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1.
EJNMMI Phys ; 7(1): 56, 2020 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-32915344

RESUMO

BACKGROUND: The Bayesian penalized likelihood (BPL) algorithm Q.Clear (GE Healthcare) allows fully convergent iterative reconstruction that results in better image quality and quantitative accuracy, while limiting image noise. The present study aimed to optimize BPL reconstruction parameters for 18F-NaF PET/CT images and to determine the feasibility of 18F-NaF PET/CT image acquisition over shorter durations in clinical practice. METHODS: A custom-designed thoracic spine phantom consisting of several inserts, soft tissue, normal spine, and metastatic bone tumor, was scanned using a Discovery MI PET/CT scanner (GE Healthcare). The phantom allows optional adjustment of activity distribution, tumor size, and attenuation. We reconstructed PET images using OSEM + PSF + TOF (2 iterations, 17 subsets, and a 4-mm Gaussian filter), BPL + TOF (ß = 200 to 700), and scan durations of 30-120 s. Signal-to-noise ratios (SNR), contrast, and coefficients of variance (CV) as image quality indicators were calculated, whereas the quantitative measures were recovery coefficients (RC) and RC linearity over a range of activity. We retrospectively analyzed images from five persons without bone metastases (male, n = 1; female, n = 4), then standardized uptake values (SUV), CV, and SNR at the 4th, 5th, and 6th thoracic vertebra were calculated in BPL + TOF (ß = 400) images. RESULTS: The optimal reconstruction parameter of the BPL was ß = 400 when images were acquired at 120 s/bed. At 90 s/bed, the BPL with a ß value of 400 yielded 24% and 18% higher SNR and contrast, respectively, than OSEM (2 iterations; 120 s acquisitions). The BPL was superior to OSEM in terms of RC and the RC linearity over a range of activity, regardless of scan duration. The SUVmax were lower in BPL, than in OSEM. The CV and vertebral SNR in BPL were superior to those in OSEM. CONCLUSIONS: The optimal reconstruction parameters of 18F-NaF PET/CT images acquired over different durations were determined. The BPL can reduce PET acquisition to 90 s/bed in 18F-NaF PET/CT imaging. Our results suggest that BPL (ß = 400) on SiPM-based TOF PET/CT scanner maintained high image quality and quantitative accuracy even for shorter acquisition durations.

2.
Ann Nucl Med ; 2020 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-32623569

RESUMO

OBJECTIVE: Many advances in PET/CT technology can potentially improve image quality and the ability to detect small lesions. A new digital TOF-PET/CT scanner based on silicon photomultipliers (SiPM) integrated with a Bayesian penalized likelihood (BPL) PET reconstruction algorithm (Q.Clear; GE Healthcare) has been introduced into clinical practice. The present study aimed to quantify the ability of a digital TOF-PET/CT scanner combined with BPL reconstruction to detect small lesions, and to determine the optimal penalization factor (ß) in BPL to accurately detect such lesions. METHODS: All PET data were acquired from a NEMA body phantom using a Discovery MI (DMI) PET/CT system (GE Healthcare). The phantom included six spheres with diameters of 4, 5, 6, 8, 10, and 13 mm, and contained a background activity level of 5.3 kBq/mL, with target-to-background ratios (TBR) of 4:1 and 8:1. Images were reconstructed using a baseline OSEM algorithm, with OSEM + PSF, OSEM + TOF, OSEM + PSF + TOF, and BPL + PSF + TOF (ß: 50-400). The matrix size was 192 × 192 and 384 × 384. Data acquired in 100-min list mode were re-binned into acquisition times ranging from 2 to 100 min. The quantitative accuracy and detectability of small hot spheres were evaluated by physical assessment of a recovery coefficient (RC) and a detectability index (DI), as well as visual assessment of PET images at each acquisition time. RESULTS: The RC and DI of sub-centimeter spheres were improved, because the digital TOF-PET/CT scanner has a larger TOF performance gain due to better timing resolution. The RC and DI were higher with BPL in sub-centimeter spheres, than with other OSEM-based types of reconstruction. The BPL for an 8-mm sphere overestimated uptake due to edge artifact overshoot induced by PSF modeling. The variability of RC and DI for acquisition times and TBR differed considerably according to ß values. The RC for ~ 8-mm spheres were > 1 at ß values between 50 and 100, but were close to 1 at ß value of 200. The visual scores for ß = 200 in BPL were maximal, whereas those for spheres that were ≥ 6 mm exceeded the criterion of 3. CONCLUSION: The BPL in the digital TOF-PET/CT scanner improved the quantitation and detectability of sub-centimeter spheres compared with OSEM-based reconstruction. Optimization of the ß value in BPL might allow the detection of lesions ≤ 6 mm, although detectability depended on the TBR of lesions. A ß value of 200 seemed optimal for detecting sub-centimeter lesions.

