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1.
Bioeng Transl Med ; 8(2): e10381, 2023 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-36925687

RESUMEN

Antibiotic resistance ranks among the top threats to humanity. Due to the frequent use of antibiotics, society is facing a high prevalence of multidrug resistant pathogens, which have managed to evolve mechanisms that help them evade the last line of therapeutics. An alternative to antibiotics could involve the use of bacteriophages (phages), which are the natural predators of bacterial cells. In earlier times, phages were implemented as therapeutic agents for a century but were mainly replaced with antibiotics, and considering the menace of antimicrobial resistance, it might again become of interest due to the increasing threat of antibiotic resistance among pathogens. The current understanding of phage biology and clustered regularly interspaced short palindromic repeats (CRISPR) assisted phage genome engineering techniques have facilitated to generate phage variants with unique therapeutic values. In this review, we briefly explain strategies to engineer bacteriophages. Next, we highlight the literature supporting CRISPR-Cas9-assisted phage engineering for effective and more specific targeting of bacterial pathogens. Lastly, we discuss techniques that either help to increase the fitness, specificity, or lytic ability of bacteriophages to control an infection.

2.
JCI Insight ; 7(11)2022 06 08.
Artículo en Inglés | MEDLINE | ID: mdl-35674135

RESUMEN

Follicular Th (Tfh) cells are key CD4+ Th cells involved in regulating B cell differentiation in the germinal center. Mice with conditional KO (CKO) of B lymphocyte-induced maturation protein-1 (BLIMP-1) expression in CD11chi DCs (Prdm1 CKO) spontaneously develop a lupus-like disease. We found an increase in the number of Tfh cells in secondary lymphoid organs in lupus-prone Prdm1-CKO mice. B cells stimulated with Tfh cells become Ab-secreting cells (ASCs); there was an increase in ASC differentiation mediated by Tfh cells from Prdm1-CKO mice compared with Tfh cells from control mice. This was mainly due to the increased expression of molecules within the IL-23/IL-17 pathway in Tfh cells from Prdm1-CKO mice. There was an increased frequency of IL-17A+ Tfh cells in Prdm1-CKO mice. These Tfh cells secrete IL-17A and an increased amount of IL-21. Furthermore, neutralizing IL-17A and IL-21 reduced ASC differentiation induced by Tfh cells. Lastly, we found the expression of IL-1ß and IL-6 was increased in BLIMP-1-deficient DCs, which are key for Th17 induction. Altogether, the lack of BLIMP-1 expression in DCs induces not only an increased number of Tfh cells, but also functionally distinct Tfh cells that lead to increased ASC differentiation.


Asunto(s)
Interleucina-17 , Activación de Linfocitos , Animales , Diferenciación Celular , Centro Germinal , Ratones , Células Th17
3.
Front Immunol ; 11: 1436, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32765503

RESUMEN

Proper expression of the transcription factor, Positive regulatory domain 1 (PRDM1), is required for maintaining homeostasis of human monocyte derived-dendritic cells (MO-DCs). The molecular mechanisms and gene targets of PRDM1 in B and T lymphocytes have been identified. However, the function of PRDM1 in dendritic cells (DCs) remains unclear. We investigate co-regulators of PRDM1 in MO-DCs identified by mass spectrometry (MS) and co-immunoprecipitation (Co-IP). Notably, non-POU domain-containing octamer-binding protein (NonO) was found to be a PRDM1 binding protein in the nucleus of MO-DCs. NonO is recruited to the PRDM1 binding site in the promoter region of IL-6. Knockdown of NonO expression by siRNA lessened suppression of IL-6 promoter activity by PRMD1 following LPS stimulation. While NonO binding to PRDM1 was observed in human myeloma cell lines, an effect of NonO on IL-6 expression was not observed. Thus, loss of NonO interrupted the inhibitory effect of PRDM1 on IL-6 expression in MO-DCs, but not plasma cells. Moreover, MO-DCs with low expression of PRDM1 or NonO induce an increased number of IL-21-producing TFH-like cells in vitro. These data suggest that low level of PRDM1 and NonO lead to enhanced activation of MO-DCs and the regulation of MO-DC function by PRDM1 is mediated through cell lineage-specific mechanisms.


