , Neoplasias Colorretais , Modelos Animais de Doenças , Microbioma Gastrointestinal , Inflamação , Doenças Inflamatórias Intestinais , Mucosa Intestinal/crescimento & desenvolvimento , Mucosa Intestinal/patologia , Camundongos , Proteínas Citotóxicas Formadoras de Poros/metabolismoRESUMO
Chronic inflammation is a causative factor of many cancers, although it originally acts as a protective host response to the loss of tissue homeostasis. Many inflammatory conditions predispose susceptible cells, most of which are of epithelial origin, to neoplastic transformation. There is a close correlation between digestive tract (DT) cancer and chronic inflammation, such as esophageal adenocarcinoma associated with Barrett's esophagus, helicobacter pylori infection as the cause of stomach cancer, hepatitis leading to liver cirrhosis and subsequent cancer, and colon cancer linking to inflammatory bowel diseases and schistosomiasis. A prominent feature of malignant transformation of DT tract epithelial cells is their adoption of somatic gene mutations resulting in abnormal expression of proteins that endow the cells with unlimited proliferation as well as increased motility and invasive capabilities. Many of these events are mediated by Gi-protein coupled chemoattractant receptors (GPCRs) including formyl peptide receptors (FPRs in human, Fprs in mice). In this article, we review the current understanding of FPRs (Fprs) and their function in DT cancer types as well as their potential as therapeutic targets.
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Neoplasias Gastrointestinais/genética , Receptores de Formil Peptídeo/genética , Biomarcadores Tumorais , Progressão da Doença , Suscetibilidade a Doenças , Neoplasias Gastrointestinais/metabolismo , Neoplasias Gastrointestinais/patologia , Regulação Neoplásica da Expressão Gênica , Humanos , Ligantes , Modelos Biológicos , Família Multigênica , Especificidade de Órgãos , Ligação Proteica , Receptores de Formil Peptídeo/metabolismoRESUMO
Diabetic retinopathy (DR) as a retinal neovascularization-related disease is one of the leading causes of irreversible blindness in patients. The goal of this study is to determine the role of a G-protein-coupled chemoattractant receptor (GPCR) FPR2 (mouse Fpr2) in the progression of DR, in order to identify novel therapeutic targets. We report that Fpr2 was markedly upregulated in mouse diabetic retinas, especially in retinal vascular endothelial cells, in associated with increased number of activated microglia and Müller glial cells. In contrast, in the retina of diabetic Fpr2 -/- mice, the activation of vascular endothelial cells and glia was attenuated with reduced production of proinflammatory cytokines. Fpr2 deficiency also prevented the formation of acellular capillary during diabetic progression. Furthermore, in oxygen-induced retinopathy (OIR) mice, the absence of Fpr2 was associated with diminished neovasculature formation and pathological vaso-obliteration region in the retina. These results highlight the importance of Fpr2 in exacerbating the progression of neuroglial degeneration and angiogenesis in DR and its potential as a therapeutic target.
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Although the efficacy of cancer radiotherapy (RT) can be enhanced by targeted immunotherapy, the immunosuppressive factors induced by radiation on tumor cells remain to be identified. Here, we report that CD47-mediated anti-phagocytosis is concurrently upregulated with HER2 in radioresistant breast cancer (BC) cells and RT-treated mouse syngeneic BC. Co-expression of both receptors is more frequently detected in recurrent BC patients with poor prognosis. CD47 is upregulated preferentially in HER2-expressing cells, and blocking CD47 or HER2 reduces both receptors with diminished clonogenicity and augmented phagocytosis. CRISPR-mediated CD47 and HER2 dual knockouts not only inhibit clonogenicity but also enhance macrophage-mediated attack. Dual antibody of both receptors synergizes with RT in control of syngeneic mouse breast tumor. These results provide the evidence that aggressive behavior of radioresistant BC is caused by CD47-mediated anti-phagocytosis conjugated with HER2-prompted proliferation. Dual blockade of CD47 and HER2 is suggested to eliminate resistant cancer cells in BC radiotherapy.