3.
PLoS One ; 15(2): e0229744, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32101568

RESUMO

Mast cells have secretory granules containing chemical mediators such as histamine and play important roles in the immune system. Polyamines are essential factors for cellular processes such as gene expression and translation. It has been reported that secretory granules contain both histamine and polyamines, which have similar chemical structures and are produced from the metabolism of cationic amino acids. We investigated the effect of polyamine depletion on mast cells using bone marrow-derived mast cells (BMMCs). Polyamine depletion was induced using α-difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase. DFMO treatment resulted in a significant reduction of cell number and abnormal secretory granules in BMMCs. Moreover, the cells showed a 2.3-fold increase in intracellular histamine and up-regulation of histidine decarboxylase (HDC) at the transcriptional level during BMMC differentiation. Levels of the transcription factor kruppel-like factor 4 (KLF4) greatly decreased upon DFMO treatment; however, Klf4 mRNA was expressed at levels similar to controls. We determined the translational regulation of KLF4 using reporter genes encoding Klf4-luc2 fusion mRNA, for transfecting NIH3T3 cells, and performed in vitro translation. We found that the efficiency of KLF4 synthesis in response to DFMO treatment was enhanced by the existence of a GC-rich 5'-untranslated region (5'-UTR) on Klf4 mRNA, regardless of the recognition of the initiation codon. Taken together, these results indicate that the enhancement of histamine synthesis by DFMO depends on the up-regulation of Hdc expression, achieved by removal of transcriptional suppression of KLF4, during differentiation.


Assuntos
Histamina/biossíntese , Fatores de Transcrição Kruppel-Like/metabolismo , Mastócitos/metabolismo , Animais , Medula Óssea/metabolismo , Células da Medula Óssea/metabolismo , Diferenciação Celular/fisiologia , Eflornitina/farmacologia , Feminino , Histamina/metabolismo , Histidina Descarboxilase/genética , Camundongos , Camundongos Endogâmicos C57BL , Células NIH 3T3 , Ornitina Descarboxilase/metabolismo , Poliaminas/metabolismo , Vesículas Secretórias/metabolismo , Espermidina/metabolismo
4.
Sci Rep ; 9(1): 18699, 2019 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-31822748

RESUMO

Most cancer cells rely on glycolysis to generate ATP, even when oxygen is available. However, merely inhibiting the glycolysis is insufficient for the eradication of cancer cells. One main reason for this is that cancer cells have the potential to adapt their metabolism to their environmental conditions. In this study, we investigated how cancer cells modify their intracellular metabolism when glycolysis is suppressed, using PANC-1 pancreatic cancer cells and two other solid tumor cell lines, A549 and HeLa. Our study revealed that glycolytically suppressed cells upregulated mitochondrial function and relied on oxidative phosphorylation (OXPHOS) to obtain the ATP necessary for their survival. Dynamic changes in intracellular metabolic profiles were also observed, reflected by the reduced levels of TCA cycle intermediates and elevated levels of most amino acids. Glutamine and glutamate were important for this metabolic reprogramming, as these were largely consumed by influx into the TCA cycle when the glycolytic pathway was suppressed. During the reprogramming process, activated autophagy was involved in modulating mitochondrial function. We conclude that upon glycolytic suppression in multiple types of tumor cells, intracellular energy metabolism is reprogrammed toward mitochondrial OXPHOS in an autophagy-dependent manner to ensure cellular survival.