Asunto(s)
Proteínas de Unión al ADN/metabolismo , Enfermedad de Dent/inmunología , Inflamación/metabolismo , Interleucina-6/genética , Monocitos/inmunología , Factor 1 de Unión al Dominio 1 de Regulación Positiva/metabolismo , Regiones Promotoras Genéticas/genética , Proteínas de Unión al ARN/metabolismo , Diferenciación Celular , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica , Células HEK293 , Homeostasis , Humanos , Inflamación/genética , Interleucina-6/metabolismo , Interleucinas/metabolismo , Activación de Linfocitos , Factor 1 de Unión al Dominio 1 de Regulación Positiva/genética , ARN Interferente Pequeño/genética , Proteínas de Unión al ARN/genética
4.
Nat Immunol ; 18(9): 1016-1024, 2017 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-28692065

RESUMEN

Aberrant population expansion of follicular helper T cells (TFH cells) occurs in patients with lupus. An unanswered question is whether an altered repertoire of T cell antigen receptors (TCRs) is associated with such expansion. Here we found that the transcription factor Blimp-1 (encoded by Prdm1) repressed expression of the gene encoding cathepsin S (Ctss), a cysteine protease that cleaves invariant chains and produces antigenic peptides for loading onto major histocompatibility complex (MHC) class II molecules. The increased CTSS expression in dendritic cells (DCs) from female mice with dendritic cell-specific conditional knockout of Prdm1 (CKO mice) altered the presentation of antigen to CD4+ T cells. Analysis of complementarity-determining region 3 (CDR3) regions containing the ß-chain variable region (Vß) demonstrated a more diverse repertoire of TFH cells from female CKO mice than of those from wild-type mice. In vivo treatment of CKO mice with a CTSS inhibitor abolished the lupus-related phenotype and reduced the diversity of the TFH cell TCR repertoire. Thus, Blimp-1 deficiency in DCs led to loss of appropriate regulation of Ctss expression in female mice and thereby modulated antigen presentation and the TFH cell repertoire to contribute to autoimmunity.


Asunto(s)
Catepsinas/metabolismo , Células Dendríticas/metabolismo , Lupus Eritematoso Sistémico/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Factores de Transcripción/genética , Animales , Anticuerpos Antinucleares/inmunología , Presentación de Antígeno/inmunología , Linfocitos T CD4-Positivos/inmunología , Diferenciación Celular , Proliferación Celular , ADN/inmunología , Células Dendríticas/inmunología , Ensayo de Inmunoadsorción Enzimática , Femenino , Riñón/patología , Lupus Eritematoso Sistémico/patología , Activación de Linfocitos , Ratones , Ratones Noqueados , Factor 1 de Unión al Dominio 1 de Regulación Positiva , Receptores de Antígenos de Linfocitos T alfa-beta/genética
5.
JCI Insight ; 2(1): e89569, 2017 01 12.
Artículo en Inglés | MEDLINE | ID: mdl-28097234

RESUMEN

A SNP identified as rs548234, which is found in PRDM1, the gene that encodes BLIMP1, is a risk allele associated with systemic lupus erythematosus (SLE). BLIMP1 expression was reported to be decreased in women with the PRDM1 rs548234 risk allele compared with women with the nonrisk allele in monocyte-derived DCs (MO-DCs). In this study, we demonstrate that BLIMP1 expression is regulated by the binding of Kruppel-like factor 4 (KLF4) to the risk SNP. KLF4 is highly expressed in MO-DCs but undetectable in B cells, consistent with the lack of altered expression of BLIMP1 in B cells from risk SNP carriers. Female rs548234 risk allele carriers, but not nonrisk allele carriers, exhibited decreased levels of BLIMP1 in MO-DCs, showing that the regulatory function of KLF4 is influenced by the risk allele. In addition, KLF4 directly recruits histone deacetylases (HDAC4, HDAC6, and HDAC7), established negative regulators of gene expression. Finally, the knock down of KLF4 expression reversed the inhibitory effects of the risk SNP on promoter activity and BLIMP1 expression. Therefore, the binding of KLF4 and the subsequent recruitment of HDACs represent a mechanism for reduced BLIMP1 expression in MO-DCs bearing the SLE risk allele rs548234.


Asunto(s)
Factores de Transcripción de Tipo Kruppel/metabolismo , Lupus Eritematoso Sistémico/genética , Factor 1 de Unión al Dominio 1 de Regulación Positiva/metabolismo , Adulto , Alelos , Animales , Linfocitos B , Femenino , Expresión Génica , Heterocigoto , Histona Desacetilasas/metabolismo , Humanos , Factor 4 Similar a Kruppel , Lupus Eritematoso Sistémico/metabolismo , Ratones , Ratones Noqueados , Persona de Mediana Edad , Polimorfismo de Nucleótido Simple/genética , Proteínas Represoras/metabolismo
6.
PLoS One ; 10(11): e0141523, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-26544187

