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1.
J Clin Lab Anal ; 37(21-22): e24978, 2023 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-37964630

RESUMO

BACKGROUND: At different stages of the disease, biomarkers can help to determine disease progression and recurrence and provide a personalized indicator of therapeutic effectiveness. The serological identification of antigens by recombinant cDNA expression cloning (SEREX) has identified five SEREX antigens. RESULTS: Compared with healthy donors, anti-FIRΔexon2 and anti-SOHLH antibodies (Abs) in the sera of patients with colorectal cancer (CRC) were markedly higher. Furthermore, no correlation was noted between five SEREX antigens and the three tumor markers (CEA, CA19-9, and anti-p53 Abs), indicating that anti-FIRΔexon2 Abs are an independent candidate marker for patients with CRC. Generally, the levels of anti-FIRΔexon2 Abs combined with clinically available tumor markers were determined to be significantly higher compared with CEA, CA19-9. Moreover, in early-stage CRC, the levels of anti-FIRΔexon2 Abs combined with existing tumor markers were higher than those of CEA, CA19-9. CONCLUSION: Due to the highly heterogeneous nature of CRC, a single tumor marker is unlikely to become a standalone diagnostic test due to its commonly insufficient sensitivity and/or specificity. Using a combination antibody detection approach of tumor markers for CRC diagnosis has the potential to be an effective approach. Therefore, the use of serum protein biomarker candidates holds promise for the development of inexpensive, noninvasive, and inexpensive tests for the detection of CRC.


Assuntos
Anti-Infecciosos , Neoplasias Colorretais , Humanos , Antígeno CA-19-9 , Detecção Precoce de Câncer , Neoplasias Colorretais/genética , Biomarcadores Tumorais , Anticorpos , Antígeno Carcinoembrionário
2.
Curr Res Microb Sci ; 3: 100130, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35909620

RESUMO

Horizontal gene transfer (HGT) is a bacterial evolution tool for improved survival. Although several environmental stimuli induce or promote HGT, the diversity and complexity of the environmental factors have not been sufficiently elucidated. In this study, we showed that the biofilm culture of Escherichia coli at the air-solid interface in the presence of a subminimal inhibitory concentration (sub-MIC) of ampicillin (∼0.5-4 µg/mL) and subsequent mechanical stimulation (rolling small glass balls, ø = 5-8 mm) cooperatively promoted horizontal plasmid transfer without the usual competence-inducing conditions. Either of the two treatments promoted plasmid transfer at a lower frequency than when the treatments were combined. The effect of several parameters on the two treatments was tested and then optimized, achieving a high frequency of plasmid transfer (up to 10-6 per cell) under optimal conditions. Plasmid transfer was DNase-sensitive for both treatments, demonstrating its mechanism of transformation. Plasmid transfer occurred using various E. coli strains, plasmids, ball materials, shaking conditions, and even the mechanical stimulation of brushing the biofilm with a toothbrush, indicating the conditional flexibility of this phenomenon. This is the first demonstration of the promoting effect of the combination of a sub-MIC antibiotic and mechanical stimulation on horizontal plasmid transfer between E. coli cells via transformation. Regarding environmental bacterial physiology, the aggregations or biofilms of bacterial cells may encounter both sub-MIC antibiotics and mechanical stimuli in some specific environments, therefore, this type of HGT could also occur naturally.