5.
J Infect Chemother ; 25(10): 750-757, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31235348

RESUMO

The prevalence of nonencapsulated Streptococcus pneumoniae (NESp) has increased with the introduction of pneumococcal conjugate vaccines in children; however, the bacteriological characteristics of NESp have not been sufficiently clarified. In this study, NESp strains isolated from the nasopharyngeal carriage of children from four nursery schools in Japan were analyzed for molecular type, antibiotic susceptibility, and biofilm productivity. A total of 152 putative S. pneumoniae strains were identified by optochin-susceptibility analysis, of which 21 were not serotypeable by slide agglutination, quellung reaction, or multiplex PCR. Among these 21 strains, three were lytA-negative and, therefore, not S. pneumoniae. The remaining 18 strains were positive for lytA, ply, pspK, and bile solubility and were confirmed as NESp. Therefore, the isolation rate of NESp in the S. pneumoniae strains in this study was 12.0% (18/149). Molecular-typing analyses classified five strains as two existing sequence types (STs; ST7502 and ST7786), and 13 strains formed four novel STs. Horizontal spread was suspected, because strains with the same ST were often isolated from the same nursery school. The NESp isolates were generally susceptible to most antimicrobials, with the exception of macrolides; however, all isolates possessed more than one abnormal penicillin-binding protein gene. Furthermore, NESp strains were more effective than encapsulated counterparts at forming biofilms, which showed obvious differences in morphology. These data indicated that NESp strains should be continuously monitored as emerging respiratory pathogens.


Assuntos
Antibacterianos/farmacologia , Biofilmes/efeitos dos fármacos , Infecções Pneumocócicas/terapia , Vacinas Pneumocócicas/uso terapêutico , Streptococcus pneumoniae/isolamento & purificação , Antibacterianos/uso terapêutico , Farmacorresistência Bacteriana/genética , Humanos , Lactente , Recém-Nascido , Japão/epidemiologia , Testes de Sensibilidade Microbiana , Tipagem Molecular/métodos , Mutação , Mucosa Nasal/microbiologia , Proteínas de Ligação às Penicilinas/genética , Infecções Pneumocócicas/epidemiologia , Infecções Pneumocócicas/microbiologia , Prevalência , Sorotipagem , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/imunologia , Fatores de Virulência/genética
6.
Med Mycol J ; 60(2): 29-37, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31155569

RESUMO

We previously reported that Candida albicans responded to mild heat stress in a range of temperature elevations simulating fever, and concluded that mild heat stress increases susceptibility to antifungal drugs. In this study, we show that mild heat stress causes a morphological change in hyphae during the process of biofilm formation. We found that mild heat stress extended the period of hyphal stage maintenance in C. albicans biofilm. Although the rate of hyphal change from yeast form to hyphal form reached the maximum within 3 hr, later, almost every cell quickly reverted to the yeast growth phase within 6 hr at 37°C but not at 39°C, or under mild heat stress. Electron microscopy using a smart specimen preparation technique revealed that mild heat stress significantly increased the thickness of the inner cell wall accompanied by a decrease in density of the outer cell wall in the hyphae of C. albicans biofilm. To identify the gene responsible for the morphological changes associated with mild heat stress, we performed microarray gene expression analysis. Eleven genes were upregulated and 17 genes were downregulated under mild heat stress in biofilm cells. The increased PHR1 gene expression in response to mild heat stress was confirmed in quantitative RT-PCR analysis. The mutant upregulated PHR1 expression showed the same sensitivity against antifungal drug micafungin as dependent on mild heat stress. Our findings point to possible therapeutic effects of hyperthermia as well as to the effect of fever during infections.