RESUMEN

Mad2, a key component of the spindle checkpoint, is closely associated with chromosomal instability and poor prognosis in cancer. p31comet is a Mad2-interacting protein that serves as a spindle checkpoint silencer at mitosis. In this study, we showed that p31comet-induced apoptosis and senescence occur via counteraction of Mad2 activity. Upon retroviral transduction of p31comet, the majority of human cancer cell lines tested lost the ability to form colonies in a low-density seeding assay. Cancer cells with p31comet overexpression underwent distinct apoptosis and/or senescence, irrespective of p53 status, confirming the cytotoxicity of p31comet. Interestingly, both cytotoxic and Mad2 binding activities were eliminated upon deletion of the C-terminal 30 amino acids of p31comet. Point mutation or deletion of the region affecting Mad2 binding additionally abolished cytotoxic activity. Consistently, wild-type Mad2 interacting with p31comet, but not its non-binding mutant, inhibited cell death, indicating that the mechanism of p31comet-induced cell death involves Mad2 inactivation. Our results clearly suggest that the regions of p31comet affecting interactions with Mad2, including the C-terminus, are essential for induction of cell death. The finding that p31comet-induced cell death is mediated by interactions with Mad2 that lead to its inactivation is potentially applicable in anticancer therapy.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Apoptosis , Proteínas de Ciclo Celular/metabolismo , Proteínas Mad2/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Línea Celular Tumoral , Supervivencia Celular , Senescencia Celular , Células Clonales/citología , Humanos , Puntos de Control de la Fase M del Ciclo Celular , Ratones , Proteínas Nucleares/química , Proteínas Nucleares/genética , Unión Proteica , Eliminación de Secuencia
7.
PLoS One ; 10(11): e0143240, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-26606454

RESUMEN

The purpose of this research is to develop a method to screen a large number of potential driver mutations of acute myeloid leukemia (AML) using a retroviral cDNA library and murine bone marrow transduction-transplantation system. As a proof-of-concept, murine bone marrow (BM) cells were transduced with a retroviral cDNA library encoding well-characterized oncogenes and homeobox genes, and the virus-transduced cells were transplanted into lethally irradiated mice. The proto-oncogenes responsible for leukemia initiation were identified by PCR amplification of cDNA inserts from genomic DNA isolated from leukemic cells. In an initial screen of ten leukemic mice, the MYC proto-oncogene was detected in all the leukemic mice. Of ten leukemic mice, 3 (30%) had MYC as the only transgene, and seven mice (70%) had additional proto-oncogene inserts. We repeated the same experiment after removing MYC-related genes from the library to characterize additional leukemia-inducing gene combinations. Our second screen using the MYC-deleted proto-oncogene library confirmed MEIS1and the HOX family as cooperating oncogenes in leukemia pathogenesis. The model system we introduced in this study will be valuable in functionally screening novel combinations of genes for leukemogenic potential in vivo, and the system will help in the discovery of new targets for leukemia therapy.


Asunto(s)
Genes Homeobox , Leucemia/genética , Proto-Oncogenes , Animales , Células de la Médula Ósea/metabolismo , Células de la Médula Ósea/patología , Trasplante de Médula Ósea , Línea Celular , Transformación Celular Neoplásica/genética , Modelos Animales de Enfermedad , Epistasis Genética , Femenino , Eliminación de Gen , Expresión Génica , Biblioteca de Genes , Vectores Genéticos/genética , Inmunofenotipificación , Leucemia/mortalidad , Leucemia/patología , Ratones , Familia de Multigenes , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas c-myc/genética , Retroviridae/genética , Transducción Genética
8.
Mol Cells ; 34(2): 201-8, 2012 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-22843119

RESUMEN

The hematopoietic cell malignancy is one of the most prevalent type of cancer and the disease has multiple pathologic molecular signatures. Research on the origin of hematopoietic cancer stem cells and the mode of subsequent maintenance and differentiation needs robust animal models that can reproduce the transformation and differentiation event in vivo. Here, we show that co-transduction of MYC and PIM2 proto-oncogenes into mouse bone marrow cells readily establishes permanent cell lines that can induce lethal myeloid sarcoma in vivo. Unlike the previous doubly transgenic mouse model in which coexpression of MYC and PIM2 transgenes exclusively induced B cell lymphoma, we were able to show that the same combination of genes can also transform primary bone marrow myeloid cells in vitro resulting in permanent cell lines which induce myeloid sarcoma upon in vivo transplantation. By inducing cancerous transformation of fresh bone marrow cells in a controlled environment, the model we established will be useful for detailed study of the molecular events involved in initial transformation process of primary myeloid bone marrow cells and provides a model that can give insight to the molecular pathologic characteristics of human myeloid sarcoma, a rare presentation of solid tumors of undifferentiated myeloid blast cells associated with various types of myeloid leukemia.