3.
J Neurosci ; 42(23): 4607-4618, 2022 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-35504726

RESUMO

Ubiquitin-specific protease 2 (USP2) participates in glucose metabolism in peripheral tissues such as the liver and skeletal muscles. However, the glucoregulatory role of USP2 in the CNS is not well known. In this study, we focus on USP2 in the ventromedial hypothalamus (VMH), which has dominant control over systemic glucose homeostasis. ISH, using a Usp2-specific probe, showed that Usp2 mRNA is present in VMH neurons, as well as other glucoregulatory nuclei, in the hypothalamus of male mice. Administration of a USP2-selective inhibitor ML364 (20 ng/head), into the VMH elicited a rapid increase in the circulating glucose level in male mice, suggesting USP2 has a suppressive role on glucose mobilization. ML364 treatment also increased serum norepinephrine concentration, whereas it negligibly affected serum levels of insulin and corticosterone. ML364 perturbated mitochondrial oxidative phosphorylation in neural SH-SY5Y cells and subsequently promoted the phosphorylation of AMP-activated protein kinase (AMPK). Consistent with these findings, hypothalamic ML364 treatment stimulated AMPKα phosphorylation in the VMH. Inhibition of hypothalamic AMPK prevented ML364 from increasing serum norepinephrine and blood glucose. Removal of ROS restored the ML364-evoked mitochondrial dysfunction in SH-SY5Y cells and impeded the ML364-induced hypothalamic AMPKα phosphorylation as well as prevented the elevation of serum norepinephrine and blood glucose levels in male mice. These results indicate hypothalamic USP2 attenuates perturbations in blood glucose levels by modifying the ROS-AMPK-sympathetic nerve axis.SIGNIFICANCE STATEMENT Under normal conditions (excluding hyperglycemia or hypoglycemia), blood glucose levels are maintained at a constant level. In this study, we used a mouse model to identify a hypothalamic protease controlling blood glucose levels. Pharmacological inhibition of USP2 in the VMH caused a deviation in blood glucose levels under a nonstressed condition, indicating that USP2 determines the set point of the blood glucose level. Modification of sympathetic nervous activity accounts for the USP2-mediated glucoregulation. Mechanistically, USP2 mitigates the accumulation of ROS in the VMH, resulting in attenuation of the phosphorylation of AMPK. Based on these findings, we uncovered a novel glucoregulatory axis consisting of hypothalamic USP2, ROS, AMPK, and the sympathetic nervous system.


Assuntos
Glicemia , Neuroblastoma , Sistema Nervoso Simpático , Ubiquitina Tiolesterase , Núcleo Hipotalâmico Ventromedial , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Glicemia/metabolismo , Glucose/metabolismo , Humanos , Masculino , Camundongos , Norepinefrina/metabolismo , Fosforilação Oxidativa , Espécies Reativas de Oxigênio/metabolismo , Sistema Nervoso Simpático/enzimologia , Sistema Nervoso Simpático/metabolismo , Ubiquitina Tiolesterase/metabolismo , Núcleo Hipotalâmico Ventromedial/enzimologia , Núcleo Hipotalâmico Ventromedial/metabolismo
4.
Cancer Immunol Immunother ; 71(12): 3099-3106, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-35624180

RESUMO

Near-infrared photoimmunotherapy (NIR-PIT) selectively kills tumor cells to which the photo-absorber dye IR700DX-conjugated antibodies are bound and induces a systemic anti-tumor immune response. NIR-PIT induces immunogenic cell death (ICD), releases damage-associated molecular patterns (DAMPs) molecules from dying tumor cells, and activates dendritic cells (DCs). However, it is unclear whether NIR-PIT affects migration of tumor-infiltrating (Ti)-DCs to draining lymph nodes (dLNs), where a systemic anti-tumor response is induced. Here, we utilized in vivo photolabeling of Ti-DCs in tumors in photoconvertible protein Kikume Green-Red (KikGR) mice to show that NIR-PIT enhanced migration of Ti-DCs including cDC1s, cDC2s, and CD326+ DCs to dLNs. This effect was abolished by blocking adenosine triphosphate (ATP), one of the DAMPs molecules, as well as by inhibition of Gαi signaling by pertussis toxin. Thus, ICD induction by NIR-PIT stimulates Ti-DC migration to dLNs via ATP-P2X7 receptor and Gαi protein-coupled receptor signaling pathways and may augment tumor antigen presentation to induce anti-tumor T cells in dLNs.