Assuntos
Biofilmes , Candida albicans/citologia , Candida albicans/fisiologia , Parede Celular/patologia , Febre/microbiologia , Temperatura Alta , Estresse Fisiológico/genética , Estresse Fisiológico/fisiologia , Antifúngicos/farmacologia , Candida albicans/genética , Candida albicans/ultraestrutura , Candidíase/terapia , Parede Celular/ultraestrutura , Regulação para Baixo/genética , Farmacorresistência Fúngica/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Expressão Gênica/genética , Regulação Fúngica da Expressão Gênica/genética , Hifas , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Micafungina/farmacologia , Microscopia Eletrônica , Fatores de Tempo
7.
Scand J Gastroenterol ; 54(5): 678-683, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31081408

RESUMO

Backgrounds: Recently, several studies have demonstrated the usefulness of cold polypectomy (CP), a safe and simple method for the removal of small polyps. We investigated the safety and efficacy of CP compared to that of endoscopic mucosal resection (EMR) and hot biopsy polypectomy (HB). Methods: We retrospectively examined 1713 colorectal polyps (size 1-9 mm) in 731 patients. CP, EMR, and HB were performed on 476, 997, and 240 lesions, respectively. We compared the region, size, morphology, the presence of delayed bleeding as overt bleeding 24 h after operation, number of clips, pathology, the presence of antithrombotic therapy, procedure time from detection of a polyp to resection and hemostasis, device cost including device and clips, and polyp remnants. Results: The delayed bleeding in the CP group (0/476) was significantly lower compared to that in the HB group (3/240) and EMR group (7/997). There were no cases of perforations. The procedure time was significantly shorter in the CP group than in the EMR group (91.3sec vs 290.1sec, p < .0001). The CP group had a significantly lower device cost than the HB and EMR groups (49.2USD vs 58.0 USD vs 91.3 USD, p < .0001) was not inferior in terms of polyp remnants to the EMR and HB groups. (1.4% vs 0.6% vs 6.1%, p = .1599) Conclusions: CP is a safe treatment that achieves less delayed bleeding. Moreover, CP is not inferior to other groups in terms of polyp remnants and offers a cost benefit. CP can be considered useful for colonic polypectomy.

8.
J Phycol ; 55(3): 534-542, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30715731

RESUMO

The life-cycle system of Ulotrichales, a major order of Ulvophyceae, remains controversial because it is unclear whether the Codiolum phase, a characteristic unicellular diploid generation in ulotrichalean algae, is a zygote or a sporophyte. This controversy inhibits the understanding of the diversified life cycles in Ulvophyceae. To distinguish between zygotes and sporophytes, we have to examine not only whether diploid generations function as sporophytes, but also whether mitosis occurs before meiosis in diploid generations. However, the nuclear behavior in the Codiolum phases is largely unknown, probably because no suitable methods are available. Using fluorescent microscopy with ethidium bromide and transmission electron microscopy of cell-wall-dissected specimens, we report the nuclear behavior in the Codiolum phases of an ulotrichalean alga with a representative life cycle, Monostroma angicava. Each vegetative Codiolum phase had a single polyploid nucleus due to endoreduplication, a type of mitosis without nuclear division. During zoosporogenesis, the nucleus had a structure that would be a meiosis-specific complex. We quantitatively showed that Codiolum phases grew extremely large and produced numerous zoospores. Our results suggest that an event comparable to mitosis occurs before meiosis in the Codiolum phase of M. angicava. This nuclear behavior and the functions (growth and zoospore production abilities) correspond to those of sporophytes. Therefore, the life-cycle system of M. angicava is a heteromorphic haplo-diplontic cycle. This system appears to be widely adopted among other ulotrichalean algae.


Assuntos
Clorófitas , Animais , Núcleo Celular , Diploide , Estágios do Ciclo de Vida
9.
Bioorg Med Chem Lett ; 29(4): 654-658, 2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30598349

RESUMO

The kisspeptin (Kp, Kp-54, metastin)/KISS1R system plays crucial roles in regulating the secretion of gonadotropin-releasing hormone. Continuous administration of nonapeptide Kp analogs caused plasma testosterone depletion, whereas bolus administration caused strong plasma testosterone elevation in male rats. To develop a new class of small peptide drugs, we focused on stepwise N-terminal truncation of Kp analogs and discovered potent pentapeptide analogs. Benzoyl-Phe-azaGly-Leu-Arg(Me)-Trp-NH2 (16) exhibited high agonist activity for KISS1R and excellent metabolic stability in rat serum. A single injection of a 4-pyridyl analog (19) at the N-terminus of 16 into male Sprague Dawley rats caused a robust increase in plasma luteinizing hormone levels, but unlike continuous administration of nonapeptide Kp analogs, continuous administration of 19 maintained moderate testosterone levels in rats. These results indicated that small peptide drugs can be successfully developed for treating sex hormone deficiency.