Asunto(s)
Células de la Médula Ósea/metabolismo , Proteínas Proto-Oncogénicas c-myc/biosíntesis , Sarcoma Mieloide/metabolismo , Animales , Células de la Médula Ósea/citología , Células de la Médula Ósea/patología , Diferenciación Celular/fisiología , Procesos de Crecimiento Celular/fisiología , Línea Celular , Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/patología , Modelos Animales de Enfermedad , Humanos , Ratones , Ratones Endogámicos C57BL , Proteínas Serina-Treonina Quinasas/biosíntesis , Proteínas Proto-Oncogénicas/biosíntesis , Sarcoma Mieloide/patología
9.
Mol Endocrinol ; 24(7): 1441-52, 2010 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-20444885

RESUMEN

In obesity, dysregulation of adipocytokines is involved in several pathological conditions including diabetes and certain cancers. As a member of the adipocytokines, adiponectin plays crucial roles in whole-body energy homeostasis. Recently, it has been reported that the level of plasma adiponectin is reduced in several types of cancer patients. However, it is largely unknown whether and how adiponectin affects colon cancer cell growth. Here, we show that adiponectin suppresses the proliferation of colon cancer cells including HCT116, HT29, and LoVo. In colon cancer cells, adiponectin attenuated cell cycle progression at the G(1)/S boundary and concurrently increased expression of cyclin-dependent kinase inhibitors such as p21 and p27. Adiponectin stimulated AMP-activated protein kinase (AMPK) phosphorylation whereas inhibition of AMPK activity blunted the effect of adiponectin on the proliferation of colon cancer cells. Furthermore, knockdown of adiponectin receptors such as AdipoR1 and AdipoR2 relieved the suppressive effect of adiponectin on the growth of colon cancer cells. In addition, adiponectin repressed the expression of sterol regulatory element binding protein-1c, which is a key lipogenic transcription factor associated with colon cancers. These results suggest that adiponectin could inhibit the growth of colon cancer cells through stimulating AMPK activity.


Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Adiponectina/farmacología , Proliferación Celular/efectos de los fármacos , Receptores de Adiponectina/metabolismo , Adiponectina/uso terapéutico , Animales , Apoptosis/efectos de los fármacos , Western Blotting , Células CHO , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/metabolismo , Cricetinae , Cricetulus , Células HCT116 , Células HT29 , Humanos , Reacción en Cadena de la Polimerasa , Receptores de Adiponectina/genética
10.
Mol Cancer Res ; 7(3): 371-82, 2009 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-19276188

RESUMEN

Functional suppression of spindle checkpoint protein activity results in apoptotic cell death arising from mitotic failure, including defective spindle formation, chromosome missegregation, and premature mitotic exit. The recently identified p31(comet) protein acts as a spindle checkpoint silencer via communication with the transient Mad2 complex. In the present study, we found that p31(comet) overexpression led to two distinct phenotypic changes, cellular apoptosis and senescence. Because of a paucity of direct molecular link of spindle checkpoint to cellular senescence, however, the present report focuses on the relationship between abnormal spindle checkpoint formation and p31(comet)-induced senescence by using susceptible tumor cell lines. p31(comet)-induced senescence was accompanied by mitotic catastrophe with massive nuclear and chromosomal abnormalities. The progression of the senescence was completely inhibited by the depletion of p21(Waf1/Cip1) and partly inhibited by the depletion of the tumor suppressor protein p53. Notably, p21(Waf1/Cip1) depletion caused a dramatic phenotypic conversion of p31(comet)-induced senescence into cell death through mitotic catastrophe, indicating that p21(Waf1/Cip1) is a major mediator of p31(comet)-induced cellular senescence. In contrast to wild-type p31(comet), overexpression of a p31 mutant lacking the Mad2 binding region did not cause senescence. Moreover, depletion of Mad2 by small interfering RNA induced senescence. Here, we show that p31(comet) induces tumor cell senescence by mediating p21(Waf1/Cip1) accumulation and Mad2 disruption and that these effects are dependent on a direct interaction of p31(comet) with Mad2. Our results could be used to control tumor growth.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/biosíntesis , Proteínas de Unión al Calcio/metabolismo , Proteínas de Ciclo Celular/biosíntesis , Proteínas de Ciclo Celular/metabolismo , Senescencia Celular/fisiología , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Proteínas Nucleares/biosíntesis , Proteínas Represoras/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Apoptosis/efectos de los fármacos , Apoptosis/genética , Proteínas de Unión al Calcio/antagonistas & inhibidores , Proteínas de Unión al Calcio/genética , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/genética , Línea Celular Tumoral , Proliferación Celular , Células Clonales , ADN/metabolismo , Humanos , Proteínas Mad2 , Mitosis/fisiología , Proteínas Nucleares/genética , Proteínas Represoras/antagonistas & inhibidores , Proteínas Represoras/genética , Transducción de Señal , Huso Acromático/genética , Huso Acromático/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , beta-Galactosidasa/biosíntesis
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