Assuntos
Imunoterapia , Receptores Purinérgicos P2X7 , Camundongos , Animais , Toxina Pertussis , Linhagem Celular Tumoral , Camundongos Nus , Morte Celular Imunogênica , Células Dendríticas , Trifosfato de Adenosina , Ensaios Antitumorais Modelo de Xenoenxerto
5.
Int J Mol Sci ; 22(3)2021 Jan 26.
Artigo em Inglês | MEDLINE | ID: mdl-33530560

RESUMO

Ubiquitin specific protease (USP) 2 is a multifunctional deubiquitinating enzyme. USP2 modulates cell cycle progression, and therefore carcinogenesis, via the deubiquitination of cyclins and Aurora-A. Other tumorigenic molecules, including epidermal growth factor and fatty acid synthase, are also targets for USP2. USP2 additionally prevents p53 signaling. On the other hand, USP2 functions as a key component of the CLOCK/BMAL1 complex and participates in rhythmic gene expression in the suprachiasmatic nucleus and liver. USP2 variants influence energy metabolism by controlling hepatic gluconeogenesis, hepatic cholesterol uptake, adipose tissue inflammation, and subsequent systemic insulin sensitivity. USP2 also has the potential to promote surface expression of ion channels in renal and intestinal epithelial cells. In addition to modifying the production of cytokines in immune cells, USP2 also modulates the signaling molecules that are involved in cytokine signaling in the target cells. Usp2 knockout mice exhibit changes in locomotion and male fertility, which suggest roles for USP2 in the central nervous system and male genital tract, respectively. In this review, we summarize the cellular events with USP2 contributions and list the signaling molecules that are upstream or downstream of USP2. Additionally, we describe phenotypic differences found in the in vitro and in vivo experimental models.


Assuntos
Suscetibilidade a Doenças , Transdução de Sinais , Ubiquitina Tiolesterase/metabolismo , Animais , Apoptose/genética , Autofagia/genética , Biomarcadores , Ciclo Celular/genética , Relógios Circadianos/genética , Metabolismo Energético , Regulação da Expressão Gênica , Humanos , Especificidade de Órgãos/genética , Ubiquitina Tiolesterase/genética
6.
Cell Mol Life Sci ; 78(6): 2929-2948, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33104844

RESUMO

Macrophages are innate immune cells that contribute to classical immune functions and tissue homeostasis. Ubiquitin-specific protease 2 (USP2) controls cytokine production in macrophages, but its organ-specific roles are still unknown. In this study, we generated myeloid-selective Usp2 knockout (msUsp2KO) mice and specifically explored the roles of testicular macrophage-derived USP2 in reproduction. The msUsp2KO mice exhibited normal macrophage characteristics in various tissues. In the testis, macrophage Usp2 deficiency negligibly affected testicular macrophage subpopulations, spermatogenesis, and testicular organogenesis. However, frozen-thawed sperm derived from msUsp2KO mice exhibited reduced motility, capacitation, and hyperactivation. In addition, macrophage Usp2 ablation led to a decrease in the sperm population exhibiting high intracellular pH, calcium influx, and mitochondrial membrane potential. Interrupted pronuclei formation in eggs was observed when using frozen-thawed sperm from msUsp2KO mice for in vitro fertilization. Administration of granulocyte macrophage-colony stimulating factor (GM-CSF), whose expression was decreased in testicular macrophages derived from msUsp2KO mice, restored mitochondrial membrane potential and total sperm motility. Our observations demonstrate a distinct role of the deubiquitinating enzyme in organ-specific macrophages that directly affect sperm function.