Assuntos
Gônadas/efeitos dos fármacos , Hipotálamo/efeitos dos fármacos , Kisspeptinas/agonistas , Hipófise/efeitos dos fármacos , Animais , Hormônio Liberador de Gonadotropina/metabolismo , Masculino , Ratos , Ratos Sprague-Dawley
10.
Front Microbiol ; 9: 1992, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30258411

RESUMO

A series of structome analyses, that is, quantitative and three-dimensional structural analysis of a whole cell at the electron microscopic level, have already been achieved individually in Exophiala dermatitidis, Saccharomyces cerevisiae, Mycobacterium tuberculosis, Myojin spiral bacteria, and Escherichia coli. In these analyses, sample cells were processed through cryo-fixation and rapid freeze-substitution, resulting in the exquisite preservation of ultrastructures on the serial ultrathin sections examined by transmission electron microscopy. In this paper, structome analysis of non pathogenic Mycolicibacterium smegmatis, basonym Mycobacterium smegmatis, was performed. As M. smegmatis has often been used in molecular biological experiments and experimental tuberculosis as a substitute of highly pathogenic M. tuberculosis, it has been a task to compare two species in the same genus, Mycobacterium, by structome analysis. Seven M. smegmatis cells cut into serial ultrathin sections, and, totally, 220 serial ultrathin sections were examined by transmission electron microscopy. Cell profiles were measured, including cell length, diameter of cell and cytoplasm, surface area of outer membrane and plasma membrane, volume of whole cell, periplasm, and cytoplasm, and total ribosome number and density per 0.1 fl cytoplasm. These data are based on direct measurement and enumeration of exquisitely preserved single cell structures in the transmission electron microscopy images, and are not based on the calculation or assumptions from biochemical or molecular biological indirect data. All measurements in M. smegmatis, except cell length, are significantly higher than those of M. tuberculosis. In addition, these data may explain the more rapid growth of M. smegmatis than M. tuberculosis and contribute to the understanding of their structural properties, which are substantially different from M. tuberculosis, relating to the expression of antigenicity, acid-fastness, and the mechanism of drug resistance in relation to the ratio of the targets to the corresponding drugs. In addition, data obtained from cryo-transmission electron microscopy examination were used to support the validity of structome analysis. Finally, our data strongly support the most recent establishment of the novel genus Mycolicibacterium, into which basonym Mycobacterium smegmatis has been classified.

11.
Sci Rep ; 8(1): 7054, 2018 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-29728586

RESUMO

We experimentally demonstrated a narrowband acoustic phonon source with simultaneous tunabilities of the centre frequency and the spectral bandwidth in the GHz-sub THz frequency range based on photoacoustic excitation using intensity-modulated optical pulses. The centre frequency and bandwidth are tunable from 65 to 381 GHz and 17 to 73 GHz, respectively. The dispersion of the sound velocity and the attenuation of acoustic phonons in silicon dioxide (SiO2) and indium tin oxide (ITO) thin films were investigated using the acoustic phonon source. The sound velocities of SiO2 and ITO films were frequency-independent in the measured frequency range. On the other hand, the phonon attenuations of both of SiO2 and ITO films showed quadratic frequency dependences, and polycrystalline ITO showed several times larger attenuation than those in amorphous SiO2. In addition, the selective excitation of mechanical resonance modes was demonstrated in nanoscale tungsten (W) film using acoustic pulses with various centre frequencies and spectral widths.