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Neoplasias da Mama/metabolismo , Antígeno CD47/metabolismo , Tolerância a Radiação , Receptor ErbB-2/metabolismo , Animais , Neoplasias da Mama/patologia , Antígeno CD47/genética , Proliferação de Células , Células Clonais , Feminino , Humanos , Células MCF-7 , Macrófagos/metabolismo , Camundongos , Modelos Biológicos , NF-kappa B/metabolismo , Fagocitose , Transdução de Sinais , Transcrição Gênica , Carga TumoralRESUMO
Whole-cell biocatalysts could be used in wide-ranging applications. In this study, a new kind of whole-cell biocatalyst was successfully constructed by genetically immobilizing soybean seed coat peroxidase (SBP) on the cell surface of Yarrowia lipolytica Po1h, using a new integrative surface display expression vector (pMIZY05). The coding sequence of SBP was optimized and chemically synthesized, then inserted into pMIZY05 to generate expression plasmid pMIZY05-oEp. A DNA fragment corresponding to SBP and selection marker expression cassettes, without bacterial sequences, was released from pMIZY05-oEp by enzyme digestion and used to transform host yeast cells. A transformant (CM11) with a high recombinant SBP activity of 1571.9 U/mL was obtained, and recombinant SBP was proved to be successfully anchored on cell surface by testing the activities of different cellular fractions. After optimization of culture conditions, the recombinant SBP activity of CM11 was increased to 4187.8 U/mL. Afterwards, biochemical properties of the recombinant SBP were determined: optimum catalytic conditions were 37.5â at pH 3.5, and recombinant SBP exhibited high stability during thermal or acidic treatment. Recombinant activity of cell-displayed SBP was re-examined at optimum temperature and pH, which promoted an increase up to 4432.5 U/mL. To our knowledge, this represents the highest activity ever reported for heterologous expression of SBP. This study also provides a useful strategy for heterologous expression of proteins which could be toxic to intracellular content of host cells.
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Peroxidases/genética , Proteínas de Soja/genética , Yarrowia/genética , Biocatálise , Clonagem Molecular , Estabilidade Enzimática , Expressão Gênica , Peroxidases/química , Peroxidases/metabolismo , Plasmídeos/genética , Plasmídeos/metabolismo , Proteínas de Soja/química , Proteínas de Soja/metabolismo , Yarrowia/metabolismoRESUMO
Formyl peptide receptors (FPRs, mouse Fprs) belong to the G protein-coupled receptor superfamily and mediate phagocyte migration in response to bacteria- and host-derived chemoattractants; however, knowledge about their in vivo roles in bacterial pathogenesis is limited. In this study, we investigated the role of Fpr1 and Fpr2 in host defense against Escherichia coli infection. In vitro, we found that supernatants from E. coli cultures induced chemotaxis of wild-type (WT) mouse bone marrow-derived neutrophils and that the activity was significantly reduced in cells genetically deficient in either Fpr1 or Fpr2 and was almost absent in cells lacking both receptors. Consistent with this, E. coli supernatants induced chemotaxis and MAPK phosphorylation in HEK293 cells expressing either recombinant Fpr1 or Fpr2 but not untransfected parental cells. WT bone marrow -derived neutrophils could actively phagocytose and kill E. coli, whereas both activities were diminished in cells lacking Fpr1 or Fpr2; again, an additive effect was observed in cells lacking both receptors. In vivo, Fpr1 and Fpr2 deficiency resulted in reduced recruitment of neutrophils in the liver and peritoneal cavity of mice infected with inactivated E. coli Moreover, Fpr1-/- and Fpr2-/- mice had significantly increased mortality compared with WT mice after i.p. challenge with a virulent E. coli clinical isolate. These results indicate a critical role of Fprs in host defense against E. coli infection.