Assuntos
Macrófagos/metabolismo , Capacitação Espermática/fisiologia , Motilidade dos Espermatozoides/fisiologia , Espermatozoides/metabolismo , Ubiquitina Tiolesterase/metabolismo , Animais , Cálcio/metabolismo , Regulação para Baixo/efeitos dos fármacos , Fertilização in vitro , Congelamento , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Concentração de Íons de Hidrogênio , Macrófagos/citologia , Macrófagos/imunologia , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Motilidade dos Espermatozoides/efeitos dos fármacos , Testículo/anatomia & histologia , Testículo/fisiologia , Testosterona/metabolismo , Tretinoína/metabolismo , Ubiquitina Tiolesterase/deficiência , Ubiquitina Tiolesterase/genética
7.
Physiol Rep ; 7(14): e14193, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31353872

RESUMO

Ubiquitin-specific protease 2 (USP2) is considered to participate in the differentiation of myoblasts to myotubes, however, its functions in myoblasts under growth conditions remain elusive. In this study, we analyzed the physiological roles of USP2 in myoblasts using Usp2 knockout (KO) C2C12 cells as well as a USP2 specific inhibitor. In addition to the disruption of differentiation, clustered regularly interspaced short palindromic repeats/Cas9-generated Usp2KO cells exhibited inhibition of proliferation compared to parental C2C12 cells. Usp2KO cells reduced the accumulation of intracellular adenosine triphosphate (ATP) content and oxygen consumption. Moreover, Usp2KO cells had fragmented mitochondria, suggesting that mitochondrial respiration was inactive. The deficiency of Usp2 did not affect the enzymatic activities of respiratory chain complexes I, III, IV, and V. However, mitochondrial membrane permeability-evaluated using calcein AM-cobalt staining-was increased in Usp2KO cells. The membrane potential of Usp2KO cells was clearly decreased. Usp2KO cells accumulated reactive oxygen species (ROS) in the mitochondria. The USP2-selective inhibitor ML364 also increased the levels of mitochondrial ROS, and modulated the membrane potential and morphology of the mitochondria. These effects were followed by a decrement in the intracellular content of ATP. Based on these findings, we speculate that USP2 may be involved in maintaining the integrity of the mitochondrial membrane. This process ensures the supply of ATP in myoblasts, presumably leading to proliferation and differentiation.


Assuntos
Trifosfato de Adenosina/metabolismo , Mitocôndrias Musculares/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Ubiquitina Tiolesterase/metabolismo , Animais , Linhagem Celular , Inibidores Enzimáticos/farmacologia , Potencial da Membrana Mitocondrial , Camundongos , Mitocôndrias Musculares/efeitos dos fármacos , Mioblastos/efeitos dos fármacos , Mioblastos/metabolismo , Fosforilação Oxidativa , Ubiquitina Tiolesterase/antagonistas & inibidores , Ubiquitina Tiolesterase/genética
8.
Biochem Biophys Res Commun ; 515(1): 196-200, 2019 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-31138439

RESUMO

Bacteria continuously change their genetic characteristics to adapt to the changing environment by means of horizontal gene transfer. Although three conventional mechanisms of horizontal gene transfer are well known (transformation, transduction, and conjugation), new variations of these mechanisms have also been described. We previously reported that DNase-sensitive cell-to-cell transfer of non-conjugative plasmids, termed as "cell-to-cell transformation," occurs between the cells of two Escherichia coli strains in a co-culture. In this study, to further investigate the mechanism of cell-to-cell transformation, we constructed a new experimental system for cell-to-cell transformation. By using this system, we found that high temperatures of approximately 41ºC-45 °C significantly promote cell-to-cell plasmid transformation. This transfer was much more frequent in solid-air biofilms than in liquid culture, suggesting an importance of biofilm environment. Plasmid transfer frequency reached over 10-7/cell under the optimal strain-plasmid combination and conditions tested. DNase sensitivity test and plasmid isolation from the transformants confirmed the horizontal transfer of full-length plasmids via transformation. Comparative natural transformation experiments, which used similar strains and plasmids under equivalent culture conditions, revealed that cell-to-cell transformation occurs approximately 103 times more frequently than natural transformation, indicating the uniqueness and effectiveness of the cell-to-cell transformation mechanism. As temperatures of approximately 41ºC-45 °C are common in the avian intestines and under some other environmental situations, the phenomenon demonstrated here can occur efficiently in such locations. To the best of our knowledge, this is the first study to demonstrate the enhancing effect of high temperatures on cell-to-cell plasmid transformation in E. coli.