12.
Med Mycol J ; 59(1): E1-E6, 2018.
Artigo em Japonês | MEDLINE | ID: mdl-29491337

RESUMO

This article presents the ultrastructural patterns of interactions between the murine lung macrophages and cells of low- (RKPGY-881, -1165, -1178) and high-virulence (RKPGY-1090, -1095, -1106) strains of Cryptococcus neoformans at the seventh post-experimental day. It was found that if macrophages ingest living yeast cells, the latter can: 1) become completely free from polysaccharide capsules, after that their contents undergo lysis, and cell wall debris are extruded from the macrophage (first scenario); 2) become partly free from their capsules, destroy the phagosomal plasma membrane and induce destructive processes inside the macrophage causing their death (second scenario); or 3) not lose their capsules and localize inside macrophage in latent state (third scenario). Macrophages can also ingest senescent and dead C. neoformans cells surrounded by capsules that are lost at the ingesting and phagosome stages (fourth scenario). The study revealed the dependence of cell-mediated immunity on the stage of development of ingested C. neoformans yeast cells. Here we describe a new mechanism of capsular polysaccharide elimination of C. neoformans yeast cells by murine macrophages.


Assuntos
Cryptococcus neoformans/imunologia , Cryptococcus neoformans/ultraestrutura , Macrófagos Alveolares/imunologia , Macrófagos Alveolares/ultraestrutura , Fagocitose , Animais , Cryptococcus neoformans/metabolismo , Cryptococcus neoformans/patogenicidade , Cápsulas Fúngicas/metabolismo , Cápsulas Fúngicas/ultraestrutura , Polissacarídeos Fúngicos/metabolismo , Imunidade Celular/imunologia , Masculino , Camundongos , Fagossomos , Virulência
13.
J Vis Exp ; (131)2018 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-29364224

RESUMO

Observing cells and cell components in three dimensions at high magnification in transmission electron microscopy requires preparing serial ultrathin sections of the specimen. Although preparing serial ultrathin sections is considered to be very difficult, it is rather easy if the proper method is used. In this paper, we show a step-by-step procedure for safely obtaining serial ultrathin sections of microorganisms. The key points of this method are: 1) to use the large part of the specimen and adjust the specimen surface and knife edge so that they are parallel to each other; 2) to cut serial sections in groups and avoid difficulty in separating sections using a pair of hair strands when retrieving a group of serial sections onto the slit grids; 3) to use a 'Section-holding loop' and avoid mixing up the order of the section groups; 4) to use a 'Water-surface-raising loop' and make sure the sections are positioned on the apex of the water and that they touch the grid first, in order to place them in the desired position on the grids; 5) to use the support film on an aluminum rack and make it easier to recover the sections on the grids and to avoid wrinkling of the support film; and 6) to use a staining tube and avoid accidentally breaking the support films with tweezers. This new method enables obtaining serial ultrathin sections without difficulty. The method makes it possible to analyze cell structures of microorganisms at high resolution in 3D, which cannot be achieved by using the automatic tape-collecting ultramicrotome method and serial block-face or focused ion beam scanning electron microscopy.


Assuntos
Eucariotos/ultraestrutura , Técnicas Histológicas/métodos , Microscopia Eletrônica de Transmissão/métodos , Microtomia/métodos , Células Procarióticas/ultraestrutura , Técnicas Histológicas/instrumentação , Microscopia Eletrônica de Transmissão/instrumentação , Microtomia/instrumentação
16.
Cancer Sci ; 109(1): 121-131, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29121435

RESUMO

Tyrosine kinase inhibitors (TKI), including imatinib (IM), improve the outcome of CML therapy. However, TKI treatment is long-term and can induce resistance to TKI, which often leads to a poor clinical outcome in CML patients. Here, we examined the effect of continuous IM exposure on intracellular energy metabolism in K562 cells, a human Philadelphia chromosome-positive CML cell line, and its subsequent sensitivity to anti-cancer agents. Contrary to our expectations, we found that continuous IM exposure increased sensitivity to TKI. Cancer energy metabolism, characterized by abnormal glycolysis, is linked to cancer cell survival. Interestingly, glycolytic activity was suppressed by continuous exposure to IM, and autophagy increased to maintain cell viability by compensating for glycolytic suppression. Notably, increased sensitivity to TKI was not caused by glycolytic inhibition but by altered intracellular signaling, causing glycolytic suppression and increased autophagy, as evidenced by suppression of p70 S6 kinase 1 (S6K1) and activation of AMP-activated protein kinase (AMPK). Using another human CML cell line (KCL22 cells) and BCR/ABL+ Ba/F3 cells (mimicking Philadelphia chromosome-positive CML cells) confirmed that suppressing S6K1 and activating AMPK increased sensitivity to TKI. Furthermore, suppressing S6K1 and activating AMPK had a synergistic anti-cancer effect by inhibiting autophagy in the presence of TKI. The present study provides new insight into the importance of signaling pathways that affect cellular energy metabolism, and suggests that co-treatment with agents that disrupt energy metabolic signaling (using S6K1 suppressors and AMPK activators) plus blockade of autophagy may be strategies for TKI-based CML therapy.


Assuntos
Proteínas Quinases Ativadas por AMP/genética , Mesilato de Imatinib/farmacologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases S6 Ribossômicas 70-kDa/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sinergismo Farmacológico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glicólise/efeitos dos fármacos , Humanos , Células K562 , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Transdução de Sinais/efeitos dos fármacos
17.
Cell Microbiol ; 20(3)2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29113011

RESUMO

Aspergillus fumigatus is an important fungal pathogen of humans. Inhaled conidia of A. fumigatus adhere to pulmonary epithelial cells, causing opportunistic infection. However, little is known about the molecular mechanism of the adherence of resting conidia. Fungal molecules adhesive to host cells are presumed to be displayed on the conidial surface during conidial formation as a result of changes in gene expression. Therefore, we exhaustively searched for adhesion molecules by comparing the phenotypes and the gene expression profiles of A. fumigatus strains that have conidia showing either high or low adherence to human pulmonary A549 cells. Morphological observation suggested that strains that produce conidia of reduced size, hydrophobicity, or number show decreased adherence to A549 cells. K-means cluster analyses of gene expression revealed 31 genes that were differentially expressed in the high-adherence strains during conidial formation. We knocked out three of these genes and showed that the conidia of AFUA_4G01030 (encoding a hypothetical protein) and AFUA_4G08805 (encoding a haemolysin-like protein) knockout strains had significantly reduced adherence to host cells. Furthermore, the conidia of these knockout strains had lower hydrophobicity and fewer surface spikes compared to the control strain. We suggest that the selectively expressed gene products, including those we identified experimentally, have composite synergistic roles in the adhesion of conidia to pulmonary epithelial cells.


Assuntos
Aspergillus fumigatus/genética , Aspergillus fumigatus/metabolismo , Proteínas Fúngicas/genética , Células A549 , Análise por Conglomerados , Proteínas Fúngicas/metabolismo , Perfilação da Expressão Gênica , Humanos , Análise de Componente Principal , Esporos Fúngicos/genética , Esporos Fúngicos/metabolismo
18.
Oncotarget ; 8(55): 94271-94285, 2017 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-29212227

RESUMO

Acute lymphoblastic leukemia (ALL) is the most common childhood malignancy. Treatments include glucocorticoids (GCs) such as dexamethasone (Dex) and prednisolone, which may be of value when used alongside cytotoxic anti-cancer drugs. To predict therapeutic efficacy of GCs, their activity against ALL cells is usually examined prior to chemotherapy; however, few studies have examined their effects when used in combination with other drugs. The paradox is that cytotoxic anti-cancer drugs that are effective against proliferating cancer cells show synergistic effects when used with GCs that prevent cell proliferation. To address this point, we investigated intracellular energy metabolism in ALL CCRF-CEM cell clones classified according to their sensitivity to Dex and cytotoxic anti-cancer drugs in bulk cultures of mixed cells. We found that Dex suppressed glycolysis, the most important metabolic system in cancer cells, in cells that were damaged by etoposide (a cytotoxic anti-cancer drug), and the cells showed a concomitant increase in mitochondrial oxidative phosphorylation. Furthermore, autophagy, an intracellular bulk degradation system, regulated mitochondrial viability. We also found that mitochondria, whose function is enhanced by Dex, were susceptible to anti-cancer drugs that inhibit respiratory complexes (e.g., etoposide and daunorubicin), resulting in increased production of reactive oxygen species and subsequent cytotoxicity. Taken together, the present study points the way toward a more accurate prediction of the sensitivity of ALL cells to the combined action of anti-cancer drugs and GCs, by taking into consideration the shift in intracellular energy metabolism caused by GCs: namely, from glycolysis to mitochondrial oxidative phosphorylation mediated by autophagy.