Assuntos
Escherichia coli/genética , Transferência Genética Horizontal , Temperatura Alta , Plasmídeos/genética , Transformação Bacteriana/genética , Técnicas Bacteriológicas/métodos , Biofilmes , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Desoxirribonucleases/metabolismo , Escherichia coli/classificação , Escherichia coli/fisiologia
9.
Chem Asian J ; 14(3): 471-479, 2019 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-30600912

RESUMO

In this study, mechanochromic luminescence was induced in a complex of mechano-inactive compounds. Dye/acid complexes containing the same π-conjugated backbones were prepared. While the luminophore showed blue and red shifts in photoluminescence spectra when combined with different acids by grinding, it exhibited slight mechanoresponsiveness itself. Also, compounds with similar molecular backbones to the dye/acid complex were synthesized to clarify the color change mechanism. The compounds showed both blue and red shifts in photoluminescence and diffuse reflectance spectra upon grinding, indicating that mechanochromic luminescence in the hydrogen-bonded complex is like its monomeric analogue and that aggregation structure plays an important role in mechanoresponsive behavior rather than the π-conjugated structure. It was shown that a color change can be mechanically induced by imitating the solid-state aggregation structure of other mechanoresponsive compounds without synthetic modification.

10.
Biomed Res ; 39(2): 95-104, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29669988

RESUMO

Recently, gene-editing using the clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR-associated protein 9 (Cas9) technique has attempted to utilize fibroblasts of livestock animals for somatic cell nuclear transfer. In this study, we establish the procedure for preparing skin fibroblast clones whose genes were edited by the CRISPR/Cas9 technique. After isolating fibroblasts from earlobes of Japanese Black cattle, subsequent collagenase-digestion and extensive wash procedures enabled us to avoid contamination of fungi. Electroporation using NEPA21, rather than lipofection using commercially available liposome reagents, allowed us to perform more efficient transfection of plasmid constructs. Although bovine ear-derived fibroblasts were not able to proliferate in single cell cultures in Dulbecco's modified Eagle medium containing 10% fetal calf serum, supplementation with insulin-transferrin-selenium mixture, human recombinant epidermal growth factor, or human recombinant basic fibroblast growth factor promoted proliferation of the cells, even in a single cell culture. Taking advantage of our established protocol, we eventually obtained eight ear-derived fibroblast clones with a recessive mutation in the isoleucyl-tRNA synthetase gene corrected by the CRISPR/Cas9 technique.


Assuntos
Fibroblastos/metabolismo , Edição de Genes , Técnicas de Transferência Nuclear , Animais , Sistemas CRISPR-Cas , Bovinos , Células Clonais , Edição de Genes/métodos , Loci Gênicos , Genótipo , Células HeLa , Humanos , Mutação , Proteínas Tirosina Fosfatases Classe 2 Semelhantes a Receptores/genética , Reprodutibilidade dos Testes , Transfecção
11.
Chem Asian J ; 9(11): 3188-95, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25197034

RESUMO

Mechanoresponsive luminophores containing different substituted pyridine rings at the molecular terminus are synthesized and their photoluminescence properties are investigated. The solid chromophore with a 4-substituted pyridine ring exhibits a reversible photoluminescent color change, while the 2-substituted chromophore shows only a small change in luminescence, and the 3-substituted chromophore displays an irreversible photoluminescent color change with mechanical grinding. A change of the sample color in response to mechanical grinding is also achieved for a dye-dispersed poly(vinyl alcohol) film. Furthermore, a simultaneous acid and mechanoresponsive photoemission color change is achieved in the dye-dispersed film.

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