19.
Microscopy (Oxf) ; 66(4): 283-294, 2017 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-28854579

RESUMO

Structome analysis, the quantitative three-dimensional structural analysis of whole cells at the electron microscopic level, of Exophiala dermatitidis (black yeast), Saccharomyces cerevisiae, Mycobacterium tuberculosis (MTB) and Myojin spiral bacteria (MSB) have already been reported. Here, the results of the structome analysis of Escherichia coli cells based on transmission electron microscope observation of serial ultrathin sections was reported, and compared with the data obtained from phase contrast microscopy and scanning electron microscopy. On average, the cells had 0.89 µm in diameter, 2.47 µm in length and 1.16 fl (µm3) in cell volume in the structome analysis. Furthermore, E. coli cells had 26 100 ribosomes per whole cell with density of 2840 per 0.1 fl cytoplasm. The total ribosome number per cell was 15 times larger than that of MTB and about one-eighth of those of the yeast cells above. On the other hand, the ribosome density of E. coli cells are more than 13 times, 4 times, 2.5-times and 1.5-times higher than MSB, MTB, E. dermatitidis and S. cerevisiae, respectively. Finally, our ribosome enumeration data were compared between the structome-analyzed species and the relationship between the ribosome density and the growth rate among these species was discussed.


Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/ultraestrutura , Ribossomos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/biossíntese , Processamento de Imagem Assistida por Computador , Microscopia Eletrônica de Transmissão/métodos
20.
Curr Genet ; 63(6): 1093-1104, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28560585

RESUMO

We constructed deletion mutants of Cryptococcus neoformans var neoformans (serotype D) genes encoding late ergosterol biosynthetic pathway enzymes and found that the mutations enhanced susceptibility to various drugs including micafungin, one of the echinocandins, to which wild-type Cryptococcus strains show no susceptibility. Furthermore, through isolation of a mutant resistant to micafungin from a micafungin-sensitive erg mutant and genetic analysis of it, we found that the responsible mutation occurred in the hotspot 2 of FKS1 encoding ß-1, 3-glucan synthase, indicating that micafungin inhibited the growth of the erg mutant via inhibiting Fks1 activity. Addition of ergosterol to the culture of the erg mutants recovered the resistance to micafungin, suggesting that the presence of ergosterol in membrane inhibits the accession of micafungin to its target. We found that a loss of one of genes encoding subunits of v-ATPase, VPH1, made Cryptococcus cells sensitive to micafungin. Our observation that the erg2 vph1 double mutant was more sensitive to micafungin than either single mutant suggests that these two genes act differently in becoming resistant to micafungin. The erg mutants allowed us to study the physiological significance of ß-1, 3-glucan synthesis in C. neoformans; the inhibition of ß-1, 3-glucan synthesis induced cell death and changes in cellular morphology. By observing the erg mutant cells recovering from the growth inhibition imposed by micafungin, we recognized ß-1, 3-glucan synthesis would suppress filamentous growth in C. neoformans.


Assuntos
Cryptococcus neoformans/genética , Farmacorresistência Fúngica/genética , Equinocandinas/farmacologia , Regulação Fúngica da Expressão Gênica , Glucosiltransferases/genética , Lipopeptídeos/farmacologia , ATPases Vacuolares Próton-Translocadoras/genética , Antifúngicos/farmacologia , Cryptococcus neoformans/efeitos dos fármacos , Cryptococcus neoformans/enzimologia , Cryptococcus neoformans/crescimento & desenvolvimento , Ergosterol/biossíntese , Ergosterol/farmacologia , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Engenharia Genética , Glucosiltransferases/deficiência , Micafungina , Testes de Sensibilidade Microbiana , Mutação , Subunidades Proteicas/deficiência , Subunidades Proteicas/genética , ATPases Vacuolares Próton-Translocadoras/deficiência
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